Dr Rose is a full Professor at the Department of Medicine at the Université de Montréal and researcher at CRCHUM since 2006. He's also affiliated to pharmacology, neuroscience and nutrition departments.
Full Professor, Departments of medicine, pharmacology, neurosciences and nutrition, Université de Montréal
2006 - now
Social media profiles :
Career
- 2006-now: Research professor at Université de Montréal.
- 2003-2006: Post-doctoral Fellow (Canadian Institutes of Health Research),
Laboratory of Neurobiology, Valencia, Spain. - 2001-2003: Post-doctoral Fellow (Foundation Alexander von Humboldt),
Max-Delbrück Center for Molecular Medicine, Cellular Neuroscience, Berlin, Germany. - 1994-2000: Ph.D., Université de Montréal, under the supervision of Dr Roger Butterworth
Related Publications
Lessons on brain edema in HE: from cellular to animal models and clinical studies.
Katarzyna Pierzchala, Anna Hadjihambi, Jessie Mosso, Rajiv Jalan, Christopher F. Rose, Cristina Cudalbu.
Brain edema is considered as a common feature associated with hepatic encephalopathy (HE). However, its central role as cause or consequence of HE and its implication in the development of the neurological alterations linked to HE are still under debate. It is now well accepted that type A and type C HE are biologically and clinically different, leading to different manifestations of brain edema. As a result, the findings on brain edema/swelling in type C HE are variable and sometimes controversial. In the light of the changing natural history of liver disease, better description of the clinical trajectory of cirrhosis and understanding of molecular mechanisms of HE, and the role of brain edema as a central component in the pathogenesis of HE is revisited in the current review. Furthermore, this review highlights the main techniques to measure brain edema and their advantages/disadvantages together with an in-depth description of the main ex-vivo/in-vivo findings using cell cultures, animal models and humans with HE. These findings are instrumental in elucidating the role of brain edema in HE and also in designing new multimodal studies by performing in-vivo combined with ex-vivo experiments for a better characterization of brain edema longitudinally and of its role in HE, especially in type C HE where water content changes are small.
Measuring and Interpreting Ammonia Levels in Cirrhosis
Maria Pilar Ballester, Christopher F. Rose, Juan-Antonio Carbonell-Asins, Rajiv Jalan.
175 Years of Bilirubin Testing: Ready for Point-of-Care?
Natalie Guirguis, Antoine-Xavier Bertrand, Christopher F. Rose, Simon Matoori.
Bilirubin was first detected in blood in 1847 and since then has become one of the most widely used biomarkers for liver disease. Clinical routine bilirubin testing is performed at the hospital laboratory, and the gold standard colorimetric test is prone to interferences. The absence of a bedside test for bilirubin delays critical clinical decisions for patients with liver disease. This clinical care gap has motivated the development of a new generation of bioengineered point-of-care bilirubin assays. In this Perspective, recently developed bilirubin assays are critically discussed, and their translational potential evaluated.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, André Marette, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome arising from chronic liver disease (CLD). HE manifests with symptoms such as poor memory, impairment in motor coordination, lethargy and coma. The gut microbiota has been shown to influence neurological functions via various mediators such as cytokines or bacterial metabolites, many studies have demonstrated the gut-brain axis is altered in liver disease. Faecal matter transplantation (FMT) in patients with cirrhosis has revealed beneficial effects yet many limitations of these studies render the results inconclusive. Purpose: The aim of this study is to explore the impact of FMT on gut microbiota and the beneficial effects on neuro behaviour in bile-duct ligated (BDL) rats. Method: Male Sprague-Dawley rats were randomly assigned to one of three groups; SHAM, BDL-VEH (vehicle) and BDL-FMT (who received FMT daily from pooled faeces from SHAM rats). After five weeks, behaviour tests were performed to evaluate short- and long-term memory (Novel Object Recognition), anxiety (Open Field and Elevated Plus Maze) and motor coordination (Rotarod). Plasmatic parameters such as cytokines, short chain fatty acids (SCFA) and liver impairment markers were measured by ELISA, LC-MS/MS and using Cobas respectively. Finally, faeces were collected for bacterial sequencing and SCFA analysis. Results: FMT did not alter degree of liver disease in BDL rats. BDL-VEH developed a loss of short/long term memory and motor coordination compared to SHAM rats. However, alterations in neurological dysfunction were prevented in the BDL-FMT group. FMT did not impact microbiota α-diversity in BDL rats and β-diversity of microbiota was significantly different between all groups. The genera Provotellaceae UCO-001 significantly increased only in SHAM and BDL-FMT rats and Clostridium Senso Stricto 1 significantly increased only in BDL-VEH compared to SHAMs. Finally, Rombustia was only present in SHAM. Plasma pro-inflammatory cytokines (TNF-α & IL-1β) increase in both BDL groups compared to the control group and no difference for the anti-inflammatory cytokine IL-10 was noted. Analysis of short-chain fatty acids in faeces and plasma showed a variation in propionate and butyrate between both BDL groups. Plasma propionate significantly positively correlated with behavioural results. Conclusion: Our results demonstrate that FMT leads to improvement in memory and motor coordination in BDL rats. The microbiota profile was different between BDL-VEH and SHAM, and FMT lead to further alterations on microbiota. The fact that FMT did not normalize microbiota profile compared to SHAM, suggests BDL-FMT leads to a novel specific microbiota profile which in turn protects the brain. The protective effect of plasma propionate needs to be further explored to define its impact on the brain and possible therapeutic application.
Farzaneh Tamnanloo, Xiaoru Chen, Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a debilitating neurological complication of chronic liver disease. Alcohol is a major etiological factor known to induce liver injury and disease. However, excessive alcohol consumption has been shown to also induce atrophy of the cerebellum and cerebellar degeneration. To date, the role of alcohol in the development of HE remains unclear. Here we examined the effects of constant alcohol consumption on the neurological decline in rats with chronic liver disease induced following bile duct ligation (BDL). Method: 5-week BDL rats and Sham-operated controls (Sham) were used. Starting day 7 after surgery, rats were gavaged twice a day (3 hours apart) with alcohol at a dose of 3g/kg, 5 days per week, for 4 weeks. Motor coordination was assessed using Rotarod every week until week 5. At the end of the model (day 40), anxiety-like behavior was assessed using the open field (OF) and elevated plus maze (EPM). Upon sacrifice, brains were collected, and western blot and immunohistochemical (IHC) analyses were used to investigate the neuronal integrity as well as assess apoptosis and necroptosis pathways in the cerebellum. Results: Alcohol worsened motor coordination performance in weeks 2, 3, 4, and 5 in BDL-alcohol rats (p<0.01 vs respective shams). Anxiety-like behavior significantly increased in BDL-alcohol rats, with an increase in time spent in the closed arms of EPM and a decrease in time spent in the center of the open field (p<0.05 vs respective shams). These impairments were associated with decreased neuronal markers of NeuN and SMI311 (p<0.01 and p<0.05, respectively), increased apoptotic markers of cleaved/pro-Caspase3 and Bax/Bcl2 ratio (p<0.001 and p<0.01 respectively), increased necroptosis markers of pRIP3 and pMLKL (p<0.01 and p<0.001, respectively), decreased total antioxidant capacity (p<0.001) and increased oxidative stress marker of 4-HNE (p<0.05) in the cerebellum of BDL-alcohol rats when compared to respective controls. IHC results confirmed the colocalization of apoptotic marker (cleaved Caspase3) and necroptosis marker (pMLKL) in the granular and Purkinje layer neurons of the cerebellum of BDL-alcohol rats. Conclusion: Constant alcohol consumption exacerbates HE by worsening motor coordination impairment and increasing anxiety in BDL rats. Furthermore, our results show neuronal loss through apoptosis and necroptosis in the cerebellum of BDL-alcohol rats. Additionally, higher levels of oxidative stress marker of 4-HNE and decreased total antioxidant capacity in the cerebellum of BDL-alcohol rats suggest that oxidative stress is the triggering factor of apoptosis and necroptosis pathway leading to neuronal loss/injury. These results demonstrate the adverse effect of constant alcohol consumption on the development of HE and neuronal integrity in chronic liver disease.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Kraine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique résultant d'une maladie du foie. Il a été démontré que le microbiote intestinal influence le cerveau et l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif : Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats mâles ont été randomisés en trois groupes : SHAM, BDL-VEH et BDL-FMT recevant quotidiennement la FMT provenant des rats SHAM. Après cinq semaines, des tests comportementaux sont effectués pour évaluer la mémoire à court/long terme, l'anxiété et la coordination motrice. Les fèces et plasma ont été collectés pour séquençage bactérien et analyses. Résultats: Les BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice comparée aux rats SHAM. Cependant, les altérations neurologiques sont prévenues dans le groupe BDL-FMT. Une modulation du microbiote a été constatée pour les rats BDL-FMT comparé aux BDL-VEH. Les cytokines (TNF-α et IL-1β) ne varient entre les groupes BDL. L'analyse des acides gras à chaîne courte dans les fèces et le plasma a montré une variation du propionate et du butyrate entre les groupes BDL. Le propionate plasmatique est positivement corrélé aux scores de comportement. Discussion: Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT conduit à un nouveau profil spécifique du microbiote avec la présence du propionate comme métabolite à explorer.
Sydnée L’Écuyer, Alexandra Matesan, Farzaneh Tamnanloo, Mariana Oliveira, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique résultant d'une maladie du foie. Il a été démontré que le microbiote intestinal influence le cerveau et l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif : Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats mâles ont été randomisés en trois groupes : SHAM, BDL-VEH et BDL-FMT recevant quotidiennement la FMT provenant des rats SHAM. Après cinq semaines, des tests comportementaux sont effectués pour évaluer la mémoire à court/long terme, l'anxiété et la coordination motrice. Les fèces et plasma ont été collectés pour séquençage bactérien et analyses. Résultats: Les BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice comparée aux rats SHAM. Cependant, les altérations neurologiques sont prévenues dans le groupe BDL-FMT. Une modulation du microbiote a été constatée pour les rats BDL-FMT comparé aux BDL-VEH. Les cytokines (TNF-α et IL-1β) ne varient entre les groupes BDL. L'analyse des acides gras à chaîne courte dans les fèces et le plasma a montré une variation du propionate et du butyrate entre les groupes BDL. Le propionate plasmatique est positivement corrélé aux scores de comportement. Discussion: Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT conduit à un nouveau profil spécifique du microbiote avec la présence du propionate comme métabolite à explorer.
Farzaneh Tamnanloo, Xiaoru Chen, Mélanie Tremblay, Christopher F. Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique résultant d'une maladie du foie. Il a été démontré que le microbiote intestinal influence le cerveau et l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif : Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats mâles ont été randomisés en trois groupes : SHAM, BDL-VEH et BDL-FMT recevant quotidiennement la FMT provenant des rats SHAM. Après cinq semaines, des tests comportementaux sont effectués pour évaluer la mémoire à court/long terme, l'anxiété et la coordination motrice. Les fèces et plasma ont été collectés pour séquençage bactérien et analyses. Résultats: Les BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice comparée aux rats SHAM. Cependant, les altérations neurologiques sont prévenues dans le groupe BDL-FMT. Une modulation du microbiote a été constatée pour les rats BDL-FMT comparé aux BDL-VEH. Les cytokines (TNF-α et IL-1β) ne varient entre les groupes BDL. L'analyse des acides gras à chaîne courte dans les fèces et le plasma a montré une variation du propionate et du butyrate entre les groupes BDL. Le propionate plasmatique est positivement corrélé aux scores de comportement. Discussion: Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT conduit à un nouveau profil spécifique du microbiote avec la présence du propionate comme métabolite à explorer.
Nutritional education strategies for patients with cirrhosis: A narrative review.
Manila Sophasath, Alexandre Brisset, Christopher F. Rose, Chantal Bémeur.
Patients with cirrhosis suffer from many complications, including malnutrition, which must be managed promptly and effectively by the healthcare team. Educating patients about their medical condition, the risk of malnutrition and other complications of cirrhosis, could contribute to optimal nutritional status, quality of life and general health. This review provides an overview of the literature on a variety of nutritional education strategies used with patients suffering from cirrhosis. This review also identifies barriers and facilitators which impact the adherence in using these strategies. A patient-partner contributed to this review by providing insights on different issues and concerns that patients with cirrhosis might ask themselves regarding nutritional education strategies. The patient-partner was also involved in the overall revision of the review. Articles published between the years 2000-2023 focusing on nutritional education strategies in patients living with cirrhosis were identified using Google Scholar and PubMed and were screened for inclusion in the study. All selected studies were intervention studies. A quality assessment of the included studies was conducted using the Mixed Methods Appraisal Tool (MMAT). Only a few nutritional education strategies in patients with cirrhosis were documented in the literature. The strategies ranged from using traditional printed materials to advanced technologies. These strategies may prove beneficial in complementing routine interventions provided by health professionals, such as registered dietitians, in their clinical practice. This narrative review clearly highlights the need for further research to elaborate and evaluate nutritional education strategies for people living with cirrhosis. Elaborating and evaluating educational strategies in nutrition for patients living with cirrhosis will be an adjuvant to health professionals and dietitians in their clinical practice by providing them, and the patients, with targeted education resources.
Sydnée L’Écuyer, Alexandra Matesan, Farzaneh Tamnanloo, Mariana Oliveira, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.
Introduction- Environ 40% des patients cirrhotiques souffrent de complications cognitives réduisant leur qualité de vie. Nous nous intéressons à la contribution de l’acide urique (AU), un médiateur reconnu pour ses effets cérébraux. Notre modèle murin démontre comment la cirrhose et l’hyperuricémie peuvent influencer les changements de comportement et les lésions cérébrales. Méthodes- Nos animaux sont divisés en 4 groupes expérimentaux prenant en compte la cirrhose (SHAM (contrôle) ou BDL (ligature de la voie biliaire)) et l’hyperuricémie (diète régulière (RD) ou diète 3% AU (HUAD)) : (1) SHAM+RD, (2) SHAM+HUAD, (3) BDL+RD et (4) BDL+HUAD. Des prélèvements sanguins sont effectués aux jours 0, 7, 14, 21 et 28 pour mesurer l’AU plasmatique. La mémoire et l’anxiété sont évaluées aux jours 14 et 28. Au jour 33, les animaux sont sacrifiés et le cortex frontal, l’amygdale et l’hippocampe sont isolés pour analyser la mort cellulaire (caspase-3 et caspase-1). Résultats- L’AU circulant augmente chez les BDL+HUAD à partir du jour 7. Au jour 14, la mémoire à court terme diminue chez les BDL+HUAD et l’anxiété augmente dans les groupes HUAD. Au jour 28, la mémoire à long terme diminue chez les SHAM+HUAD. La caspase-3 augmente dans le cortex frontal pour les animaux SHAM+HUAD, dans l’amygdale pour les animaux BDL+HUAD et dans l’hippocampe pour les HUAD. La caspase-1 augmente significativement dans l’amygdale et l’hippocampe des animaux HUAD. Conclusion- Chez les animaux cirrhotiques, l’hyperuricémie entraîne une perte cellulaire dans l’amygdale et l’hippocampe, suggérant un lien avec les comportements anxieux et la perte de mémoire.
Farzaneh Tamnanloo, Xiaoru Chen, Mélanie Tremblay, Christopher F. Rose.
Contexte : L'encéphalopathie hépatique (EH) est une complication neurologique débilitante de la maladie hépatique chronique (MHC). L'alcool est un facteur étiologique majeur connu pour induire des dommages hépatiques, mais son rôle dans le développement de l'EH reste flou. Nous avons examiné les effets de la consommation constante d'alcool sur le déclin neurologique chez les rats atteints de MHC. Méthode : Des rats opérés avec une ligation de voie biliaire (BDL) et des témoins (Sham) après 5 semaines ont été utilisés. Au jour 7, les rats ont été gavés deux fois par jour avec de l'éthanol (3 g/kg, 5 j/sem, pendant 4 sem). La coordination motrice a été évaluée par Rotarod et le comportement anxieux par champ ouvert et labyrinthe surélevé. Nous avons effectué des immunobuvardages afin d'étudier l'intégrité neuronale dans le cervelet. Résultats : L'alcool a aggravé la coordination motrice et augmenté le comportement de type anxieux chez les rats BDL-alcool. Les déficiences étaient associées à une diminution des marqueurs neuronaux de NeuN et SMI311, une augmentation des marqueurs apoptotiques pro-Caspase3/clivée et Bax/Bcl2 et de nécroptose de pRIP3 et pMLKL, une diminution de la capacité antioxydante totale et une augmentation d’un marqueur de stress oxydatif, le 4-HNE, dans le cervelet des rats BDL-alcool par rapport aux témoins respectifs. Conclusion : Les niveaux plus élevés de 4-HNE et la diminution de la capacité antioxydante dans le cervelet des rats BDL-alcool suggèrent que le stress oxydatif est le facteur déclenchant de l'apoptose et de la nécroptose entraînant une perte/lésion neuronale.
Sydnée L’Écuyer, Alexandra Matesan, Farzaneh Tamnanloo, Mariana Oliveira, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.
Background: Over 40% of patients with liver cirrhosis also suffer from cognitive impairments, mainly from hepatic encephalopathy (HE). This reversible and episodic syndrome leads to gross disorientation, asterixis, memory impairments, and death and is associated with an increase in blood ammonia. However, some patients with cirrhosis present with cognitive alterations in absence of increased ammonia circulation. This justifies investigating other factors that may induce further cerebral lesions than what is seen in HE. With this project, we focus on the role of uric acid (UA) and hyperuricemia in inducing cognitive impairments in cirrhosis. Hyperuricemia is associated with metabolic syndrome and chronic alcohol consumption which are known precipitating factors of CLD. Furthermore, uric acid (UA) has been reported to be involved in the induction of neuroinflammation and cell loss by apoptosis and pyroptosis, leading to anxiety-like behavior and memory impairment. This project uses a joint murine model of chronic liver disease and hyperuricemia to investigate behavioral alterations and cerebral lesions. Method: To induce liver cirrhosis we used the well-characterized model of bile-duct ligation (BDL) knowing that these animals also develop symptoms of ammonia-related HE. Fifty-two male Sprague-Dawley rats (200-240 g) were randomly assigned to a control (SHAM) or a BDL surgery. To induce hyperuricemia, half of the animals were fed a high uric acid diet (HUAD) (3% uric acid), dividing our rats into 4 groups: (1) SHAM+regular diet (RD), (2) SHAM+HUAD, (3) BDL+RD and (4) BDL+HUAD. Plasma UA measurements were made on days 0, 7, 14, 21, and 28. Behavior tests were conducted to measure anxiety (open-field test and elevated plus-maze) and short and long-term memory (novel-object recognition) at days 14 and 28. At day 33, the rats were sacrificed, their brains collected, and the frontal cortex, hippocampus, and amygdala were isolated to assess cell death by measuring caspase-3 (apoptosis) and caspase-1 (pyroptosis) activity. Result(s): HUAD significantly increased circulating UA in BDL rats. This was associated with a significant decrease in short-term memory without changes in anxiety-like behavior for HUAD rats compared to their RD counterparts in both SHAMs and BDLs. For long-term memory, BDLs were shown to be significantly impaired compared to SHAMs. A significantly higher degree of impairment was seen in BDL+HUAD rats compared to BDL+RD. Cell death (apoptosis and pyroptosis) was then evaluated in the limbic system to explain some of the behavioral changes. In the frontal cortex, we can see a significant increase of caspase-3 activity in the SHAM+HUAD animals when compared to the SHAM+RD rats and no changes for caspase-1 activity. In the amygdala, we see a significant increase of caspase-3 activity in the BDL+HUAD rats when compared to all three other groups and a significant increase of caspase-1 in the SHAM+HUAD and BDL+HUAD groups when compared to their RD counterparts. In the hippocampus, we see a significant increase of caspase-3 activity for the SHAM+HUAD and BDL+HUAD groups when compared to their RD counterparts. We also detect a significant increase of caspase-1 activity in both HUAD groups. Conclusion(s): In SHAM rats, diet-induced hyperuricemia shows a significant increase in circulating uric acid concentration as well as short-term memory loss associated with apoptosis (caspase-3 activity) in the frontal cortex and the hippocampus. In BDL rats, hyperuricemia induces long-term memory impairments and apoptosis in the frontal cortex as well as pyroptosis in the hippocampus. The results from this study show the merit of further investigating the role of hyperuricemia in cognitive impairment in the context of liver cirrhosis.
Farzaneh Tamnanloo, Xiaoru Chen, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a debilitating neurological complication of chronic liver disease with alcohol being a common etiological factor. However, excessive alcohol consumption has been shown to impact neurological integrity. To date, the influence of alcohol in the development of HE remains unclear. Therefore, we examined the effect of constant alcohol consumption on neurological decline in rats with chronic liver disease induced via bile-duct ligation (BDL). Method: 5-week BDL rats and Sham-operated controls were used. Day 7 after surgery, rats were administered alcohol twice a day (dose of 3g/kg, via gavage) for 4 weeks. Motor coordination (rotarod) and anxiety-like behavior (open field (OF) and elevated plus maze (EPM)) were assessed. Upon sacrifice, brains were collected, and western blot and immunohistochemical (IHC) analyses were used to investigate neuronal integrity in frontal cortex and cerebellum. Results: Alcohol (BDL-alcohol rats) further impaired motor coordination at weeks 2, 3, 4, and 5 compared to SHAM-alcohol (p<0.01). Furthermore, BDL-alcohol rats demonstrated an increase in anxiety-like behavior; increase in time spent in the closed arms of EPM and decrease in time spent in the center of the OF (p<0.05 vs SHAM-alcohol). BDL-alcohol rats demonstrated a decrease in neuronal markers of NeuN and SMI311 (p<0.01 and p<0.05, respectively), an increase in apoptotic markers of cleaved/pro-caspase3 (p<0.001), an increase in necroptosis markers of pRIP3 and pMLKL (p<0.01 and p<0.001, respectively), a decrease in total antioxidant capacity (p<0.001) and an increase in oxidative stress marker of 4-HNE (p<0.05) in the cerebellum (not found in frontal cortex) compared to all groups. IHC results confirmed the colocalization of apoptotic marker (cleaved Caspase3) and necroptosis marker (pMLKL) in the granular and Purkinje layer neurons of the cerebellum of BDL-alcohol rats. Conclusion: Constant alcohol consumption exacerbates HE and leads to neuronal loss via apoptosis and necroptosis in the cerebellum. Additionally, higher levels of oxidative stress marker of 4-HNE and decreased total antioxidant capacity in the cerebellum of BDL-alcohol rats suggest that oxidative stress is a triggering factor leading to neuronal loss/injury. These results demonstrate an adverse effect of constant alcohol consumption on the development of HE and neuronal integrity in chronic liver disease.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome arising from chronic liver disease (CLD). HE manifests with symptoms such as poor memory, impairment in motor coordination, lethargy and coma. The gut microbiota has been shown to influence neurological functions via various mediators such as cytokines or bacterial metabolites, many studies have demonstrated the gut-brain axis is altered in liver disease. Faecal matter transplantation (FMT) in patients with cirrhosis has revealed beneficial effects yet many limitations of these studies render the results inconclusive. Purpose: The aim of this study is to explore the impact of FMT on gut microbiota and the beneficial effects on neuro behaviour in bile-duct ligated (BDL) rats. Method: Male Sprague-Dawley rats were randomly assigned to one of three groups; SHAM, BDL-VEH (vehicle) and BDL- FMT (who received FMT daily from pooled faeces from SHAM rats). After five weeks, behaviour tests were performed to evaluate short- and long-term memory (Novel Object Recognition), anxiety (Open Field and Elevated Plus Maze) and motor coordination (Rotarod). Plasmatic parameters such as cytokines, short chain fatty acids (SCFA) and liver impairment markers were measured by ELISA, LC-MS/MS and using Cobas respectively. Finally, faeces were collected for bacterial sequencing and SCFA analysis. Result(s): FMT did not alter degree of liver disease in BDL rats. BDL-VEH developed a loss of short/long term memory and motor coordination compared to SHAM rats. However, alterations in neurological dysfunction were prevented in the BDL-FMT group. FMT did not impact microbiota α-diversity in BDL rats and β-diversity of microbiota was significantly different between all groups. The genera Bifidobacterium, Lactobacillus and Akkermansia significantly increased in both BDL groups, while Provotellaceae UCO-001 significantly increased only in SHAM and BDL-FMT rats and Clostridium Senso Stricto 1 significantly increased only in BDL-VEH compared to SHAMs. Finally, Rombustia was only present in SHAM. Plasma pro-inflammatory cytokines (TNF-α & IL-1β) increase in both BDL groups compared to the control group and no difference for the anti-inflammatory cytokine IL-10 was noted. Analysis of short-chain fatty acids in faeces and plasma showed a variation in propionate and butyrate between both BDL groups. Plasma propionate significantly positively correlated with behavioural results.
Manila Sophasath, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Le guide La nutrition pour la cirrhose a été élaboré par une équipe nationale d’experts et de patients. Objectifs: 1)Évaluer l’impact chez les patients cirrhotiques du Guide sur: i) le risque nutritionnel; ii) les connaissances en nutrition; iii) la qualité de vie. 2) Sonder l’appréciation du Guide. Méthodologie: Une étude randomisée contrôlée incluant 100 patients cirrhotiques du CHUM divisés en 2 groupes: Intervention (Guide+) et Contrôle (Guide-). Le risque nutritionnel, les connaissances en nutrition et la qualité de vie sont évalués à 0, 3 et 6 mois. Cinq groupes de discussion de 3 patients sont constitués afin d’évaluer l’appréciation générale du Guide. Résultats préliminaires: 42 patients sont inclus dans l'étude à ce jour: Guide+ (n=21) et Guide- (n=21). À ce jour, 21 patients ont complété l'étude: Guide+ (n=10) et Guide- (n=11). Une tendance d’amélioration des connaissances des patients Guide+ (76,0 % à 80,4 %) après 3 mois n’est pas maintenue à 6 mois (76,4 % ; p>0,05). Le risque nutritionnel a tendance à s’améliorer dans le groupe Guide+ (50% des patients initialement dénutris, 40% à 3 mois et 20% à 6 mois; p>0,05). Le risque nutritionnel est plus élevé dans le groupe Guide- (de 36,4% à 56,5% à 3 mois, à 45,5% à 6 mois; p>0,05). Les résultats préliminaires des groupes de discussion suggèrent une satisfaction globale du contenu, mais un besoin d'alléger et mieux diviser les concepts. Discussion: Ces résultats préliminaires démontrent une tendance positive d’amélioration des connaissances nutritionnelles des patients utilisant le Guide sur 3 mois non maintenue à 6 mois. Une amélioration du risque nutritionnel à 6 mois est observée. Les résultats de ce projet permettront d'optimiser la qualité des soins et de l’éducation nutritionnelle prodiguées aux patients atteints de cirrhose.
Isaac Ruiz, Mélanie Tremblay, Crystèle Hogue, Amal Trigui, Geneviève Huard, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Background. The global obesity epidemic continues to progress at an alarming rate. Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, is becoming the most common cause of end-stage liver disease (ESLD) and indication for liver transplantation (LT). Documenting the prevalence of overweight and obesity in patients on the waiting list for LT is imperative to make recommendations and improve management. Objective. The aim of this study was to evaluate the prevalence of overweight and obesity in patients on the waiting list at the moment of LT. Methodology. This is a retrospective analysis including all consecutive patients who underwent LT at the Centre hospitalier de l'Université de Montréal (CHUM) between 2019 and 2021. Only patients with biological, clinical and or radiological confirmed cirrhosis were included. Overweight and obesity was defined as body mass index (BMI) equal or superior to 25.0 and 30.0, respectively. Results. During the study period, 181 patients were transplanted at the CHUM. 155 patients with cirrhosis were included in this study. Among them, 104/155 (67.1%) were men, mean age was 52 years old (range 18-69). At the moment of transplantation, overall median BMI was 27.7 (range 16.9 – 44.5, mean 28.1). Overweight was present in 58/155 (37.4%), and obesity was present in 47/155 (30.3%). Results of BMI corrected by the presence of ascites and/or oedema will be presented. Figure 1 shows the prevalence of overweight and obesity overall (panel A), and according to the main etiology of chronic liver disease (panel B). Table 1 shows the median BMI in groups divided by the main etiology of cirrhosis. Discussion / Conclusion. At the moment of LT, almost 1/3 of patients display overweight and nearby 1/3 obesity. This study demonstrates the high prevalence of overweight and obesity in cirrhotic patients on the LT waiting list. Therapeutic and management strategies pre- and post-LT are mandatory using a multidisciplinary team, including nutritional intervention, while emphasizing weight stigma awareness.
Amal Trigui, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Après la transplantation hépatique (TH), la malnutrition, la sarcopénie et la fragilité sont associées à des résultats cliniques défavorables et à une diminution de la qualité de vie. Objectif : Déterminer la prévalence de la malnutrition, de la sarcopénie et de la fragilité à un an, deux ans et trois ans après la TF et décrire la fonction musculaire, la qualité de vie et le statut de l'emploi chez ces patients. Méthodologie : Une étude transversale incluant 80 patients transplantés entre 2019 et 2021. Une seule rencontre virtuelle est réalisée avec chaque patient afin évalués le risque nutritionnel (outil canadien de dépistage nutritionnel), la sarcopénie (questionnaire SARC-F), la fragilité (questionnaire FRAIL), la fonction musculaire (test d’élévation de la chaise), la qualité de vie (SF-36) et le statut d’emploi. Résultats : 57 (29 en 2019, 12 en 2020 et 16 en 2021) patients ont terminé l'étude. 10,5 % des patients sont à risque de malnutrition, 19,3 % sont à risque de sarcopénie tandis que 43,9 % et 19,3 % sont pré-fragiles et fragiles. Ces prévalences sont restées inchangées jusqu'à 3 ans après la transplantation. La fonction musculaire est altérée après la TH (15,8 ± 5,5 s contre 12,6 s chez les personnes en bonne santé). En ce qui concerne la qualité de vie, le score de santé physique (62,6 % ± 22,1) est légèrement inférieur au score normale et ce score reste inchangé jusqu'à 3 ans après la transplantation. 68,4% des patients sont au chômage dont 46,2% sont en retraite anticipée pour une cause liée à la maladie du foie. Discussion: Jusqu'à 3 ans après la TH, les patients sont toujours à risque de malnutrition, de sarcopénie et de fragilité. Le score de la composante physique de leur qualité de vie est inférieur à celui de la population générale.
Myriam Mireault, Constantine J. Karvellas, Christopher F. Rose, Lekha Sleno.
Acetaminophen (APAP) is a very common analgesic used worldwide to relieve pain and decrease fever. Unfortunately, when taken excessively, this drug can cause acute liver failure, through the formation of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI). This reactive species binds to liver proteins leading to liver failure. This affects hundreds of thousands of people each year in North America and constitutes the main cause of acute liver failure in the Western world. Previous studies have shown that APAP can influence the level of circulating bile acids by disrupting the blood-bile barrier. Increased levels of bile acids is a marker of liver disease. To investigate the effects of acetaminophen on circulating bile acid profiles, metabolites were extracted from human serum samples from patients with APAP-related acute liver failure and healthy controls. Bile acids were analysed by LC-MS/MS and all peaks detectable in samples from a standard mix of 46 bile acids were assigned based on accurate mass and retention time. Then, potential bile acid isomers not contained in the standard mix, as well as putative glucuronide and sulfate conjugates of these bile acids were assessed by accurate mass filtering. Our results showed that acetaminophen significantly influenced the levels of bile acids and conjugates. Several peaks varied significantly between healthy controls and ALF patients, as well as some statistically relevant changes based on patient outcome.
Farzaneh Tamnanloo, Xiaoru Chen, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a debilitating neurological complication of chronic liver disease with alcohol being a common etiological factor. However, excessive alcohol consumption has been shown to impact neurological integrity. To date, the influence of alcohol in the development of HE remains unclear. Therefore, we examined the effect of constant alcohol consumption on neurological decline in rats with chronic liver disease induced via bile-duct ligation (BDL). Method: 5-week BDL rats and Sham-operated controls were used. Day 7 after surgery, rats were administered alcohol twice a day (dose of 3g/kg, via gavage) for 4 weeks. Motor coordination (rotarod) and anxiety-like behavior (open field (OF) and elevated plus maze (EPM)) were assessed. Upon sacrifice, brains were collected, and western blot and immunohistochemical (IHC) analyses were used to investigate neuronal integrity in frontal cortex and cerebellum. Results: Alcohol (BDL-alcohol rats) further impaired motor coordination at weeks 2, 3, 4, and 5 compared to SHAM-alcohol (p<0.01). Furthermore, BDL-alcohol rats demonstrated an increase in anxiety-like behavior; increase in time spent in the closed arms of EPM and decrease in time spent in the center of the OF (p<0.05 vs SHAM-alcohol). BDL-alcohol rats demonstrated a decrease in neuronal markers of NeuN and SMI311 (p<0.01 and p<0.05, respectively), an increase in apoptotic markers of cleaved/pro-caspase3 (p<0.001), an increase in necroptosis markers of pRIP3 and pMLKL (p<0.01 and p<0.001, respectively), a decrease in total antioxidant capacity (p<0.001) and an increase in oxidative stress marker of 4-HNE (p<0.05) in the cerebellum (not found in frontal cortex) compared to all groups. IHC results confirmed the colocalization of apoptotic marker (cleaved Caspase3) and necroptosis marker (pMLKL) in the granular and Purkinje layer neurons of the cerebellum of BDL-alcohol rats. Conclusion: Constant alcohol consumption exacerbates HE and leads to neuronal loss via apoptosis and necroptosis in the cerebellum. Additionally, higher levels of oxidative stress marker of 4-HNE and decreased total antioxidant capacity in the cerebellum of BDL-alcohol rats suggest that oxidative stress is a triggering factor leading to neuronal loss/injury. These results demonstrate an adverse effect of constant alcohol consumption on the development of HE and neuronal integrity in chronic liver disease.
Le guide La nutrition pour la cirrhose : une ressource éducative adéquate pour nos patients ?
Manila Sophasath, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Problématique: La malnutrition est l’une des complications les plus prévalentes de la cirrhose (maladie du foie chronique) et affecte la qualité de vie des patients. Objectifs: Le projet vise l’évaluation d’un Guide éducatif en nutrition pour patients avec une cirrhose en 2 volets: 1)Évaluer le guide La nutrition pour la cirrhose sur les connaissances en nutrition, qualité de vie et état nutritionnel sur 6 mois. 2)Évaluer la satisfaction du Guide par les patients. Méthode: 1)100 patients suivis au CHUM randomisés en deux groupes égaux: Intervention (Guide+) et Contrôle (Guide-). Les évaluations de l’état nutritionnel, connaissances en nutrition et qualité de vie sont réalisées à 0, 3 et 6 mois. 2)5 groupes de discussion (3 patients/groupe) pour évaluer l’appréciation du Guide. Résultats prélim. (volet 1): À ce jour, 31 patients (Guide+; n=16 et Guide-; n=15) ont complété l’évaluation à 3 mois. Les résultats démontrent une tendance d’amélioration des connaissances (de 75,4% à 79,6%) pour le groupe Guide+ vs Guide-(de 73,7% à 74,6%), (p=0.212). L’évaluation à 6 mois a été complétée par 21 patients (Guide+; n=10 et Guide-; n=11). Les résultats préliminaires démontrent que la tendance d’amélioration de connaissances du groupe Guide+ après 3 mois n’est pas maintenue après 6 mois : de 80,4% (3 mois) à 76.4% (6 mois). Discussion: Le Guide ne semble pas permettre une rétention de l’information à long terme. Élaborer une ressource éducative plus adaptée serait envisageable.
Amal Trigui, Joann Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
La malnutrition est la complication la plus fréquente chez les patients atteints de maladies hépatiques chroniques (cirrhose) en attente d’une transplantation hépatique (TH) et elle pourrait mener à l’apparition d’autres complications avant et après la TH, incluant la sarcopénie (perte de masse et fonction musculaire). Objectifs: Chez les patients cirrhotiques en attente d’une TH : 1) Évaluer les changements longitudinaux du risque nutritionnel, de la fonction musculaire et de la qualité de vie. 2) Identifier les facteurs pouvant entraîner une détérioration des trois variables évaluées. Méthode: Les patients atteints d'une cirrhose et en attente d’une TH au CHUM sont inclus. Le risque nutritionnel (Liver Disease Undernutrition Screening Tool), la fonction musculaire (Chair Stand Test), et la qualité de vie (SF-36) sont évalués tous les 3 mois avant la TH. Résultats: Actuellement, 36 patients en attente d’une TH ont été inclus. L'âge moyen est de 51,3 ± 11,6 ans. L'étiologie la plus fréquente est l'alcoolisme (47 %) suivie par la cholangite sclérosante primitive (25%). 86 % (31/36) des patients sont à risque de malnutrition, situation qui demeure inchangée jusqu'à 18 mois sur la liste d’attente. Parmi les 5 patients qui n'étaient pas à risque de malnutrition, 60% sont devenus à risque. À l'inclusion, 73 % (26/36) des patients présentaient une faible force musculaire (score moyen de 18,9 ± 7,8 s vs 12,6 s chez les patients sains ; p < 0,001) qui diminuait avec le temps d’attente pour une TH. En ce qui concerne la qualité de vie, les scores moyens de santé physique (44,6% ± 20,4) et de santé mentale (53,5% ± 26,1) étaient inférieurs aux scores des Canadiens du même âge considérés en bonne santé. Le score de santé physique avait tendance à diminuer davantage avec le temps. Conclusion: La majorité des patients en attente d'une TH sont à risque de malnutrition, présentent une fonction musculaire altérée qui se détériore avec le temps. La qualité de vie est également diminuée tandis que le score de santé physique a tendance à diminuer avec le temps.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique causé par une maladie du foie. Il a été démontré que le microbiote intestinal influence les fonctions neurologiques via divers médiateurs (cytokines/métabolites bactériens), tandis que l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif: Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats mâles ont été randomisés en trois groupes : Contrôle (SHAM), BDL-véhicule (VEH) et BDL-FMT qui ont reçu quotidiennement la FMT provenant des rats contrôles. Après cinq semaines, des tests comportementaux ont été effectués pour évaluer la mémoire à court/long terme, l'anxiété et la coordination motrice. Des paramètres cliniques et paracliniques ont été mesurés et les fèces collectées pour séquençage bactérien. Résultats: Les rats BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice comparativement aux rats contrôles. Cependant, les altérations neurologiques ont été prévenues chez les rats BDL-FMT. Le genre Provotellaceae UCO-001 est présent uniquement chez les rats SHAM et BDL-FMT. A l’inverse, le genre Clostridium SS 1 est uniquement présent chez les rats BDL-VEH. L’analyse plasmatique des cytokines ne montre aucune différence entre les rats BDL. Les concentrations d’acides gras à chaines courtes butyrate et propionate au niveau des fèces et du plasma varie entre les rats BDL. Le propionate plasmatique ressort de par les corrélations positives avec les scores de comportement. Discussion et perspectives : Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT n'a pas normalisé le profil du microbiote comparé aux rats SHAM, ce qui suggère qu’elle conduit à un nouveau profil spécifique du microbiote semblant protéger le cerveau. La présence de propionate plasmatique doit être explorée pour définir son impact sur le cerveau.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, André Marette, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome arising from chronic liver disease (CLD). The gut microbiota has been shown to influence neurological functions via various mediators such as cytokines or bacterial metabolites. Faecal matter transplantation (FMT) in patients with cirrhosis has revealed beneficial effects on behaviour yet many limitations of these studies render the results inconclusive. Purpose: The aim of this study was to explore the impact of FMT on gut microbiota and neuro behaviour in bile-duct ligated (BDL) rats. Method: Male SpragueDawley rats were randomly assigned to one of three groups; SHAM, BDL-VEH (vehicle) and BDL-FMT (who received FMT daily from pooled faeces from SHAM rats). After five weeks, behaviour tests were performed to evaluate short- and longterm memory, anxiety and motor coordination. Plasmatic parameters including cytokines, short chain fatty acids (SCFA) and liver biomarkers were measured. Finally, faeces were collected for bacterial sequencing, SCFA and tissues for analysis after the sacrifice. Results: BDL-VEH developed a loss of short- and long-term memory and motor coordination compared to SHAM rats. However, neurological dysfunction was prevented in the BDL-FMT group. FMT impacted microbiota composition as the genera Bifidobacterium, Lactobacillus and Akkermansia significantly increased in both BDL groups, while Provotellaceae UCO-001 significantly increased only in SHAM and BDL-FMT rats. Clostridium Senso Stricto 1 significantly increased only in BDL-VEH compared to SHAMs. Plasma pro-inflammatory cytokines (TNF-α & IL-1β) increase in both BDL groups compared to SHAM and no difference in anti-inflammatory cytokine IL-10 was noted. Analysis of SCFA in plasma and faeces showed a variation in propionate and butyrate between both BDL groups. Conclusion: Our results demonstrate that FMT leads to improvement in memory and motor coordination in BDL rats. The microbiota profile was different between BDL-VEH and SHAM whereas FMT led to further microbiota alterations. BDL-FMT led to a novel specific microbiota profile which in turn protected the brain.
Amal Trigui, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Background: The liver is the second most transplanted organ worldwide. Despite the high survival rate after liver transplant (LT), malnutrition, sarcopenia and frailty are present in 47%, 80% and 59%, respectively, of patients 3 months after surgery. These three complications are associated with adverse clinical outcomes and decreased quality of life after LT. However, there are no data regarding malnutrition, sarcopenia, and frailty in the long term after LT. Purpose: The primary objective is to determine the prevalence of malnutrition, sarcopenia, and frailty starting from 1 year after LT. The secondary objectives aim to describe muscle function, quality of life and employment status in LT recipients. Methods: Cross-sectional observational study is conducted including 80 patients transplanted between 2019 and 2021. A single virtual meeting is performed with each patient during which nutritional risk (Canadian Nutrition Screening Tool), sarcopenia (SARC-F questionnaire), frailty (FRAIL questionnaire), muscle function (chair stand test), quality of life (SF36) and employment status are assessed. Results: To date, 57 (29 in 2019, 12 in 2020 and 16 in 2021) patients have completed the study (34 men and 23 women). The mean age is 58.9 ± 10.5 years and 10.5% of patients were at risk of malnutrition, 19.3% of patients were at risk of sarcopenia whereas 43.9% and 19.3% were prefrail and frail. The prevalence of the risk of malnutrition, sarcopenia, and frailty (prefrail and frail) remained unchanged until 3 years after LT. Muscle function was impaired after LT (15.8 ± 5.5 s vs. 12.6 s in healthy people). Regarding quality of life, the score of physical heath (62.6% ± 22.1) is slightly below normal and the scores remain unchanged until 3 years after LT. 68.4% of patients are unemployed of which 46.2% are in early retirement for a liver disease-related cause. Conclusion: Up to 3 years after LT, patients are still at risk of malnutrition, sarcopenia, and frailty. The physical component score of their quality-of-life score is below the score of the general population. The results of this project may help identify appropriate interventions in the long term after LT.
Manila Sophasath, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Background: Liver disease affects 1 in 4 Canadians. One of the most common complications of chronic liver disease is malnutrition, associated with decreased quality of life. Very few studies have focused on nutritional education resources for this population rendering their development and evaluation essential. Purpose: The general objective is to assess the potential impact of the evidence-based Nutrition in Cirrhosis Guide on cirrhotic patients at the CHUM’s liver outpatient clinic. The first specific objective is to quantitatively assess nutritional knowledge, quality of life and nutritional status. The second is to qualitatively assess the patients’ satisfaction of the Guide. Methods: A randomized controlled study including 100 cirrhotic patients in 2 groups: Guide+ (n = 50) and Guide− (n = 50), is on-going. Patients are assessed for nutrition knowledge, quality of life and presence of malnutrition at baseline, 3 and 6 months. The Guide is taught to Guide+ patients for 6 months. Guide− patients do not use the Guide. The qualitative part evaluates patients’ satisfaction of the Guide through 5 focus groups (3 patients) to assess appreciation, complexity and applicability. A patient-partner participates in focus groups. Results: To date, 21 patients have completed the study: Guide+ (n = 10) and Guide− (n = 11). The preliminary results show a trend of improvement of nutrition knowledge for Guide+ patients (from 76.0% to 80.4%) after 3 months, not maintained at 6 months (down to 76.4%; p > 0.05). The Guide− patients’ knowledge remains unchanged throughout the study. There is a trend of improvement in malnutrition in the group Guide+ (50% of patients initially malnourished, 40% at 3 months and 20% at 6 months; p > 0.05), which worsened in the group Guide− (from 36.4% to 56.5% at 3 months, to 45.5% at 6 months). Preliminary results from focus groups suggest an overall satisfaction towards the content, but a need to lighten and better divide the concepts. Conclusion: A trend of improvement is denoted in patients’ nutritional knowledge over 3 months, not maintained at 6 months, and a decrease in the presence of malnutrition after 6 months. The results of this project will help optimize the cirrhotic patients’ quality of care.
Nutritional education strategies for cirrhotic patients : Evaluation of a nutrition guide.
Manila Sophasath, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
BACKGROUND: In Canada, liver disease affects 1 in 4 persons. One of the most common complications of cirrhosis is malnutrition, associated with an increased risk of mortality and a decrease in the quality of life of patients. Adherence to nutrition guidelines appears to be difficult. Very few studies have focused on the development and evaluation of nutritional education resources adapted to this population. Thus, the development and evaluation of such strategies is essential. PURPOSE: The general objective of the study is to assess the potential impact of the evidence-based Nutrition in Cirrhosis Guide on cirrhotic patients followed at the CHUM’s liver outpatient clinic through a mixed-design study. The first specific objective is to quantitatively assess nutritional knowledge, quality of life and nutritional status and the second specific objective is to qualitatively assess the patients’ satisfaction of the Guide. METHOD: A randomized controlled study including 100 cirrhotic patients divided in 2 groups: Intervention (Guide+, n=50) and Control (Guide-, n=50), is on-going. All patients are assessed for nutrition knowledge (literacy questionnaire), quality of life (Chronic Liver Disease Questionnaire) and presence of malnutrition (Liver Disease Undernutrition Screen Tool). The Guide is taught to Guide+ patients for 6 months. Guide- patients do not use the Guide. At times T=0, 3 and 6 months, the same evaluations are carried out. The qualitative part evaluates patients’ satisfaction of the Guide through 5 focus groups of 3 patients each to assess general appreciation, complexity and applicability of the Guide. A patient-partner participates in focus groups. RESULT(S): 42 patients are included in the study so far: Guide+ (n=21) and Guide- (n=21). The groups are comparable in age (mean = 59.0 and 55.0 in Guide+ and Guide-, respectively; p=0.28). To date, 21 patients have completed the study: Guide+ (n=10) and Guide- (n=11). The preliminary results show a trend of improvement of nutrition knowledge for Guide+ patients (from 76.0% to 80.4%) after 3 months, which is not maintained at 6 months (down to 76.4%; p>0.05). The Guide- patients’ knowledge remains unchanged throughout the study (from 74.2% to 74.5% after 3 months, to 77.8% after 6 months; p>0.05). As for the presence of malnutrition, a trend of improvement is denoted in the group Guide+ (50% of patients were initially malnourished, 40% at 3 months and 20% at 6 months; p>0.05). Presence of malnutrition worsened in the group Guide- (from 36.4% to 56.5% at 3 months, to 45.5% at 6 months). Preliminary results from focus groups suggest an overall satisfaction towards the content, but a need to lighten and better divide the concepts. CONCLUSION(S): The preliminary results demonstrate a trend of improvement in patients’ nutritional knowledge over 3 months, which is not maintained at 6 months, and a decrease in the presence of malnutrition after 6 months. The results of this project will help optimize the quality of care for people suffering from cirrhosis.
Amal Trigui, Joann Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
In Canada, more than 400 liver transplantation (LT) are performed every year. However, organ availability continues to be a major issue in LT, as the waiting time for a deceased liver donor ranges from months to years. In patients with end-stage liver disease waiting for LT, protein-energy malnutrition is the most common complication (> 80%) and one of the main risk factors for the onset and progression of sarcopenia (loss of muscle mass and function or quality). Previous clinical studies have shown that sarcopenia and malnutrition are associated with a prolonged hospital stay, increased mortality and higher rate of infections as well as a decrease in quality of life. Pre-operative malnutrition could also impact negatively on the post-transplant outcomes. Screening for the nutritional risk for patients waiting for LT is the first step to address adequate nutritional therapy. However, since the delay to receive a new liver could be long, following the patients longitudinally could help to detect the deterioration in nutritional status and muscle function at an advanced stage as well as to identify factors that could lead to this deterioration. Purpose: In patients with cirrhosis waiting for LT: 1) Assess longitudinal changes in nutritional risk, muscle function and quality of life. 2) Identify factors that could lead to deterioration of nutritional status, muscle function and quality of life. Method: Patients with end-stage liver disease waiting for LT at the CHUM were included. Muscle function (Chair Stand Test), nutritional risk (Liver Disease Undernutrition Screening Tool), and quality of life (SF-36) are assessed every 3 months prior to LT. Biochemical and anthropometric data, medications as well as complications are collected at each follow-up. Results: Currently, 36 patients awaiting LT have been included. The mean age is 51.3 ± 11,6 years. The most common etiology is alcohol (33 %). At enrollment, 86 % (31/36) of patients are at risk of malnutrition, which remain unchanged with follow-ups ranging to 18 months while on the LT waiting list. Among the 5 patients who were not at risk of malnutrition and enrollment, 60% were defined at risk with follow-ups. Based on the EWGSOP-2 diagnostic criteria, at enrollment, 73% (26/36) of patients had a low muscle strength with a mean score of 18,9s ± 7,8 s (vs 12,6 s in healthy patients; p < 0,001) which decreased with time while waiting for an LT. Regarding quality of life, the mean score of physical heath (44,6% ± 20,4) and mental health (53,5% ± 26,1) were below normal scores for healthy Canadian in the same age. The physical health score tended to further decrease over time. Conclusion: A majority of patients with cirrhosis placed on the LT list are at risk of malnutrition, have an impaired muscle function which decreases with time. Moreover, quality of life is impacted, and the physical health component tended to decrease with time.
Farzaneh Tamnanloo, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome arising from chronic liver disease (CLD). HE manifests with symptoms such as poor memory, impairment in motor coordination, lethargy and coma. The gut microbiota has been shown to influence neurological functions via various mediators such as cytokines or bacterial metabolites, many studies have demonstrated the gut-brain axis is altered in liver disease. Faecal matter transplantation (FMT) in patients with cirrhosis has revealed beneficial effects yet many limitations of these studies render the results inconclusive. Purpose: The aim of this study is to explore the impact of FMT on gut microbiota and the beneficial effects on neuro behaviour in bile-duct ligated (BDL) rats. Method: Male Sprague-Dawley rats were randomly assigned to one of three groups; SHAM, BDL-VEH (vehicle) and BDLFMT (who received FMT daily from pooled faeces from SHAM rats). After five weeks, behaviour tests were performed to evaluate short- and long-term memory (Novel Object Recognition), anxiety (Open Field and Elevated Plus Maze) and motor coordination (Rotarod). Plasmatic parameters such as cytokines, short chain fatty acids (SCFA) and liver impairment markers were measured by ELISA, LC-MS/MS and using Cobas respectively. Finally, faeces were collected for bacterial sequencing and SCFA analysis. Result(s): FMT did not alter degree of liver disease in BDL rats. BDL-VEH developed a loss of short/long term memory and motor coordination compared to SHAM rats. However, alterations in neurological dysfunction were prevented in the BDL-FMT group. FMT did not impact microbiota α-diversity in BDL rats and β-diversity of microbiota was significantly different between all groups. The genera Bifidobacterium, Lactobacillus and Akkermansia significantly increased in both BDL groups, while Provotellaceae UCO-001 significantly increased only in SHAM and BDL-FMT rats and Clostridium Senso Stricto 1 significantly increased only in BDL-VEH compared to SHAMs. Finally, Rombustia was only present in SHAM. Plasma pro-inflammatory cytokines (TNF-α & IL-1) increase in both BDL groups compared to the control group and no difference for the anti-inflammatory cytokine IL-10 was noted. Analysis of short-chain fatty acids in faeces and plasma showed a variation in propionate and butyrate between both BDL groups. Plasma propionate significantly positively correlated with behavioural results.Conclusion(s): Our results demonstrate that FMT leads to improvement in memory and motor coordination in BDL rats. The microbiota profile was different between BDL-VEH and SHAM, and FMT lead to further alterations on microbiota. The fact that FMT did not normalize microbiota profile compared to SHAM, suggests BDL-FMT leads to a novel specific microbiota profile which in turn protects the brain. The protective effect of plasma propionate needs to be further explored to define its impact on the brain and possible therapeutic application.
Sydnée L'Écuyer, Alexandra Matesan, Farzaneh Tamnanloo, Mariana Oliveira, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a debilitating complication of cirrhosis that affects over 40% of chronic liver disease (CLD) patients, leading to gross disorientation, asterixis, memory impairments, and death. An increase in blood ammonia has been coined the major factor in the pathogenesis of HE. However, the severity of symptoms does not always correlate with blood ammonia concentrations. This implies other factors may be involved. Hyperuricemia is associated with metabolic syndrome and chronic alcohol consumption which are known precipitating factors of CLD. Furthermore, uric acid (UA) has been reported to be involved in the induction of neuroinflammation and cell loss by apoptosis and pyroptosis, leading to anxiety-like behavior and memory impairment. Purpose: This study investigates the impact of hyperuricemia on cognitive impairment in a rat model of chronic liver disease. Method: We used the bile-duct ligation (BDL) model; a well-characterized rat model of chronic liver disease and HE. Fifty-two male Sprague-Dawley rats (200-240 g) were randomly assigned to a control (SHAM) or a BDL surgery. To induce hyperuricemia, half of the animals were fed a high uric acid diet (HUAD) (3% uric acid), dividing our rats into 4 groups: (1) SHAM+regular diet (RD), (2) SHAM+HUAD, (3) BDL+RD and (4) BDL+HUAD. During 5 weeks, weekly plasma UA measurements were made and different behavior tests were conducted (anxiety (open-field) and short and long-term memory (novel-object recognition)) during the 5th week. At the end of week 5, rats were sacrificed, their brains collected, and the frontal cortex, hippocampus, and amygdala were isolated to assess cell death (caspase-3 (apoptosis) and caspase-1 (pyroptosis) activity). Result(s): HUAD significantly increased circulating UA in both SHAM and BDL rats. This was associated with a significant decrease in short-term memory without changes in anxiety-like behaviour for HUAD rats compared to their RD counterparts in both SHAMs and BDLs. For long-term memory, BDLs were shown to be significantly impaired compared to SHAMs. A significantly higher degree of impairment was seen in BDL+HUAD rats compared to BDL+RD. Cell death (apoptosis and pyroptosis) was then evaluated in the limbic system to explain some of the behavioral changes. In the frontal cortex, increased caspase-3 activity was measured in both SHAM+HUAD and BDL+HUAD rats in comparison to RD animals. In the hippocampus, caspase-1 activity was significantly increased in the BDL+HUAD rats compared to the BDL+RD rats and SHAM groups. In the amygdala, no significant changes in caspase activity were detected. Conclusion(s): In SHAM rats, diet-induced hyperuricemia shows a significant increase in circulating uric acid concentration as well as short-term memory loss associated with apoptosis (caspase-3 activity) in the frontal cortex. In BDL rats, hyperuricemia induces long-term memory impairments and apoptosis in the frontal cortex as well as pyroptosis in the hippocampus. The results from this study show the merit of further investigating the role of uric acid in HE.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome arising from chronic liver disease (CLD). HE manifests with symptoms such as poor memory, impairment in motor coordination, lethargy and coma. The gut microbiota has been shown to influence neurological function and many studies have demonstrated the association between altered microbiota and liver disease. Fecal matter transplantation (FMT) has revealed beneficial effects in clinical studies yet many limitations of these studies render the results inconclusive. Purpose: The aim of this study is to explore the impact of FMT on gut microbiota and the beneficial effects on neuro behaviour in bile-duct ligated (BDL) rats. Method: Male Sprague-Dawley rats were randomly assigned to one of three groups; SHAM (N=10), BDL-VEH (vehicle) (N=9) and BDL-FMT (who received FMT daily from pooled feces from SHAM rats). After five weeks, behaviour analysis was performed to evaluate short and long-term memory (Novel Object Recognition), anxiety (Open Field and Elevated Plus Maze) and motor coordination (Rotarod). Other parameters such as body weight and composition, ascites, gastrocnemius muscle weight and markers of liver function (ALT, AST, bilirubin and NH3) were also measured. Finally, the feces were collected and 16S RNA was sequenced for all groups. Result(s): FMT did not alter body composition (weight, composition, ascites and gastrocnemius muscle weight as well as and degree of liver disease (liver damage markers) in BDL-FMT vs BDL-Vehicle. BDL-VEH developed a loss of short/longterm memory and motor coordination compared to Sham rats. However, alterations in neurological dysfunction were prevented in the BDL-FMT group. The microbiota -diversity was not significantly different between BDL-VEH and SHAM and furthermore, FMT did not impact -diversity in BDL rats. In contrast, -diversity of microbiota did significantly differ between all groups (p < 0.05). The relative abundance also was significantly different between all three groups. In the BDL-FMT group, the phylum Firmicutes was found decreased while the Bacteroidetes were increased compared to BDLVEH and SHAM. The genera Bifidobacterium and Lactobacillus significantly increased in both BDL groups, while Akkermansia and Provotellaceae UCO-001 increased only in BDL-FMT rats and Clostridium Senso Stricto increased only in BDL-VEH compared to Shams. Finally, the genus Rombustia was only present in SHAM. Conclusion(s): Our results demonstrate that FMT lead to improvement in memory and motor coordination in BDL rats. The microbiota profile was different between BDL-VEH and SHAM, and FMT lead to further alterations. The fact that FMT did not normalize microbiota profile, suggests BDL-FMT leads to a novel specific microbiota profile which in turn protects the brain. The FMT-induced increase in Akkermansia and Provotellaceae UCO-001 merits to be further investigated in regards to their beneficial neurological effect in CLD.
Amal Trigui, Joann Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
La malnutrition est la complication la plus fréquente chez les patients atteints de maladies hépatiques chroniques (cirrhose) en attente d’une transplantation hépatique (TH) et elle pourrait mener à l’apparition d’autres complications avant et après la TH, incluant la sarcopénie (perte de masse et fonction musculaire). Objectifs: Chez les patients cirrhotiques en attente d’une TH : 1) Évaluer les changements longitudinaux du risque nutritionnel, de la fonction musculaire et de la qualité de vie. 2) Identifier les facteurs pouvant entraîner une détérioration des trois variables évaluées. Méthode: Les patients atteints d'une cirrhose et en attente d’une TH au CHUM sont inclus. Le risque nutritionnel (Liver Disease Undernutrition Screening Tool), la fonction musculaire (Chair Stand Test), et la qualité de vie (SF-36) sont évalués tous les 3 mois avant la TH. Résultats: Actuellement, 36 patients en attente d’une TH ont été inclus. L'âge moyen est de 51,3 ± 11,6 ans. L'étiologie la plus fréquente est l'alcoolisme (47 %) suivie par la cholangite sclérosante primitive (25%). 86 % (31/36) des patients sont à risque de malnutrition, situation qui demeure inchangée jusqu'à 18 mois sur la liste d’attente. Parmi les 5 patients qui n'étaient pas à risque de malnutrition, 60% sont devenus à risque. À l'inclusion, 73 % (26/36) des patients présentaient une faible force musculaire (score moyen de 18,9 ± 7,8 s vs 12,6 s chez les patients sains ; p < 0,001) qui diminuait avec le temps d’attente pour une TH. En ce qui concerne la qualité de vie, les scores moyens de santé physique (44,6% ± 20,4) et de santé mentale (53,5% ± 26,1) étaient inférieurs aux scores des Canadiens du même âge considérés en bonne santé. Le score de santé physique avait tendance à diminuer davantage avec le temps. Conclusion: La majorité des patients en attente d'une TH sont à risque de malnutrition, présentent une fonction musculaire altérée qui se détériore avec le temps. La qualité de vie est également diminuée tandis que le score de santé physique a tendance à diminuer avec le temps.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique causé par une maladie du foie. Il a été démontré que le microbiote intestinal influence les fonctions neurologiques via divers médiateurs (cytokines/métabolites bactériens), tandis que l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif: Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats mâles ont été randomisés en trois groupes : Contrôle (SHAM), BDL-véhicule (VEH) et BDL-FMT qui ont reçu quotidiennement la FMT provenant des rats contrôles. Après cinq semaines, des tests comportementaux ont été effectués pour évaluer la mémoire à court/long terme, l'anxiété et la coordination motrice. Des paramètres cliniques et paracliniques ont été mesurés et les fèces collectées pour séquençage bactérien. Résultats: Les rats BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice comparativement aux rats contrôles. Cependant, les altérations neurologiques ont été prévenues chez les rats BDL-FMT. Le genre Provotellaceae UCO-001 est présent uniquement chez les rats SHAM et BDL-FMT. A l’inverse, le genre Clostridium SS 1 est uniquement présent chez les rats BDL-VEH. L’analyse plasmatique des cytokines ne montre aucune différence entre les rats BDL. Les concentrations d’acides gras à chaines courtes butyrate et propionate au niveau des fèces et du plasma varie entre les rats BDL. Le propionate plasmatique ressort de par les corrélations positives avec les scores de comportement. Discussion et perspectives : Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT n'a pas normalisé le profil du microbiote comparé aux rats SHAM, ce qui suggère qu’elle conduit à un nouveau profil spécifique du microbiote semblant protéger le cerveau. La présence de propionate plasmatique doit être explorée pour définir son impact sur le cerveau.
Amal Trigui, Christopher F. Rose, Chantal Bémeur.
Persisting or newly developed malnutrition and sarcopenia after liver transplant (LT) are correlated with adverse health outcomes. This narrative review aims to examine the literature regarding nutrition strategies to manage malnutrition and sarcopenia after LT. The secondary aims are to provide an overview of the effect of nutrition strategies on the incidence of infections, hospital length of stay (LOS), acute cellular rejection (ACR), and mortality after LT. Four databases were searched. A total of 25 studies, mostly of mid-high quality, were included. Six studies found a beneficial effect on nutritional parameters using branched-chain amino acids (BCAA), immunomodulating diet (IMD), or enteral nutrition (EN) whereas two studies using beta-hydroxy-beta-methylbutyrate (HMB) found a beneficial effect on muscle mass and function. Fourteen studies using pre- or pro-biotics, IMD, and EN were effective in lowering infection and six studies using IMD, BCAA or HMB reported reduced hospital LOS. Finally, four studies using HMB and vitamin D were effective in reducing ACR and one study reported reduced mortality using vitamin D after LT. In conclusion, nutritional intervention after LT has different beneficial effects on malnutrition, sarcopenia, and other advert outcomes. Additional large and well-constructed RCTs using validated tools to assess nutritional status and sarcopenia are warranted to ensure more robust conclusions.
Sydnée L'Écuyer, Emmanuel Charbonney, François Martin Carrier, Christopher F. Rose.
The incidence of chronic liver disease is on the rise. One of the primary causes of hospital admissions for patients with cirrhosis is hepatic encephalopathy (HE), a debilitating neurological complication. HE is defined as a reversible syndrome, yet there is growing evidence stating that, under certain conditions, HE is associated with permanent neuronal injury and irreversibility. The pathophysiology of HE primarily implicates a strong role for hyperammonemia, but it is believed other pathogenic factors are involved. The fibrotic scarring of the liver during the progression of chronic liver disease (cirrhosis) consequently leads to increased hepatic resistance and circulatory anomalies characterized by portal hypertension, hyperdynamic circulatory state and systemic hypotension. The possible repercussions of these circulatory anomalies on brain perfusion, including impaired cerebral blood flow (CBF) autoregulation, could be implicated in the development of HE and/or permanent brain injury. Furthermore, hypotensive insults incurring during gastrointestinal bleed, infection, or liver transplantation may also trigger or exacerbate brain dysfunction and cell damage. This review will focus on the role of hypotension in the onset of HE as well as in the occurrence of neuronal cell loss in cirrhosis.
Amal Trigui, Joan Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
In patients living with cirrhosis and waiting for liver transplantation (LT), protein-energy malnutrition is the most common complication (> 80%). After LT, nutritional status can worsen rapidly leading to sarcopenia (loss of muscle mass and function). Objectives: 1) Assess longitudinal changes in nutritional risk, muscle function and quality of life in cirrhotic patients awaiting LT. 2) Evaluate the effect of early nutritional supplementation rich in protein, beta-hydroxy-beta-methylbutyrate (HMB) and energy, after LT, on muscle mass and function, nutritional risk and quality of life. Method: A randomized controlled pilot study is conducted including 30 patients going through LT. Muscle mass (CT scan), muscle function (chair stand test), nutritional risk (liver disease undernutrition screening tool) and quality of life (SF-36) are assessed every 3 months before LT, immediately after discharge from hospital and 12 weeks post-LT. At LT, participants are randomized in: (1) intervention group (n=15) receiving standard nutritional care as well as supplements rich in protein, HMB and energy for 12 weeks and (2) control group (n=15) receiving standard nutritional care. Results: Currently, 26 patients awaiting LT are included. One participant was transplanted. The mean age is 51.3 ± 12.9 years. The most common etiology is alcohol (33 %). 84 % of patients are at risk of malnutrition which remain unchanged within up to one year on the waitlist. Muscle function is impaired (18.1 ± 11.0 s vs. 12.6 s in healthy patients; p < 0,001) and decreased with time on waitlist. Regarding quality of life, the score of physical heath (41.8% ± 18.6) and mental health (52.1% ± 25) are below normal. The quality of life tended to decrease over time before LT. Conclusion: The majority of patients waiting for a LT are at risk of malnutrition, display altered muscle function, and a tendency of decreased quality of life. These preliminary data support the need for early nutritional support after LT.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major complication of chronic liver disease (CLD). Defined as a metabolic disorder, HE should resolve following liver transplantation (LT). However, persisting neurological complications have been reported in up to 47% of LT recipients. Retrospective studies have demonstrated an association between a history of HE episodes and neu-rological deficits post-LT. Our aim was to evaluate the impact of multiple episodes (induced by ammonia) on neurological status, integrity and injury in rats with CLD. Methods: 5-week bile-duct ligation (BDL) rats were in-jected (ip) with ammonium acetate (BDL-Ammonia), pre-cipitating an overt episode of HE (loss of righting reflex). Episodes of HE were induced every 4 days from week 3 post-BDL surgery (total; 4 episodes). Sham rats were also injected with a similar dose of ammonia (Sham-Ammonia) and respective controls were injected with sa-line. Three days after the last episode, long-term mem-ory (LTM) was assessed. Upon sacrifice, plasma and brains were collected for oxidative stress measurements and western blot analysis for NeuN and SMI311 (neuro-nal markers), caspase-3 and Bax/Bcl2 ratio (markers of apoptosis), GFAP (astrocyte marker) and 4-HNE (marker of oxidative stress) in frontal cortex, cerebellum and hip-pocampus. Results: LTM was found to be impaired in both BDL-Saline and BDL-Ammonia groups vs respec-tive controls. However, LTM impairment was further ag-gravated in BDL-Ammonia rats. In BDL-Ammonia, higher protein levels of GFAP and apoptotic markers were found in the hippocampus, whereas NeuN and SMI311 levels were reduced compared to all other experimental groups. BDL-Ammonia rats showed increased levels of plasma oxidative stress compared to respective Sham and BDL rats. Decreased levels of total antioxidant capacity as well as increased 4-HNE were found solely in the hip-pocampus (not frontal cortex or cerebellum) of the BDL-Ammonia group compared to controls. Conclusion:Multiple episodes of overt HE leads to a worsening of neurological impairment in BDL rats. LTM impairment in the BDL-Ammonia rats was associated with increased oxidative stress, apoptotic markers and decreased neu-ronal markers in the hippocampus, supporting neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuate gliosis, possibly a result of neuronal loss. These results suggest that repeated HE episodes may cause permanent cell damage which will less likely be revers-ible following LT.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome arising from chronic liver disease (CLD). HE manifests with symptoms such as poor memory, impairment in motor coordination, lethargy and coma. The gut microbiota has been shown to influence neurological function and many studies have demonstrated the association between altered microbiota and liver disease. Fecal matter transplantation (FMT) has revealed beneficial effects in clinical studies yet many limitations of these studies render the results inconclusive. Purpose: The aim of this study is to explore the impact of FMT on gut microbiota and the beneficial effects on neuro behaviour in bile-duct ligated (BDL) rats. Method: Male Sprague-Dawley rats were randomly assigned to one of three groups; SHAM (N=10), BDL-VEH (vehicle) (N=9) and BDL-FMT (who received FMT daily from pooled feces from SHAM rats). After five weeks, behaviour analysis was performed to evaluate short and long-term memory (Novel Object Recognition), anxiety (Open Field and Elevated Plus Maze) and motor coordination (Rotarod). Other parameters such as body weight and composition, ascites, gastrocnemius muscle weight and markers of liver function (ALT, AST, bilirubin and NH3) were also measured. Finally, the feces were collected and 16S RNA was sequenced for all groups. Result(s): FMT did not alter body composition (weight, composition, ascites and gastrocnemius muscle weight as well as and degree of liver disease (liver damage markers) in BDL-FMT vs BDL-Vehicle. BDL-VEH developed a loss of short/longterm memory and motor coordination compared to Sham rats. However, alterations in neurological dysfunction were prevented in the BDL-FMT group. The microbiota -diversity was not significantly different between BDL-VEH and SHAM and furthermore, FMT did not impact α-diversity in BDL rats. In contrast, β-diversity of microbiota did significantly differ between all groups (p < 0.05). The relative abundance also was significantly different between all three groups. In the BDL-FMT group, the phylum Firmicutes was found decreased while the Bacteroidetes were increased compared to BDLVEH and SHAM. The genera Bifidobacterium and Lactobacillus significantly increased in both BDL groups, while Akkermansia and Provotellaceae UCO-001 increased only in BDL-FMT rats and Clostridium Senso Stricto increased only in BDL-VEH compared to Shams. Finally, the genus Rombustia was only present in SHAM. Conclusion(s): Our results demonstrate that FMT lead to improvement in memory and motor coordination in BDL rats. The microbiota profile was different between BDL-VEH and SHAM, and FMT lead to further alterations. The fact that FMT did not normalize microbiota profile, suggests BDL-FMT leads to a novel specific microbiota profile which in turn protects the brain. The FMT-induced increase in Akkermansia and Provotellaceae UCO-001 merits to be further investigated in regards to their beneficial neurological effect in CLD.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique résultant d'une maladie du foie. Il a été démontré que le microbiote intestinal influence les fonctions neurologiques et l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif : Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats Sprague-Dawley mâles ont été randomisés en trois groupes : Contrôle (N=10), BDL-VEH (véhicule) (N=9) et BDL-FMT et ont reçu quotidiennement la FMT provenant des rats contrôles. Après cinq semaines, des tests comportementaux sont effectués pour évaluer la mémoire à court et long terme, l'anxiété et la coordination motrice. Le poids, la composition corporelle, la masse musculaire et les marqueurs hépatiques (ALT, AST, bilirubine). Enfin, les fèces ont été collectées et l’ADN bactérien a été séquencé. Résultats: La FMT n'a pas modifié la composition corporelle et la fonction hépatique chez les BDL-FMT par rapport aux BDL-VEH. Les BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice par rapport aux rats contrôles. Cependant, les altérations neurologiques ont été prévenues dans le groupe BDL-FMT. Concernant le microbiote, l’α-diversité n'était pas significativement différente entre tous les groupes contrairement à la β-diversité. Les genres Bifidobacterium et Lactobacillus sont augmentés de manière significative dans les groupes BDL, tandis que Akkermansia et Provotellaceae UCO-001 sont augmentés uniquement dans les rats BDL-FMT. Discussion: Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT n'a pas normalisé le profil du microbiote dans le groupe BDL-FMT, ce qui suggère qu’elle conduit à un nouveau profil spécifique du microbiote qui, à son tour, semble protéger en partie le cerveau. L'augmentation d’Akkermansia et de Provotellaceae dans le groupe BDL-FMT mérite d'être étudiée plus en détails.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background/Aims: Hepatic encephalopathy (HE) is a neuro¬psychiatric syndrome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypothesize that episodes of HE will accelerate neurological deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neurological status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days by injection of ammonium acetate starting week 3 post-BDL. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis of NeuN, SMI311, caspase-3, Bax/Bcl2 and GFAP. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP, cleaved-caspase-3 and Bax/Bcl2 protein expression were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and Bax/Bcl2 and a decrease in neuronal markers of NeuN and SMI311 in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis because of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Amal Trigui, Joan Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
La malnutrition est la complication la plus fréquente chez les patients atteints de maladies hépatiques chroniques (cirrhose) en attente d’une transplantation hépatique (TH) et elle pourrait mener à l’apparition d’autres complications cliniques avant et après la TH, incluant la sarcopénie (perte de masse et fonction musculaire). Objectifs: Évaluer les changements longitudinaux de l'état nutritionnel, de la qualité de vie et de la fonction musculaire des patients cirrhotiques en attente d’une TH. Méthode: 30 patients cirrhotique en attente d’une TH sont inclus dans cette étude. La fonction musculaire (Test d'élévation de la chaise), l'état nutritionnel (Liver Disease Undernutrition Screening Tool) et la qualité de vie (SF-36) sont évalués chaque 3 mois avant la TH jusqu’à la transplantation. Résultats: 29 patients en attente de TH sont inclus. L'âge moyen des patients est 51,1 ± 11,6 ans. L'étiologie la plus fréquente est l’alcoolisme (41 %). 83 % des patients sont à risque de malnutrition. La fonction musculaire est altérée (18,9 ± 7,8 s vs 12,6 s chez les patients sains ; p < 0,001) et diminue avec le temps d’attente. Concernant la qualité de vie, les scores de la santé physique (44,6% ± 20,4) et de santé mentale (53,5% ± 26,1) sont inférieurs aux scores normaux des patients en bonne santé (p < 0,001). La qualité de vie avait tendance à diminuer avec le temps d’attente. Conclusion: La majorité des patients en attente d'une TH sont à risque de malnutrition, présentent une fonction musculaire altérée qui diminue avec le temps d’attente et des scores de qualité de vie diminués.
Amal Trigui, Joan Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
La malnutrition est la complication la plus fréquente chez les patients atteints de cirrhose de foie et en attente d’une transplantation hépatique (TH) et elle pourrait mener à une perte de masse et de fonction musculaire (sarcopénie). Objectifs: 1) Évaluer les changements longitudinaux de l'état nutritionnel, de la qualité de vie et de la fonction musculaire des patients cirrhotiques en attente d’une TH. 2) Évaluer l’effet de la supplémentation précoce après la TH, riche en protéines et en énergie sur les mêmes paramètres. Méthode: Une étude pilote randomisée est menée auprès de 30 patients en attente d’une TH randomisés entre (1) un groupe intervention (n=15) qui reçoit des suppléments nutritionnels pendant 12 semaines et (2) un groupe contrôle (n=15) qui reçoit les soins nutritionnels standards. La fonction musculaire, l'état nutritionnel et la qualité de vie sont évalués chaque 3 mois avant la TH, à la sortie de l'hôpital et après 12 semaines. Résultats: 26 patients en attente de TH sont inclus dont un participant est transplanté. L'âge moyen des patients est 51,3 ± 12,9 ans. L'étiologie la plus fréquente est l’alcoolisme (33 %). 84 % des patients sont à risque de malnutrition. La fonction musculaire est altérée (18,1 ± 11,0 s vs 12,6 s chez les patients sains ; p < 0,001) et diminue avec le temps d’attente. Concernant la qualité de vie, les scores de la santé physique (41,8% ± 18,6) et de santé mentale (52,1% ± 25,0) sont inférieurs aux scores normaux des patients en bonne santé (p < 0,001). La qualité de vie avait tendance à diminuer avec le temps d’attente. Conclusion: La majorité des patients en attente d'une TH sont à risque de malnutrition, présentent une fonction musculaire altérée et des scores de qualité de vie diminués. Ces données préliminaires confirment la nécessité d'un soutien nutritionnel précoce après la TH.
Mariana Macedo de Oliveira, Alexis Monnet-Aimard, Cristina R. Bosoi, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a debilitating neurological complication of chronic liver disease (CLD). Hyperammonemia plays an important role in HE's pathogenesis, acting synergistically with systemic oxidative stress. During CLD, muscle plays a compensatory role in detoxifying ammonia, and therefore muscle loss leads to an increase in the risk of developing HE. With most animal studies involving males, sex's impact on the development of CLD and associated complications such as HE and muscle loss remains unknown. Therefore, we aimed to identify the impact of sex on CLD, HE, and muscle mass loss in a rodent model of CLD. Liver injury markers, hyperammonemia, oxidative stress, muscle mass, and ammonia clearance were measured in female and male bile-duct ligated (BDL) rats. In addition, covert HE was assessed in females while ammonia-precipitated severe HE was assessed in female and male BDL rats, and male BDL rats treated with allopurinol (100 mg/kg), an antioxidant (xanthine oxidase inhibitor). Female BDL developed CLD and HE (impaired motor coordination and night activity) compared to respective SHAM. Hyperammonemia and muscle ammonia clearance were similar between female and male BDL. However, only female BDL rats did not develop muscle loss, brain edema, and short-term memory impairment (vs. female SHAM) and systemic oxidative stress and decreased albumin levels (vs. male BDL). Furthermore, both female BDL and allopurinol-treated male BDL rats were protected against ammonia-induced overt HE. In conclusion, female and male BDL rats develop distinct features of CLD and HE, with systemic oxidative stress playing a pivotal role in the susceptibility to ammonia-precipitated overt HE.
Amal Trigui, Joan Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Introduction: In cirrhosis patients awaiting liver transplantation (LT), protein-energy malnutrition is the most common complication (> 80%) and one of the main risk factors for the onset and progression of sarcopenia, defined as a loss of muscle mass, quality, and function. Previous clinical studies have shown that malnutrition and sarcopenia are associated with negative outcomes, such as increased infections and mortality, as well as decreased quality of life. Objectives: Assess longitudinal changes in nutritional risk, muscle function and quality of life in cirrhotic patients awaiting LT. Method: A randomized controlled pilot study is conducted including 30 cirrhotic patients on the waiting list for LT. Nutritional risk (Liver Disease Undernutrition Screening Tool), muscle function (chair stand test), and quality of life (SF-36) are assessed every 3 months before and until LT. Results: Currently, 25 patients awaiting LT are included. The mean age of the patients is 51.3 ± 12.9 years. The most common etiology is alcoholism (33 %). 84 % of patients were at risk of malnutrition and with follow-ups up to a year on the waitlist, the percentage has not changed. Muscle function is impaired in those patients (18.1 ± 11.0 s vs. 12.6 s in healthy patients) (p < 0,001) and it decreased with the follow-ups (p < 0,05). Regarding quality of life, the score of physical heath was 41.8% ± 18.6 and mental health was 52.1% ± 25. With the follow-ups, the quality of life tended to decrease. Conclusion: The majority of patients waiting for a LT are at risk of malnutrition, display altered muscle function, and a tendency of decreased quality of life.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background/Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypothesize that episodes of HE will accelerate neurological deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neurological status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days by injection of ammonium acetate starting week 3 post-BDL. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis of NeuN, SMI311, caspase-3, Bax/Bcl2 and GFAP. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP, cleaved-caspase-3 and Bax/Bcl2 protein expression were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and Bax/Bcl2 and a decrease in neuronal markers of NeuN and SMI311 in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis because of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Implication de l’acide urique dans la pathophysiologie de l’encéphalopathie hépatique.
Sydnée L'Écuyer, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.
La maladie hépatique chronique (MHC) entraîne des complications graves dont l’encéphalopathie hépatique (EH). Les manifestations cliniques de l’EH sont des troubles de mémoire et d’apprentissage évoluant vers l’astérixis et le coma. L’EH se présente sous forme d’épisodes qui seraient médiés par la circulation d’ammoniaque. Par contre, les traitements diminuant l’ammoniaque ne permettent pas une réduction complète des déficits cognitifs. Ce projet suggère l’implication d’un nouveau facteur : l’acide urique (AU). L’hyperuricémie causée par une augmentation de l’AU circulant est considérée comme un facteur causal de la perte cellulaire et l’inflammation au niveau de plusieurs organes incluant le cerveau. Considérant que, dans le contexte de la MHC, l’hyperuricémie pourrait induire de la neuroinflammation, de la perte neuronale et des altérations comportementales, notre hypothèse est que l’AU participe à la pathophysiologie de l’EH. Pour confirmer l’hypothèse, nous utiliserons un modèle murin bien établi de cirrhose par ligation du canal biliaire (LCB), chez lequel nous induirons une hyperuricémie par l’administration d’une diète contenant 3% d’AU (HUAD). Pour mesurer l’effet sur la cognition, nous utilisons des tests comportementaux mesurant l’anxiété et la mémoire. Nous mesurerons également la neuroinflammation et la perte neuronale. Nos résultats préliminaires indiquent que, chez des animaux sains, la diète HUAD induit des troubles de mémoire ainsi qu’une augmentation de l’apoptose cérébrale. Chez les LCB, la diète HUAD induit des déficits de mémoires de manière précoce. En conclusion, l’AU, par ses effets sur le cerveau, induit des déficits cognitifs et une perte cellulaire au niveau cérébral.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique résultant d'une maladie du foie. Il a été démontré que le microbiote intestinal influence les fonctions neurologiques et l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif : Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats Sprague-Dawley mâles ont été randomisés en trois groupes : Contrôle (N=10), BDL-VEH (véhicule) (N=9) et BDL-FMT et ont reçu quotidiennement la FMT provenant des rats contrôles. Après cinq semaines, des tests comportementaux sont effectués pour évaluer la mémoire à court et long terme, l'anxiété et la coordination motrice. Le poids, la composition corporelle, la masse musculaire et les marqueurs hépatiques (ALT, AST, bilirubine). Enfin, les fèces ont été collectées et l’ADN bactérien a été séquencé. Résultats: La FMT n'a pas modifié la composition corporelle et la fonction hépatique chez les BDL-FMT par rapport aux BDL-VEH. Les BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice par rapport aux rats contrôles. Cependant, les altérations neurologiques ont été prévenues dans le groupe BDL-FMT. Concernant le microbiote, l’α-diversité n'était pas significativement différente entre tous les groupes contrairement à la β-diversité. Les genres Bifidobacterium et Lactobacillus sont augmentés de manière significative dans les groupes BDL, tandis que Akkermansia et Provotellaceae UCO-001 sont augmentés uniquement dans les rats BDL-FMT. Discussion: Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT n'a pas normalisé le profil du microbiote dans le groupe BDL-FMT, ce qui suggère qu’elle conduit à un nouveau profil spécifique du microbiote qui, à son tour, semble protéger en partie le cerveau. L'augmentation d’Akkermansia et de Provotellaceae dans le groupe BDL-FMT mérite d'être étudiée plus en détails.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major complication of chronic liver disease (CLD). HE is defined as a metabolic syndrome and therefore should resolve following liver transplantation (LT). However, persisting neurological complications and poor quality of life have been reported in up to 47% of LT recipients. Several retrospective studies have demonstrated an association between a history of HE episodes and neurological deficits following LT. However, the impact of HE episodes on the brain remains unknown. Purpose: Our aim was to evaluate the impact of multiple episodes (induced by ammonia) on neurological status, integrity and brain injury in cirrhotic rats. Method: 5-week bile-duct ligation (BDL) rats and Sham-operated controls (Sham) were used. BDL rats were injected (ip) with ammonium acetate (BDL-Ammonia), precipitating an overt episode of HE (pre-coma; loss of righting reflex) every 4 days from week 3 post-BDL surgery (total; 4 episodes). Sham rats were also injected with ammonia (Sham-Ammonia) and BDL and Sham rats were injected with saline as controls. Three days after the last episode, both short- and long-term memory (LTM) were assessed. Upon sacrifice, plasma and brains were collected for oxidative stress measurements and western blot analysis for Neuronal nuclei (NeuN and SMI311), caspase-3 (apoptotic marker), Bax/Bcl2 ratio (apoptotic marker), GFAP (astrocyte marker) and 4-HNE (oxidative stress marker) in frontal cortex, cerebellum and hippocampus. Result(s): LTM was found to be impaired in both BDL-Saline and BDL-Ammonia groups vs respective Sham controls. However, LTM impairment was further aggravated in BDL-Ammonia rats. In BDL-Ammonia, higher protein levels of GFAP and apoptotic markers were found in the hippocampus, whereas NeuN and SMI311 levels were reduced compared to all other experimental groups. BDL-Ammonia rats showed increased levels of plasma oxidative stress compared to respective Sham and BDL rats. Decreased levels of total antioxidant capacity and increased 4-HNE (oxidative stress marker) were found in the hippocampus (not in frontal cortex or cerebellum) of the BDL-Ammonia group compared to respective sham and BDL rats. Conclusion(s): Multiple episodes of overt HE lead to a worsening of the neurological impairments in BDL rats. LTM impairment in the BDL-Ammonia rats was associated with increased oxidative stress, apoptotic markers (caspase-3, Bax/Bcl2) and decreased neuronal markers (NeuN, SMI311) in the hippocampus, suggesting neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuate gliosis, possibly a result of neuronal loss. These results suggest that cumulative HE episodes may cause permanent cell damage and, therefore, will less likely reversible following LT. The impact of HE on neurological outcomes following LT merits further investigation.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome arising from chronic liver disease (CLD). HE manifests with symptoms such as poor memory, impairment in motor coordination, lethargy and coma. The gut microbiota has been shown to influence neurological function and many studies have demonstrated the association between altered microbiota and liver disease. Fecal matter transplantation (FMT) has revealed beneficial effects in clinical studies yet many limitations of these studies render the results inconclusive. Purpose: The aim of this study is to explore the impact of FMT on gut microbiota and the beneficial effects on neuro behaviour in bile-duct ligated (BDL) rats. Method: Male Sprague-Dawley rats were randomly assigned to one of three groups; SHAM (N=10), BDL-VEH (vehicle) (N=9) and BDL-FMT (who received FMT daily from pooled feces from SHAM rats). After five weeks, behaviour analysis was performed to evaluate short and long-term memory (Novel Object Recognition), anxiety (Open Field and Elevated Plus Maze) and motor coordination (Rotarod). Other parameters such as body weight and composition, ascites, gastrocnemius muscle weight and markers of liver function (ALT, AST, bilirubin and NH3) were also measured. Finally, the feces were collected and 16S RNA was sequenced for all groups. Result(s): FMT did not alter body composition (weight, composition, ascites and gastrocnemius muscle weight as well as and degree of liver disease (liver damage markers) in BDL-FMT vs BDL-Vehicle. BDL-VEH developed a loss of short/longterm memory and motor coordination compared to Sham rats. However, alterations in neurological dysfunction were prevented in the BDL-FMT group. The microbiota -diversity was not significantly different between BDL-VEH and SHAM and furthermore, FMT did not impact -diversity in BDL rats. In contrast, -diversity of microbiota did significantly differ between all groups (p < 0.05). The relative abundance also was significantly different between all three groups. In the BDL-FMT group, the phylum Firmicutes was found decreased while the Bacteroidetes were increased compared to BDLVEH and SHAM. The genera Bifidobacterium and Lactobacillus significantly increased in both BDL groups, while Akkermansia and Provotellaceae UCO-001 increased only in BDL-FMT rats and Clostridium Senso Stricto increased only in BDL-VEH compared to Shams. Finally, the genus Rombustia was only present in SHAM. Conclusion(s): Our results demonstrate that FMT lead to improvement in memory and motor coordination in BDL rats. The microbiota profile was different between BDL-VEH and SHAM, and FMT lead to further alterations. The fact that FMT did not normalize microbiota profile, suggests BDL-FMT leads to a novel specific microbiota profile which in turn protects the brain. The FMT-induced increase in Akkermansia and Provotellaceae UCO-001 merits to be further investigated in regards to their beneficial neurological effect in CLD.
Mariana M. Oliveira, Ole-Martin Fuskevåg, Mélanie Tremblay, Christopher Rose.
Background: The liver plays a major role in regulating ammonia levels in the blood. Therefore, liver disease and the loss of hepatic function lead to hyperammonemia, increased brain ammonia and consequently hepatic encephalopathy (HE). Hence, ammonia-lowering strategies remain the mainstay therapeutic strategy. Ammonia, both as an ion (NH 4+ ) and gas (NH 3 ), easily crosses all plasma membranes, including the blood-brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme that in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. Purpose: Therefore, we aimed to evaluate the presence and metabolism of GS in endothelial cells of the BBB. Method: Using primary rat brain microvascular endothelial cells (ECs) and isolated cerebral microvessels (CMV) from naïve rats (from frontal cortex and cerebellum), the presence of GS was assessed using rtPCR, western blot, and activity assay. In addition, to evaluate ammonia metabolism and potential GS targets in ECs, we exposed ECs to 5- 13 C glutamate, 5- 13 C ornithine and 5- 13 C -ketoglutarate, with and without ammonium chloride (0.5 mM and 1 mM) and measured the production of 5- 13 C glutamine by GS for up to 24h using LC-MS/MS. Result(s): In vitro , ECs were found to express GS mRNA and protein, along with GS activity. GS expression was also found in CMVs (similar levels between frontal cortex and cerebellum). 3D reconstruction using confocal microscopy showed that GS was co-localized with ECs in brain slices, although at lower levels than those expressed in astrocytes (p<0.001). Generation of 5- 13 C glutamine was the highest with 5- 13 C ornithine and 5- 13 C glutamate vs. 5- 13 C -ketoglutarate at all time points (p<0.01). Ammonia administration to cultured ECs did not lead to an increase of 5- 13 C glutamine generation in all groups. Conclusion(s): These results demonstrate for the first time that GS is present in ECs in both in vitro and ex-vivo . The lower expression of the enzyme compared to that found in the astrocytes in vitro and brain slices could explain why GS has never been reported in these cells. Both ornithine and glutamate have the potential to increase GS activity in ECs. Ammonia administration did not increase glutamine production by GS, likely due to insufficient substrates for glutamine synthesis. Therefore, the beneficial effect of ammonia scavenger therapies such as L-Ornithine L-Aspartate (LOLA) or Ornithine Phenylacetate (OP) might include stimulation of GS in the BBB.
Impact of sex on muscle mass loss and hepatic encephalopathy in rats with chronic liver disease.
Mariana M. Oliveira, Alexis Monnet-Aimard, Cristina Bosoi, Mélanie Tremblay, Christopher Rose.
Background: In chronic liver disease (CLD) loss of muscle mass (sarcopenia) is highly prevalent which leads to an increased risk of hepatic encephalopathy (HE). Muscle plays a compensatory role during CLD in clearing ammonia since it expresses glutamine synthetase (GS). Therefore, diminished muscle mass in CLD leads to a further reduced capacity to clear ammonia. Male rats with CLD due to bile-duct ligation (BDL) have been shown to result in a loss of muscle in association with hyperammonemia and HE. However, these complications have not been explored in female CLD rats. Purpose: Our aim was to identify whether female sex impacts muscle mass loss, blood ammonia levels and HE in ratswith CLD. Method: Five weeks after either BDL (n=8) or Sham (n=8) surgery in male and female rats, we assessed markers of liver injury (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)) and function (albumin and bilirubin). Neurophenotyping was achieved using the open field test and the elevated plus maze test for anxiety, the rota-rod test for motor coordination, the novel object recognition for short-term memory and the nighttime activity test. In addition, brain edema was assessed using the gravimetric technique. Body parameters (weight, composition (MRI)) and muscle (gastrocnemius weight and circumference and grip strength) were also evaluated. In addition, muscle GS activity, ammonia clearance as well as glutamine generation (femoral venous-arterial difference)were evaluated in female vs. male BDL rats. Result(s): Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin and albumin (p<0.001)) and both developed HE (impaired motor-coordination and night activity (p<0.05)) when compared to respective Shams. However, female BDL rats did not develop brain edema and did not have loss of short-term memory. Male BDL rats experienced loss of lean mass as well as reduced muscle circumference, weight and strength (p<0.01) compared to Sham rats, while similar differences between female BDL vs. Sham rats were not found. Male and female BDL rats had comparable blood ammonia levels as well as similar muscle ammonia clearance and glutamine production. However, GS activity was lower in female vs. male BDL rats (p<0.01). Conclusion(s): Our results demonstrate that following BDL surgery, female rats develop similar degrees of CLD compared to male rats. As in male BDL rats, female rats also develop HE but female BDL rats acquire unique features (not observed in males) such as lack of brain edema and intact short-term memory. Contrary to males, female BDL rats did not develop sarcopenia compared to respective controls. However, preserved muscle mass in female BDL did not result in increased muscle ammonia clearance and glutamine production. Therefore, the similar degrees of hyperammonemia in males vs. females may be due to an upregulation in GS found in the muscle of male BDL rats. Thus, the female-induced protection against brain edema and short-term memory in BDL rats likely involves additional factors besides ammonia.
Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher Rose.
Problématique: L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique résultant d'une maladie du foie. Il a été démontré que le microbiote intestinal influence les fonctions neurologiques et l'association entre l'altération du microbiote et les maladies hépatiques ressort dans de nombreuses études. Objectif : Explorer l'impact de la transplantation de microbiote fécal (FMT) sur le développement de l’EH de rats ayant subi une ligature du canal biliaire (BDL). Méthodologie: Des rats Sprague-Dawley mâles ont été randomisés en trois groupes : Contrôle (N=10), BDL-VEH (véhicule) (N=9) et BDL-FMT et ont reçu quotidiennement la FMT provenant des rats contrôles. Après cinq semaines, des tests comportementaux sont effectués pour évaluer la mémoire à court et long terme, l'anxiété et la coordination motrice. Le poids, la composition corporelle, la masse musculaire et les marqueurs hépatiques (ALT, AST, bilirubine). Enfin, les fèces ont été collectées et l’ADN bactérien a été séquencé. Résultats: La FMT n'a pas modifié la composition corporelle et la fonction hépatique chez les BDL-FMT par rapport aux BDL-VEH. Les BDL-VEH ont développé une perte de mémoire à court/long terme et une perte de coordination motrice par rapport aux rats contrôles. Cependant, les altérations neurologiques ont été prévenues dans le groupe BDL-FMT. Concernant le microbiote, l’α-diversité n'était pas significativement différente entre tous les groupes contrairement à la β-diversité. Les genres Bifidobacterium et Lactobacillus sont augmentés de manière significative dans les groupes BDL, tandis que Akkermansia et Provotellaceae UCO-001 sont augmentés uniquement dans les rats BDL-FMT. Discussion: Nos résultats démontrent que la FMT améliore la mémoire et la coordination motrice chez les rats BDL. La FMT n'a pas normalisé le profil du microbiote dans le groupe BDL-FMT, ce qui suggère qu’elle conduit à un nouveau profil spécifique du microbiote qui, à son tour, semble protéger en partie le cerveau. L'augmentation d’Akkermansia et de Provotellaceae dans le groupe BDL-FMT mérite d'être étudiée plus en détails.
Amal Trigui, Yvette Mukaneza, Mélanie Tremblay, Joann Maxwell, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Problématique : La malnutrition est la complication la plus fréquente chez les patients atteints de maladies hépatiques chroniques (cirrhose) en attente d’une transplantation hépatique (TH) et elle pourrait avoir un impact négatif sur les issues cliniques avant et après la TH. La malnutrition peut également entraîner la sarcopénie (perte de masse et fonction musculaires). L’objectif est d’évaluer les changements longitudinaux de l'état nutritionnel, de la qualité de vie et de la fonction musculaire des patients cirrhotiques en attente d’une TH. Après la TH, notre objectif est d’évaluer l’effet de la supplémentation précoce, riche en protéines, en bêta-hydroxy-bêta-méthylbutyrate et en énergie sur les mêmes paramètres qu’avant la TH. Méthodologie : Une étude pilote randomisée est menée auprès de 30 patients ayant reçu une TH, (1) groupe intervention (n=15) qui reçoit des suppléments nutritionnels pendant 12 semaines et (2) un groupe témoin (n=15) qui reçoit les soins nutritionnels standards. La masse musculaire (CT-scan), la fonction musculaire (Test d'élévation de la chaise), l'état nutritionnel (Liver Disease Undernutrition Screening Tool) et la qualité de vie (SF-36) sont évalués avant la TH, à la sortie de l'hôpital et après 12 semaines. Résultats et discussion : 24 patients en attente de TH sont présentement inclus. L’âge moyen des patients est 54,7 ± 8,0 ans. Avant la TH, 83.3% des patients souffrent de malnutrition. Les scores de la santé physique (43,5% ± 20,4) et mentale (53,8% ± 25,6) du SF-36 sont inférieurs aux scores des patients en bonne santé (81,60% et 78,58 %; p < 0,001). La fonction musculaire est altérée chez ces patients (19,3 ± 7,5 s vs 12,6 s chez des patients en bonne santé) (p < 0,001). La qualité de vie a légèrement diminué avec le temps (p>0,05). Conclusion : Ces données préliminaires appuient la nécessité d’un soutien nutritionnel précoce après la TH.
Manila Sophasath, Yvette Mukaneza, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Problématique: La malnutrition est l’une des complications les plus prévalentes de la cirrhose et affecte la qualité de vie des patients et des proches aidants. Objectifs: Le projet vise l’évaluation de ressources éducatives nutritionnelles auprès de patients cirrhotiques, en plusieurs volets. 1)Évaluer le Guide de nutrition pour la cirrhose sur les connaissances en nutrition, qualité de vie et état nutritionnel sur 6 mois. 2)Évaluer l’impact du Guide sur la qualité de vie et perception de fardeau des proches aidants sur 6 mois. 3)Évaluer la satisfaction du Guide des patients. 4)Évaluer la réceptivité des patients à une éducation nutritionnelle via les plateformes technologiques. Méthodologie: 1)100 patients de la clinique d’hépatologie du CHUM divisés en deux groupes (n=50/groupe): Intervention (Guide) et Contrôle (sans Guide). Les évaluations de l’état nutritionnel, les connaissances en nutrition, la qualité de vie et la fonction hépatique sont réalisées à 0, 3 et 6 mois. 2)La qualité de vie et perception de fardeau des proches aidants sont évaluées à 0 et 6 mois. 3)5 groupes de discussion (4 patients/groupe) permettront d’évaluer leur appréciation du Guide. 4)100 patients qui rempliront des questionnaires (transversal) sur leur utilisation, compétences et préférences en matière de technologies. Résultats préliminaires (volet 1): 31 patients ont complété l’évaluation de 3 mois à ce jour : intervention (n=16) et contrôle (n=15). Ces résultats démontrent une tendance d’amélioration des connaissances (4,2%; de 75,4% à 79,6%) pour le groupe intervention, versus 0,9% pour le groupe contrôle (de 73,7% à 74,6%), (p=0.212). Maintenant, 18 patients ont complété l’évaluation de 6 mois: intervention (n=9) et contrôle (n=9). Les résultats préliminaires démontrent que la tendance d’amélioration de connaissances dénotée après 3 mois n’est pas maintenue après 6 mois (groupe intervention): de 75,4% à 79,6% (3 mois), à 74.4% (6 mois). Discussion: Les résultats de cette étude devraient permettre d’optimiser la qualité des soins chez les gens souffrant de cirrhose.
Ammonia-induced episodic HE leads to neuronal cell injury in bile-duct ligated rats
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background/Aims: Hepatic encephalo¬pathy (HE) is a neuro¬psychiatric syn¬drome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypo¬thesize that episodes of HE will accelerate neuro¬logical deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neuro¬logical status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days by injection of ammonium acetate starting week 3 post-BDL. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis of NeuN, SMI311, caspase-3, Bax/Bcl2 and GFAP. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP, cleaved-caspase-3 and Bax/Bcl2 protein expression were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and Bax/Bcl2 and a decrease in neuronal markers of NeuN and SMI311 in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis because of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Implication de l’acide urique dans la pathophysiologie de l’encéphalopathie hépatique.
Sydnée L'Écuyer, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.
Problématique-La maladie hépatique chronique (MHC) entraîne des complications graves dont l’encéphalopathie hépatique (EH). Ses manifestations cliniques sont des troubles de mémoire et d’apprentissage évoluant vers l’astérixis et le coma. L’EH se présente sous forme d’épisodes médiés par la circulation d’ammoniaque. Par contre, les concentrations plasmatiques d’ammoniaque ne corrèlent pas avec la sévérité des symptômes. Ce projet vise à vérifier l’implication d’un nouveau facteur dans l’EH: l’acide urique (AU). L’augmentation de l’AU en circulation sanguine cause une perte cellulaire et l’inflammation de plusieurs organes incluant le cerveau. Considérant que l’hyperuricémie pourrait induire de la neuroinflammation, de la perte neuronale et des altérations comportementales, notre hypothèse est que l’AU contribue à la pathophysiologie de l’EH. Méthodes- Pour confirmer l’hypothèse, nous utiliserons un modèle murin de cirrhose par ligation du canal biliaire (LCB),avec hyperuricémie induite par l’administration d’une diète contenant 3% d’AU (HUAD). Pour mesurer l’effet sur la cognition, nous utilisons des tests comportementaux mesurant l’anxiété (test du labyrinthe en croix surélevé et du champ ouvert) et la mémoire (test de reconnaissance d’un nouvel objet). Une analyse de la perte neuronale sera également complétée par mesure de l’activité de la caspase-3 (C3) et expression de la protéine NeuN. Résultats- Nos résultats préliminaires indiquent que, chez des animaux sains, la diète HUAD induit des troubles de mémoire avec une diminution significative du temps d’exploration du nouvel objet et une activité doublée de la C3 en comparaison au groupe contrôle sur diète régulière. Chez les LCB, la diète HUAD induit ces déficits de mémoire de manière précoce, à la 3e semaine, mais n’induit pas une augmentation de l’apoptose. Conclusion- En conclusion, dans le contexte de la MHC, l’AU induit des troubles de mémoire et une perte cellulaire au niveau cérébral. Subventions: Ferring Pharmaceuticals, CRCHUM et Département de Pharmacologie et Physiologie de l'Université de Montréal
The effect of early nutritional intervention on muscle mass following liver transplantation.
Amal Trigui, Joann Maxwell, Geneviève Huard, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
After liver transplantation (LT), nutritional status can worsen rapidly leading to sarcopenia (loss of muscle mass and function) and altered quality of life. Few studies have focused on preventing malnutrition and sarcopenia after LT whereas guidelines for the management of sarcopenia after LT are lacking. The objectives of our study are to evaluate the effect of early nutritional supplementation, after LT, rich in protein, beta-hydroxy-beta-methylbutyrate (HMB, active metabolite of leucine) and energy on muscle mass and function, nutritional status, quality of life and development of complications. Method: A randomized controlled pilot study is conducted including 30 patients who have undergone LT: (1) intervention group (n=15) receiving, in addition to the standard nutritional care, supplements rich in protein, HMB and energy for 12 weeks and (2) control group (n=15) receiving standard nutritional care. Muscle mass (computed tomography scan), muscle function (chair stand test), nutritional status (liver disease undernutrition screening tool) and quality of life (SF-36) are assessed every 3 months before LT, after LT on discharge from hospital and after 12 weeks. Dietary protein intake and physical activity are assessed once a month. Adherence to treatment is monitored by phone calls, by a logbook and by measuring urinary HMB once a month. Preliminary results: Currently, 23 patients awaiting liver transplantation are included. Once transplanted, the patient is randomized into one of the two groups. The mean age of the patients is 53.9 ± 12.0 years. Before LT, 82 % of patients were classified as malnourished. Regarding quality of life, the score of physical (47.4% ± 28.6) and mental (51.1% ± 28.8) health of the SF-36 are significantly lower than the scores of healthy patients of the same age (81.60% and 78.58%, respectively) (p < 0,001). Muscle function is also impaired in those patients (25.1 ± 11.0 s vs. 12.6 s in healthy patients) (p < 0,001). Conclusion: These preliminary data support the need for early nutritional support after LT. Our study could significantly improve the health and quality of life of patients after LT through the primary and secondary prevention of malnutrition and sarcopenia.
Mimosa Nguyen, Yvette Mukaneza, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
Liver transplantation (LT) is the only curative treatment for cirrhosis. However, the presence of complications can impact outcomes following LT. Sarcopenia, or muscle mass loss, is highly prevalent in patients with cirrhosis and is associated with longer hospitalization stays and a higher infection rate post-surgery. We aimed to identify patients at higher risk of early sarcopenia post-LT. This retrospective study included 79 cirrhotic patients who underwent LT. Muscle mass was evaluated using the third lumbar spine vertebra skeletal muscle index (SMI) and sarcopenia was defined using established cut-off values. Computerized tomography (CT) scans performed within a six-month peri-operative period (three months pre- and post-LT) were included in the study. Complications and comorbidities were collected and correlated to SMI post-LT and predictive models for SMI post-LT were constructed. The overall prevalence of sarcopenia was 46% and 62% before and after LT, respectively. Newly developed sarcopenia was found in 42% of patients. Post-LT sarcopenia was associated with longer hospital stays (54±37 versus 29±10 days, = 0.002), higher number of infection (3±1 versus 1±2, = 0.027), and greater number of complications (5±2 versus 3±2, < 0.001) compared to absence of sarcopenia. Multivariate analyses showed that the SMI post-LT was independently associated with pre-LT renal function markers, the glomerular filtration rate (GFR) and creatinine (Model 1, GFR: = 0.33; 95% CI 0.04-0.17; = 0.003; Model 2, Creatinine: = -0.29; 95% CI -0.10 to -0.02; = 0.009). The present study highlights the potential role of renal dysfunction in the development and persistence of sarcopenia after LT.
Amino acids, ammonia, and hepatic encephalopathy.
Katerina Kroupina, Chantal Bémeur, Christopher F. Rose.
Hepatic encephalopathy (HE) is a decline in brain function arising due to liver insufficiency. The liver's diminished capacity to clear ammonia, and the subsequent accumulation of it, is highly implicated in pathogenesis of HE. Ammonia is endogenously generated from the catabolism of amino acids derived from dietary protein intake. Therefore, a conflict arises in cirrhosis where dietary protein intake may increase ammonia and precipitate HE, and at the same time, cirrhotic patients require high daily protein intake due to altered nutrient metabolism. A nutritional solution is needed to deliver sufficient doses of protein to patients without increasing the risk of HE. In order to address this issue, this review will discuss the catabolism of individual amino acids with a special focus on ammonia-generating steps and highlight a subset of amino acids that have the potential to generate multiple equivalents of ammonia. Following, studies investigating the effects of individual amino acids in cirrhosis on blood ammonia levels as well as development of HE will be reviewed.
Heretical thoughts into Hepatic Encephalopathy
Rajiv Jalan, Christopher F. Rose.
Clinical progress in the development of new diagnostic modalities and therapeutic strategies for the management of patients with hepatic encephalopathy has lagged behind the vast knowledge that has been generated from basic studies. In this article, we critically assess matters that should be revisited, such as definition, classification, diagnosis and grading of hepatic encephalopathy, which are difficult to apply reproducibly using the current criteria. Many lines of investigation have confirmed that hepatic encephalopathy is irreversible in many patients and suggest the need for further studies focussing on mechanisms of neuronal injury and death, to guide future drug development for these patients. The clinical evidence behind using lactulose for all severities of hepatic encephalopathy, which is currently considered the standard of care, is poor and placebo-controlled trials for hepatic encephalopathy should be considered ethically sound. This expert opinion identifies current challenges in hepatic encephalopathy and highlights areas which require further debate and investigation in order to help advance the field both scientifically and clinically.
Amal Trigui, Yvette Mukaneza, Mélanie Tremblay, Joan Maxwell, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Après la transplantation hépatique (TH), l'état nutritionnel d'un patient peut s’aggraver rapidement conduisant à une sarcopénie (perte de masse et fonction musculaire). L’objectif de notre étude est d’évaluer l’effet de la supplémentation précoce, après la TH, riche en protéines, en bêta-hydroxy-bêta-méthylbutyrate (HMB, métabolite actif de la leucine) et en énergie en sur (1) la masse et la fonction musculaire, (2) l'état nutritionnel, (3) la qualité de vie et (4) le développement de complications. Méthode : Une étude pilote randomisée contrôlée est menée auprès de 30 patients ayant subi une TH, (1) groupe intervention (n=15) qui reçoit des suppléments riches en protéines, en HMB et en énergie (60 ml 4 fois/jour pendant 12 semaines) et (2) un groupe témoin (n=15) qui reçoit les soins nutritionnels standards. La masse musculaire (CT-scan), la fonction musculaire (Test d'élévation de la chaise), l'état nutritionnel (Liver Disease Undernutrition Screening Tool) et la qualité de vie (SF-36) sont évalués avant la TH, après la transplantation à la sortie de l'hôpital et après 12 semaines. L'apport en protéines alimentaires et l'activité physique sont évalués une fois par mois. Résultats préliminaires et discussion : Présentement, 21 patients en attente de greffe hépatique sont inclus. L’âge moyen des patients est 53,9 ± 12,0 ans. Avant la TF, la majorité des patients (82%) souffrent de malnutrition. Concernant la qualité de vie, le score de la santé physique (47,4% ± 28,6) et mentale (51,1% ± 28,8) du SF-36 sont inférieurs aux scores des patients du même âge en bonne santé (81,60% et 78,58 %, respectivement). La fonction musculaire est altérée chez les patients (25,1 ± 11,0 s vs 12,6 s chez des patients en bonne santé). Conclusion : Ces données préliminaires appuient la nécessité d’un soutien nutritionnel précoce après la TF. Notre étude pourrait contribuer à améliorer de façon importante la santé et la qualité de vie des gens ayant reçu une TF en plus de diminuer les coûts hospitaliers, via la prévention primaire et secondaire de la malnutrition et de la sarcopénie.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD) loss of muscle mass (sarcopenia) is highly prevalent which leads to an increased risk in hepatic encephalopathy (HE). Muscle plays a compensatory role during liver disease in clearing ammonia since it contains glutamine synthetase (GS). Therefore, diminished muscle mass leads to a further reduced capacity to remove ammonia. Loss of muscle mass has been described in male rats with CLD but has not been explored in female CLD rats. Our aim was to identify whether sex has an effect on muscle mass and blood ammonia levels. Methods: Five weeks after either BDL (n=8) or Sham (n=8) surgery in male and female rats, the following were assessed; markers of liver injury and function, HE (open field test for anxiety, rota-rod test for motor coordination and night-time activity), body parameters (weight, composition (MRI) and gastrocnemius muscle weight/circumference and grip strength). In addition, muscle GS activity and muscle ammonia clearance as well as glutamine generation (femoral venous-arterial difference) were evaluated in female vs male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin and albumin (p<0.001)) and both developed HE (motor-coordination and night activity (p<0.05)) when compared to respective Shams. Male BDL rats experienced loss of lean mass, muscle weight and strength (p<0.01) while no differences were found in female BDL vs Sham rats. Male and female BDL rats had similar ammonia clearance and glutamine production by muscle, while GS activity was lower in female vs. male BDL rats (p<0.01). Discussion: Our results demonstrate that following BDL surgery, female rats develop CLD and HE comparable to male rats. However, contrary to males, female BDL rats did not develop sarcopenia compared to respective controls. Preserved muscle mass in female BDL did not result in lower blood ammonia (higher ammonia clearance) whereas muscle mass loss in male BDL rats was accompanied with an upregulation in GS which may explain the similar blood ammonia levels found in both male and female BDL rats.
Episodic hepatic encephalopathy induces neuronal cell injury in rats with chronic liver disease
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalo¬pathy (HE) is a neuro¬psychiatric syn¬drome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications which is believed to be associated with a history of HE pre-LT. We hypo¬thesize that episodes of HE will accelerate neuro¬logical deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes (ammonia induced) on neuro¬logical status and brain injury in rats with chronic liver disease (CLD). Methods: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were grouped into episodic and non-episodic groups. Episodes of HE were induced every 4 days starting the week 3 post-BDL following a reversible injection of ammonium acetate in which BDL rats fell into pre-coma (loss of righting reflex) for 20-30min. Three days following the last injection, neurobehavior was assessed. Upon sacrifice, plasma ammonia was measured, and brains were collected for western blot and immunofluorescence analysis; neuronal markers including NeuN and SMI311, astrocytic marker; GFAP and apoptotic markers such as cleaved caspase-3 and Bax and Bcl2 were measured to evaluate neurological integrity. Results: BDL rats, and not Sham rats, injected with ammonia developed overt HE. Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls. However, LTM impairment was further aggravated in episodic BDL rats. Protein expression levels of GFAP, cleaved caspase-3 and Bax/Bcl2 ratio were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL rats vs non-episodic BDL rats. Presence of cleaved caspase-3 in neuronal layer of CA1 area of hippocampus was confirmed with immunofluorescence imaging. Blood ammonia levels were found to be higher in episodic vs non-episodic BDL rats. Conclusion: Episodes of overt HE exasperate neurological impairment in BDL rats. LTM impairment was associated with an increase in apoptotic markers, decrease in neuronal markers and presence of cleaved caspase-3 in neuronal layer of CA1 area of hippocampus, suggesting neuronal injury/loss. Elevated levels of GFAP (astrocytic marker) in the hippocampus insinuates gliosis, possibly as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to therefore irreversible and persisting neurological complications post-LT.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD), the muscle plays a compensatory role in clearing ammonia and therefore muscle mass loss increases the risk of hepatic encephalopathy (HE). It has been demonstrated that systemic oxidative stress is implicated in the pathogenesis of brain edema, however only in male rats with CLD. Therefore, our aim is to identify the impact of sex on the pathogenesis of HE and brain, including ammonia-induced overt HE. Methods: Male and female bile-duct ligated (BDL) rats (and respective Shams) were evaluated for minimal HE and ammonia-precipitated overt HE (lethargy/loss of righting reflex). In addition, liver markers, hyperammonemia, systemic OS, muscle mass and ammonia clearance were evaluated. Results: Female and male BDL rats had similar liver impairment (ALP, AST, and bilirubin (p<0.001)) and HE (motor-coordination and night activity (p<0.05)) vs. respective Shams. Contrary to males, females BDLs did not develop muscle loss (p<0.05), brain edema or severe HE episode (p<0.005). Furthermore, degree of hyperammonemia as well as muscle ammonia clearance were similar between male and female BDLs but systemic OS was significantly lower in females (p<0.01). Discussion: Our findings demonstrate that female BDL rats develop CLD, HE and similar degrees of hyperammonemia compared to male rats. Differently from males, female BDL did not develop muscle mass loss, brain edema nor severe HE following ammonia challenge. The protection against brain edema in female BDLs is primarily due to low systemic OS but whether brain edema or systemic OS renders male BDLs most susceptible to ammonia-precipitated insults remains to be determined.
Hepatic Encephalopathy: From Metabolic to Neurodegenerative.
Rafael Ochoa-Sanchez, Farzaneh Tamnanloo, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome of both acute and chronic liver disease. As a metabolic disorder, HE is considered to be reversible and therefore is expected to resolve following the replacement of the diseased liver with a healthy liver. However, persisting neurological complications are observed in up to 47% of transplanted patients. Several retrospective studies have shown that patients with a history of HE, particularly overt-HE, had persistent neurological complications even after liver transplantation (LT). These enduring neurological conditions significantly affect patient's quality of life and continue to add to the economic burden of chronic liver disease on health care systems. This review discusses the journey of the brain through the progression of liver disease, entering the invasive surgical procedure of LT and the conditions associated with the post-transplant period. In particular, it will discuss the vulnerability of the HE brain to peri-operative factors and post-LT conditions which may explain non-resolved neurological impairment following LT. In addition, the review will provide evidence; (i) supporting overt-HE impacts on neurological complications post-LT; (ii) that overt-HE leads to permanent neuronal injury and (iii) the pathophysiological role of ammonia toxicity on astrocyte and neuronal injury/damage. Together, these findings will provide new insights on the underlying mechanisms leading to neurological complications post-LT.
Evaluation of the Nutrition in Cirrhosis Guide : an implementation study.
Manila Sophasath, Yvette Mukaneza, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, C. Bémeur.
Introduction: Liver disease affects over 9 million Canadians and kills 2 million people annually worldwide. Malnutrition is the most common complication of chronic liver disease and impacts the patients quality of life. This project aims to investigate the potential beneficial impact of the Nutrition in Cirrhosis Guide, an evidence-based document developed by experts and patients. Specifically, 1)Assess the short/long-term effects on i)nutritional status; ii)nutritional knowledge; iii)quality of life; iv)liver function; and, long-term only, v)complications; vi)hospital stays. 2) Determine the Guide’s impact on the caregivers’s quality of life and burden. 3)Assess the patients’ and caregivers’ satisfaction towards the Guide. 4)Elaborate implementation steps of the Guide among this population. Methods: 160 patients from the hepatology outpatients clinic from the Centre hospitalier de l’Université de Montréal in Canada will be divided in 2 groups (n=80/group): Experimental (Guide) and Control (no Guide). Assessements will be performed by a registered dietitian at T=0, 12 and 52 weeks regarding nutritional status, quality of life, liver function and nutritional knowledge. At 52 weeks, complications and hospital stays will be documented. The caregivers’ quality of life and burden will be assessed at 0, 12 and 52 weeks. Finally, three focus groups of patients and caregivers will be randomly chosen from the Experimental group to discuss the Guide’s appreciation. Preliminary results: So far, 24 patients completed the study (67% men, 60 years old in average, and etiologies 30% NASH, 25% alcohol, 12,5% HCV, HBV and mixed etiologies, and 4% others). The results show a trend of a better nutritional knowledge in the Experimental group (n=19) compared to controls (n=5) by 10% after 12 weeks. There is yet no difference in nutritional status and quality of life. Conclusion: Overall, our results should facilitate the implementation of the Guide and, importantly, optimize the quality of care in the cirrhotic community.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome observed in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids and brain lipid alterations to neurological impairment, we hypothesize that obese-induced brain lipid dysregulation increases the progression of HE in bile-duct ligated (BDL) rats. Aim: Study the impact of obesity on brain sphingolipids and the development of neurological impairment in a rat model of CLD and obesity. Method: CLD and HE model: 5-week BDL rats and Sham-controls, were used. Groups: Obese-BDL (O-BDL) and Obese-Sham (O-Sham) received high-fat diet (HFD) for 25-days pre-BDL surgery and then proceeded with high-carbohydrate diet (HCD) for 5 weeks post-BDL; Lean-BDL (L-BDL) and Lean-Sham (L-Sham) received regular-diet (RD) pre-BDL surgery and then HCD post-BDL. Body weight (BW), fat-mass, behavior, plasma liver markers and brain hippocampal sphingolipids were measured at 0 (pre-BDL), 3 and 5 weeks post-BDL. Results: Pre-BDL, HFD lead to increased BW and fat mass vs RD which was sustained with HCD in O-BDL vs L-BDL at 3 and 5 weeks. Ammonia, albumin, AST, ALT, ALP and bilirubin were impaired in O-BDL and L-BDL vs Shams at 3 and 5 weeks. High-density and low-density (LDL) lipoprotein levels were detected in pre-BDL rats which persisted in O-BDL and L-BDL vs controls at 3 and 5 weeks. LDL and total-cholesterol were higher in O-BDL vs L-BDL at 5 weeks. Short- and long-term (LTM) memory were impaired in both BDL groups at 5 weeks, whereas at 3 weeks, LTM impairment was found solely in O-BDL. Furthermore at 3 weeks, motor coordination was reduced in O-BDL, but not in L-BDL. At 5 weeks, motor coordination decreased in both BDL groups, with worse performance in O-BDL vs L-BDL. At 3 and 5 weeks, skill learning improved in L-Shams and L-BDL, but not in O-Shams and O-BDL. Muscle-strength was reduced in L-BDL but not in O-BDL vs Shams. Hippocampal sphingomyelin lipids were increased in O-BDL vs L-BDL rats at 0, 3 and 5 weeks, while ceramides were only reduced at 5 weeks. Conclusion: CLD-obesity rat model is characterized by increased fat mass and hyperlipidemia pre- and post-BDL. Neurological decline in O-BDL rats developed earlier and was more severe vs L-BDL rats. In addition, different neurological impairments developed in O-BDL vs L-BDL. Brain sphingolipid alteration in obese-cirrhotic rats may contribute to accelerate/worsen HE.
Role of sex on the development of sarcopenia and ammonia metabolism in bile-duct ligated rats.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD) loss of muscle mass (sarcopenia) is highly prevalent which leads to an increased risk in hepatic encephalopathy (HE). Muscle plays a compensatory role during liver disease in clearing ammonia since it contains glutamine synthetase (GS). Therefore, diminished muscle mass leads to a further reduced capacity to remove ammonia. Loss of muscle mass has been described in male rats with CLD but has not been explored in female CLD rats. Our aim was to identify whether sex has an effect on muscle mass and blood ammonia levels. Methods: Five weeks after either BDL (n=8) or Sham (n=8) surgery in male and female rats, the following were assessed; markers of liver injury and function, HE (open field test for anxiety, rota-rod test for motor coordination and night-time activity), body parameters (weight, composition (MRI) and gastrocnemius muscle weight/circumference and grip strength). In addition, muscle GS activity and muscle ammonia clearance as well as glutamine generation (femoral venous-arterial difference) were evaluated in female vs male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin and albumin (p<0.001)) and both developed HE (motor-coordination and night activity (p<0.05)) when compared to respective Shams. Male BDL rats experienced loss of lean mass, muscle weight and strength (p<0.01) while no differences were found in female BDL vs Sham rats. Male and female BDL rats had similar ammonia clearance and glutamine production by muscle, while GS activity was lower in female vs. male BDL rats (p<0.01). Discussion: Our results demonstrate that following BDL surgery, female rats develop CLD and HE comparable to male rats. However, contrary to males, female BDL rats did not develop sarcopenia compared to respective controls. Preserved muscle mass in female BDL did not result in lower blood ammonia (higher ammonia clearance) whereas muscle mass loss in male BDL rats was accompanied with an upregulation in GS which may explain the similar blood ammonia levels found in both male and female BDL rats.
Régime hyperprotéiné dans l'encéphalopathie hépatique : précipitant ou protecteur ?
Katerina Kroupina, Karine Dubois, Mélanie Tremblay, Christopher Rose.
Introduction : L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique qui survient à la suite d'une cirrhose. Les symptômes comprennent des troubles de la mémoire et de la coordination motrice, le coma et même la mort. L'ammoniaque, une toxine produite lors du métabolisme d’acides aminés, est impliquée dans la pathogenèse. La première ligne de défense contre l'augmentation de l'ammoniaque, le foie, ne fonctionne pas à pleine capacité lors de la cirrhose et sa détoxification diminue, augmentant les concentrations sériques. La deuxième ligne de défense est le muscle squelettique. Toutefois, les patients cirrhotiques présentent une importante perte musculaire, ce qui augmente aussi le risque d’EH. L'objectif de ce projet est de déterminer si l'excès de protéines est néfaste en raison de la production excessive d'ammoniac, ou protecteur en raison du maintien de la masse musculaire. Méthodes : Les rats cirrhotiques (ligation de voie biliaire) se nourriront d’une diète haute en protéines (36%) ou régulière (18%) pendant les 5 semaines post-chirurgie. L’ammoniaque sérique sera évaluée à 0, 3 semaines, et 5 semaines. Les tests comportementaux pour évaluer les symptômes d’EH et l’évaluation de la composition corporelle seront faits à 3 et 5 semaines. L’activité de glutamine synthétase musculaire sera évaluée pour déterminer si la diète en augmente l’activité. Résultats : Les résultats préliminaires indiquent que les rats mangeant une diète hyperprotéinée voient une amélioration de la masse musculaire et ne présentent pas d’augmentation de l’ammoniaque ou de déficience cognitive comparé aux rats sur une diète normale. Discussion : Ce projet aidera à améliorer les lignes directrices en alimentation protéique chez les patients souffrant d’EH.
Sydnée L'Écuyer, Karine Dubois, Mélanie Tremblay, Emmanuel Charbonney, Christopher Rose.
La maladie hépatique chronique (MHC) entraîne des complications graves dont l’encéphalopathie hépatique (EH). Les manifestations cliniques de l’EH sont des troubles de mémoire et d’apprentissage évoluant vers l’astérixis et le coma. Des études sur la physiopathologie de l’EH ont déterminé qu’elle arrive de manière épisodique et serait médiée par l’ammoniaque. Par contre, les traitements diminuant l’ammoniaque ne permettent pas une réduction complète des déficits cognitifs. Ce projet suggère l’implication d’un nouveau facteur : l’acide urique (AU). L’AU est une alarmine relâchée par les cellules mourantes. Dans la MHC, l’AU serait relâchée par les hépatocytes lésés et cause de la neuroinflammation, de la perte neuronale et des altérations comportementales. Notre hypothèse est que l’AU participe à la pathophysiologie de l’EH. Pour confirmer l’hypothèse, nous utiliserons un modèle murin bien établi de cirrhose de ligation du canal biliaire (LCB), chez lequel nous augmenterons la circulation d’AU par l’administration d’une diète contenant 3% d’AU (HUAD) et la diminuerons avec une uricase. Pour mesurer l’effet sur la cognition, nous utiliserons des tests comportementaux mesurant l’anxiété et les déficits de mémoire. Nous mesurerons également la neuroinflammation et perte neuronale par immunobuvardage et immunofluorescence. Des résultats préliminaires suggèrent une circulation d’AU augmentée chez les LCB comparés aux contrôles. Chez des animaux sains, la diète HUAD induit des troubles de mémoire. Chez les LCB, nous nous attendons à ce que la diète HUAD aggrave ou accélère les déficits cognitifs. En conclusion, ce projet vise à élucider le rôle d’un nouveau médiateur, l’AU, dans la pathophysiologie de l’EH.
Sex impacts muscle loss and ammonia metabolism in rats with chronic liver disease.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD), muscle wasting is highly prevalent and leads to an increased risk of hepatic encephalopathy (HE). Since muscle’s glutamine synthetase (GS) compensates for ammonia detoxification during CLD, less muscle leads to a lower capacity to remove ammonia. Muscle loss has been described in male rats but not in female rats with CLD. We aimed to identify whether sex influences muscle mass and blood ammonia levels. Methods: Five weeks after either bile duct ligation (BDL) (n=8) or Sham surgery (n=8) in male and female rats, the following were assessed; markers of liver injury and function, HE (behavioral tests), body weight, and composition (MRI) and, muscle weight, circumference and strength, as well as muscle GS and glutamine generation were evaluated in female vs. male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin, and albumin (p<0.001)) and HE (motor-coordination and activity (p<0.05)) compared to respective Shams. Male BDL, but not female, experienced loss of lean mass, muscle weight, and strength (p<0.01). Male and female BDL had similar muscle ammonia clearance and glutamine production, with lower GS activity in female vs. male BDL (p<0.01). Discussion: Our results demonstrate that although sex did not influence CLD and HE, female rats had preserved muscle mass, which did not result in lower blood ammonia. Contrarily, muscle loss in male rats upregulated GS, which may explain the similar blood ammonia levels found in both male and female BDL rats.
Sydnée L'Écuyer, Karine Dubois, Mélanie Tremblay, Emmanuel Charbonney, Christopher Rose.
La maladie hépatique chronique (MHC) entraîne des complications graves dont l’encéphalopathie hépatique (EH). Les manifestations cliniques de l’EH sont des troubles de mémoire et d’apprentissage évoluant vers l’astérixis et le coma. Des études sur la physiopathologie de l’EH ont déterminé qu’elle arrive de manière épisodique et serait médiée par l’ammoniaque. Par contre, les traitements diminuant l’ammoniaque ne permettent pas une réduction complète des déficits cognitifs. Ce projet suggère l’implication d’un nouveau facteur : l’acide urique (AU). L’AU est une alarmine relâchée par les cellules mourantes. Dans la MHC, l’AU serait relâchée par les hépatocytes lésés et cause de la neuroinflammation, de la perte neuronale et des altérations comportementales. Notre hypothèse est que l’AU participe à la pathophysiologie de l’EH. Pour confirmer l’hypothèse, nous utiliserons un modèle murin bien établi de cirrhose de ligation du canal biliaire (LCB), chez lequel nous augmenterons la circulation d’AU par l’administration d’une diète contenant 3% d’AU (HUAD) et la diminuerons avec une uricase. Pour mesurer l’effet sur la cognition, nous utiliserons des tests comportementaux mesurant l’anxiété et les déficits de mémoire. Nous mesurerons également la neuroinflammation et perte neuronale par immunobuvardage et immunofluorescence. Des résultats préliminaires suggèrent une circulation d’AU augmentée chez les LCB comparés aux contrôles. Chez des animaux sains, la diète HUAD induit des troubles de mémoire. Chez les LCB, nous nous attendons à ce que la diète HUAD aggrave ou accélère les déficits cognitifs. En conclusion, ce projet vise à élucider le rôle d’un nouveau médiateur, l’AU, dans la pathophysiologie de l’EH.
Ammonia-induced episodic HE leads to neuronal cell injury in bile-duct ligated rats.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalo¬pathy (HE) is a neuro¬psychiatric syn¬drome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypo¬thesize that episodes of HE will accelerate neuro¬logical deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neuro¬logical status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats, and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days starting the week 3 post-BDL following injection of ammonium acetate. 3 days following the last injection, neurological status was assessed. Upon sacrifice brains were collected for western blot analysis; Neuronal nuclei (NeuN), caspase-3 and GFAP were measured. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP and caspase-3 protein expression were significantly increased, whereas NeuN was significantly decreased in hippocampus of episodic BDL rats vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and a decrease in NeuN in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Amal Trigui, Yvette Mukaneza, Mélanie Tremblay, Joann Maxwell, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
La malnutrition protéino-énergétique est la complication la plus courante (> 80%) chez les patients en attente d’une transplantation hépatique (TH). Après une TH, l'état nutritionnel d'un patient peut s'aggraver conduisant à une sarcopénie (perte de masse musculaire), fortement corrélée à la morbidité et la mortalité après la TH. Objectifs : Évaluer l’effet de la supplémentation en protéines et en bêta-hydroxy-bêta-méthylbutyrate (HMB, métabolite actif de la leucine) en post-TH sur (1) la masse et la fonction musculaire, (2) l'état nutritionnel, (3) la qualité de vie et (4) le développement de complications. Méthode : Une étude pilote randomisée contrôlée, dont le recrutement est en cours, est menée auprès de 30 patients ayant subi une TH, (1) groupe intervention (n=15) qui reçoit des suppléments riches en protéines et en HMB (60 ml 4 fois/jour pendant 12 semaines), en plus de recevoir les conseils nutritionnels habituels et (2) un groupe témoin (n=15) qui reçoit les soins nutritionnels standards. La masse musculaire (CT-scan), la fonction musculaire (force de préhension), l'état nutritionnel (tableau de l’identification de la malnutrition) et la qualité de vie (SF-36) sont évalués : avant la TH, après la transplantation à la sortie de l'hôpital et au terme de 12 semaines d’intervention. L'apport en protéines alimentaires et l'activité physique sont contrôlés une fois par mois. Après la sortie de l'hôpital, l’adhérence est vérifiée par un suivi téléphonique toutes les deux semaines ainsi que par la mesure du HMB urinaire une fois par mois. Résultats attendus et impact potentiel : L’intervention nutritionnelle riche en protéines et en HMB pourrait améliorer la masse musculaire, l’état nutritionnel et la qualité de vie durant la période postopératoire et ainsi prévenir l’apparition des complications par rapport au groupe contrôle. Notre étude pourrait contribuer à améliorer la santé et la qualité de vie des gens ayant reçu une TH en plus de diminuer les coûts hospitaliers.
Manila Sophasath, Yvette Mukaneza, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Problématique: Les maladies hépatiques touchent plus de 9 millions de Canadiens. La malnutrition est l’une des complications les plus prévalentes de la cirrhose (maladie hépatique chronique) et affecte la qualité de vie des patients et des proches aidants. Objectif: L’objectif général est d’évaluer l’impact du Guide de nutrition pour la cirrhose, un document éducatif développé par des experts et patients canadiens, basé sur des données probantes. Spécifiquement, 1)Évaluer l’effet court/moyen-long terme sur i) l’état nutritionnel; ii) les connaissances en nutrition; iii) la qualité de vie; iv) la fonction hépatique et, à long terme, v) les hospitalisations (fréquence/durée). 2)Déterminer l’impact du Guide sur la qualité de vie et perception de fardeau des proches aidants. 3)Évaluer la satisfaction du Guide des patients et des proches aidants. 4)Élaborer les étapes d’implantation du Guide. Méthodologie: 100 patients de la clinique d’hépatologie du CHUM sont divisés en deux groupes (n=50/groupe) : Expérimental (Guide) et Contrôle (sans Guide). Les évaluations de l’état nutritionnel, les connaissances en nutrition, la qualité de vie et la fonction hépatique sont réalisées par une nutritionniste aux temps T=0, 3 et 6 mois. À 6 mois, les hospitalisations sont documentées. La qualité de vie et perception de fardeau des proches aidants sont évaluées à 0 et 6 mois. Finalement, 3 groupes de discussion de 10 patients et proches permettront l’évaluation qualitative de leur appréciation du Guide. Résultats préliminaires: 25 patients ont complété le suivi court terme (67% hommes, âge moyen de 60 ans, étiologies: 30% NASH, 25% alcoolique, 12,5% virale et étiologies mixtes, et 4% autres). Les résultats démontrent que le groupe expérimental (n=20) a tendance à voir une amélioration des connaissances en nutrition de 10% par rapport aux contrôles (n=5) après 3 mois. Discussion: Les résultats de cette étude devraient permettre d’optimiser la qualité des soins chez les gens souffrant de cirrhose.
La barrière hémato-encéphalique lors d'encéphalopathie hépatique épisodique.
Mihai Alexandru Popovici, Farzaneh Tamnanloo, Mélanie Tremblay, Christopher F. Rose.
L’encéphalopathie hépatique (EH) est une complication neuropsychiatrique de la maladie chronique du foie (cirrhose). Le foie endommagé n’a plus la même capacité de biotransformation qu’un foie en santé ce qui entraine une augmentation en circulation de neurotoxines comme l’ammoniaque. Les symptômes peuvent être légers (troubles cognitifs : attention, mémoire) ou sévères (confusion, désorientation ou coma). L’EH est de forme épisodique lorsque les symptômes apparaissent de façon ponctuelle. La barrière hémato-encéphalique (BHE) protège le cerveau en temps normal. Toutefois, au cours des dernière années, il a été observé que la présence de symptômes persiste malgré la résolution des épisodes. Le but du projet est d’évaluer la perméabilité de la BHE chez des modèles d’EH épisodique. Pour ce faire, des modèles de rats atteints d’EH épisodique ont été obtenus en effectuant une ligature de la voie biliaire pour provoquer une cirrhose et en injectant de l’acétate d’ammonium (3,5mmol/kg ip.) tous les quatre jours à partir de la 19e journée. Au 38e jour, les rats ont été sacrifiés et l’expression de protéines de jonctions serrées (Claudine-5, Occludine, ZO-1 et ZO-2) a ensuite été mesurée dans le cortex frontal, le cervelet et l’hippocampe par Western blot. Une diminution de l’expression de ZO-2 entre les rats contrôles et les rats épisodiques a été observée. D’autres études seront nécessaires pour confirmer ce résultat et vérifier l’implication de la BHE dans l’EH épisodique, en relation avec les changements de comportement observés (troubles de la mémoire et coordination motrice).
Régime hyperprotéiné dans l'encéphalopathie hépatique : précipitant ou protecteur ?
Katerina Kroupina, Karine Dubois, Mélanie Tremblay, Christopher F. Rose.
Introduction: Chronic liver disease adversely affects neurocognitive functioning. These cognitive and mental status changes can range from subclinical and frequently undiagnosed impairments, known as covert hepatic encephalopathy (HE), to overt HE, with requires hospitalization and increases mortality. Efficient and practical screening tests are needed to detect early cognitive alterations, to avoid the progression to advanced grades of HE. Objective: To identify patients at high risk of developing overt EH within a year, using a battery of online cognitive tests. Methods: 190 cirrhotic patients from CHUM Hepatology clinic will be recruited for a prospective 1-year longitudinal study and will perform the CBS cognitive assessment at baseline. Cambridge Brain Sciences (CBS), an online platform adapted for research, offers a 45-minute online self-assessment that evaluates 4 cognitive domains (memory, reasoning, concentration and verbal ability). Scores are compared to a large age and gender-matched population database. Patients will be followed up for 1 year with phone calls every 3 months and medical records will be checked to identify HE-related hospitalizations and mortality. Statistical analysis will be performed to study the correlation between screening test results and patient outcomes. Results: To date, 14 participants (78% men, mean age 62.42 ± 7.25) have been included. Results show that cirrhotic patients scored below average in more than 90% of cognitive tasks performed, compared to the normative database. Discussion: The expected results can provide important information for identifying patients at high risk of developing overt HE within 1 year, which can propose prophylaxis, early treatment, prevention of episodes and promote a great impact on the patient's quality of life, cost of treatments and decrease in mortality.
Sydnée L'Écuyer, Karine Dubois, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.
Introduction- La maladie hépatique chronique (MHC) entraîne des complications graves dont l’encéphalopathie hépatique (EH). Les manifestations cliniques de l’EH sont des troubles de mémoire et d’apprentissage évoluant vers l’astérixis et le coma. Des études sur la physiopathologie de l’EH ont déterminé qu’elle arrive de manière épisodique et serait médiée par l’ammoniaque. Par contre, les traitements diminuant l’ammoniaque ne permettent pas une réduction complète des déficits cognitifs. Ce projet suggère l’implication d’un nouveau facteur : l’acide urique (AU). L’AU est une alarmine relâchée par les cellules mourantes. Dans la MHC, l’AU serait relâchée par les hépatocytes lésés et cause de la neuroinflammation, de la perte neuronale et des altérations comportementales. Notre hypothèse est que l’AU participe à la pathophysiologie de l’EH. Matériel et Méthode- Pour confirmer l’hypothèse, nous utiliserons un modèle murin bien établi de cirrhose de ligation du canal biliaire (LCB), chez lequel nous augmenterons la circulation d’AU par l’administration d’une diète contenant 3% d’AU (HUAD) et la diminuerons avec une uricase. Pour mesurer l’effet sur la cognition, nous utiliserons des tests comportementaux mesurant l’anxiété et les déficits de mémoire. Nous mesurerons également la neuroinflammation et perte neuronale par immunobuvardage et immunofluorescence. Résultats- Des résultats préliminaires suggèrent une circulation d’AU augmentée chez les LCB comparés aux contrôles. Chez des animaux sains, la diète HUAD induit des troubles de mémoire. Chez les LCB, nous nous attendons à ce que la diète HUAD aggrave ou accélère les déficits cognitifs. Conclusion- Ce projet vise à élucider le rôle d’un nouveau médiateur, l’AU, dans la pathophysiologie de l’EH.
Amal Trigui, Yvette Mukaneza, Mélanie Tremblay, Joann Maxwell, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Introduction : La malnutrition protéino-énergétique est la complication la plus courante (> 80%) chez les patients en attente d’une transplantation hépatique (TH). L'état nutritionnel peut s'aggraver conduisant à une sarcopénie (perte de masse et fonction musculaire), fortement corrélée à la morbidité et la mortalité après la TH. L’objectif de notre étude est d’évaluer l’effet de la supplémentation précoce en protéines, en bêta-hydroxy-bêta-méthylbutyrate (HMB, métabolite actif de la leucine) et en énergie en post-TH sur (1) la masse et la fonction musculaire, (2) l'état nutritionnel, (3) la qualité de vie et (4) le développement de complications. Méthode : Une étude pilote randomisée contrôlée, dont le recrutement est en cours, est menée auprès de 30 patients ayant subi une TH répartis dans un groupe intervention (n=15) qui reçoit des suppléments riches en protéines, HMB et énergie (60 ml 4 fois/jour/12 semaines) et un groupe témoin (n=15) qui reçoit les soins nutritionnels standards. La masse musculaire (CT-scan), la fonction musculaire (Chair Stand Test), l'état nutritionnel (LDUST,Liver Disease Undernutrition Screening Tool) et la qualité de vie (SF-36, Short Form 36) sont évalués avant la TH, après la transplantation à la sortie de l'hôpital et après 12 semaines. L'apport en protéines alimentaires et l'activité physique sont également évalués une fois par mois. Résultats: Six patients sont présélectionnés et sont en attente de greffe. L’âge moyen des patients est 48,3 ±12,4 ans. 100% des patients sont malnutris. Concernant la qualité de vie, le score des composantes physiques est 50,6 ± 8,6 % et le score des composantes mentales est 60,4± 17,9%.[BC1] Conclusion : Notre étude pourrait améliorer considérablement la santé et la qualité de vie des personnes ayant reçu une TH en plus de réduire les coûts hospitaliers, grâce à la prévention primaire et secondaire de la malnutrition et de la sarcopénie.
Silícia Ane Tres, Mélanie Tremblay, Yvette Mukaneza, Christopher F. Rose.
Introduction: Chronic liver disease adversely affects neurocognitive functioning. These cognitive and mental status changes can range from subclinical and frequently undiagnosed impairments, known as covert hepatic encephalopathy (HE), to overt HE, with requires hospitalization and increases mortality. Efficient and practical screening tests are needed to detect early cognitive alterations, to avoid the progression to advanced grades of HE. Objective: To identify patients at high risk of developing overt EH within a year, using a battery of online cognitive tests. Methods: 190 cirrhotic patients from CHUM Hepatology clinic will be recruited for a prospective 1-year longitudinal study and will perform the CBS cognitive assessment at baseline. Cambridge Brain Sciences (CBS), an online platform adapted for research, offers a 45-minute online self-assessment that evaluates 4 cognitive domains (memory, reasoning, concentration and verbal ability). Scores are compared to a large age and gender-matched population database. Patients will be followed up for 1 year with phone calls every 3 months and medical records will be checked to identify HE-related hospitalizations and mortality. Statistical analysis will be performed to study the correlation between screening test results and patient outcomes. Results: To date, 14 participants (78% men, mean age 62.42 ± 7.25) have been included. Results show that cirrhotic patients scored below average in more than 90% of cognitive tasks performed, compared to the normative database. Discussion: The expected results can provide important information for identifying patients at high risk of developing overt HE within 1 year, which can propose prophylaxis, early treatment, prevention of episodes and promote a great impact on the patient's quality of life, cost of treatments and decrease in mortality.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD), muscle wasting is highly prevalent and leads to an increased risk of hepatic encephalopathy (HE). Since muscle’s glutamine synthetase (GS) compensates for ammonia detoxification during CLD, less muscle leads to a lower capacity to remove ammonia. Muscle loss has been described in male rats but not in female rats with CLD. We aimed to identify whether sex influences muscle mass and blood ammonia levels. Methods: Five weeks after either bile duct ligation (BDL) (n=8) or Sham surgery (n=8) in male and female rats, the following were assessed; markers of liver injury and function, HE (behavioral tests), body weight, and composition (MRI) and, muscle weight, circumference and strength, as well as muscle GS and glutamine generation were evaluated in female vs. male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin, and albumin (p<0.001)) and HE (motor-coordination and activity (p<0.05)) compared to respective Shams. Male BDL, but not female, experienced loss of lean mass, muscle weight, and strength (p<0.01). Male and female BDL had similar muscle ammonia clearance and glutamine production, with lower GS activity in female vs. male BDL (p<0.01). Discussion: Our results demonstrate that although sex did not influence CLD and HE, female rats had preserved muscle mass, which did not result in lower blood ammonia. Contrarily, muscle loss in male rats upregulated GS, which may explain the similar blood ammonia levels found in both male and female BDL rats.
Ammonia-induced episodic HE leads to neuronal cell injury in bile-duct ligated rats.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypothesize that episodes of HE will accelerate neurological deterioration. Our goal was to evaluate the impact of cumulative HE episodes on neurological status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days starting week 3 post-BDL following injection of ammonium acetate. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis; Neuronal nuclei (NeuN), caspase-3, and GFAP were measured. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP and caspase-3 protein expression were significantly increased, whereas NeuN was significantly decreased in the hippocampus of episodic BDL rats vs non-episodic BDL rats. Conclusion: HE episodes exacerbate neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and a decrease in NeuN in the hippocampus, which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Repeated hyperammonemic insults leads to irreversible brain injury in bile-duct ligated rats.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD). Defined as a metabolic disorder, HE is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications. Retrospective studies have revealed that patients with a history of overt HE are associated with a poor neurological outcome post-LT. However, the impact of HE episodes on neuro¬logical in¬tegrity is unknown. We hypothesize that episodes of HE will accelerate and/or exasperate neurological deterioration. Purpose: Our goal was to characterize an animal model of episodic HE and to evaluate the impact of cumulative episodes induced by hyperammonemia on neurological status and brain injury in cirrhotic rats. Method: For animal model of CLD and HE, 5-week bile-duct ligation (BDL) rats, and Sham-operated controls were used. Rats, both Sham and BDL, were divided in two groups, episodic and non-episodic. Injection (ip) of ammonium acetate was used to induce episodes of overt HE (pre-coma; loss of righting reflex) every 4 days starting at 3-weeks post-BDL surgery (total 4 episodes). Saline was injected as vehicle for non-episodic group. Three days following the last episode of HE, neurological status was assessed. Upon sacrifice, plasma ammonia was measured, and brains were collected for western blot analysis; Neuronal nuclei (NeuN), caspase-3 (apoptotic marker) and GFAP (astrocyte marker) were measured to evaluate neurological integrity. Results: BDL rats (vs Sham) following ammonia insult developed overt HE (loss of righting reflex). Long-term memory (LTM) was impaired in both non-episodic and episodic BDL groups vs respective controls. However, LTM impairment was further aggravated in episodic BDL rats. Both GFAP and cleaved caspase-3 protein expression were significantly increased, whereas NeuN was significantly decreased in hippocampus of episodic BDL rats vs non-episodic BDL rats. Blood ammonia levels were found to be higher in episodic vs non-episodic BDL rats. Conclusion: HE episodes exasperate neurological impairments in BDL rats. LTM impairment was associated with an increase in the apoptotic marker (caspase-3) and a decrease in neuronal marker (NeuN) in the hippocampus, suggesting neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage and therefore will less likely be reversible following LT justifying persisting neurological complications post-LT.
Dysregulation of sphingolipids in obese-cirrhotic rats aggravates hepatic encephalopathy.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome observed in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids and brain lipid alterations to neurological impairment, we hypothesize that obese-induced brain lipid dysregulation increases the progression of HE in bile-duct ligated (BDL) rats. Aim: Study the impact of obesity on brain sphingolipids and the development of neurological impairment in a rat model of CLD and obesity. Method: CLD and HE model: 5-week BDL rats and Sham-controls, were used. Groups: Obese-BDL (O-BDL) and Obese-Sham (O-Sham) received high-fat diet (HFD) for 25-days pre-BDL surgery and then proceeded with high-carbohydrate diet (HCD) for 5 weeks post-BDL; Lean-BDL (L-BDL) and Lean-Sham (L-Sham) received regular-diet (RD) pre-BDL surgery and then HCD post-BDL. Body weight (BW), fat-mass, behavior, plasma liver markers and brain hippocampal sphingolipids were measured at 0 (pre-BDL), 3 and 5 weeks post-BDL. Results: Pre-BDL, HFD lead to increased BW and fat mass vs RD which was sustained with HCD in O-BDL vs L-BDL at 3 and 5 weeks. Ammonia, albumin, AST, ALT, ALP and bilirubin were impaired in O-BDL and L-BDL vs Shams at 3 and 5 weeks. High-density (HDL) and low-density (LDL) lipoprotein levels were detected in pre-BDL rats which persisted in O-BDL and L-BDL vs controls at 3 and 5 weeks. LDL and total-cholesterol were higher in O-BDL vs L-BDL at 5 weeks. Short- and long-term (LTM) memory were impaired in both BDL groups at 5 weeks, whereas at 3 weeks, LTM impairment was found solely in O-BDL. Furthermore at 3 weeks, motor coordination was reduced in O-BDL, but not in L-BDL. At 5 weeks, motor coordination decreased in both BDL groups, with worse performance in O-BDL vs L-BDL. At 3 and 5 weeks, skill learning improved in L-Shams and L-BDL, but not in O-Shams and O-BDL. Muscle-strength was reduced in L-BDL but not in O-BDL vs Shams. Hippocampal sphingomyelin lipids were increased in O-BDL vs L-BDL rats at 0, 3 and 5 weeks, while ceramides were only reduced at 5 weeks. Conclusion: CLD-obesity rat model is characterized by increased fat mass and hyperlipidemia pre- and post-BDL. Neurological decline in O-BDL rats developed earlier and was more severe vs L-BDL rats. In addition, different neurological impairments developed in O-BDL vs L-BDL. Brain sphingolipid alteration in obese-cirrhotic rats may contribute to accelerate/worsen HE.
Sex disparity in the development of brain edema and hepatic encephalopathy severity in BDL rats.
Mariana Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome with symptoms ranging from impaired reaction time, poor memory to asterixis, gross disorientation, lethargy and coma. HE is a major complication of chronic liver disease (CLD) and develops when the hepatic capacity to detoxify ammonia is decreased. During this condition, muscle plays a compensatory role removing ammonia, but muscle mass loss further reduces the capacity to metabolise ammonia and hyperammonemia prevails. HE is marked by an increase in brain water caused via the synergistic effect of ammonia and oxidative stress (reactive oxygen species (ROS)). Brain edema and ammonia-related cognitive impairments as well as muscle mass loss have been well described in male rats with CLD but have not been explored in female CLD rats. Therefore, our aim was to identify whether sex influences brain edema and ammonia-mediated cognitive impairments. Methods: Five weeks after either bile-duct ligation (BDL) (n=8) or Sham (n=8) surgery in male and female rats, we assessed markers of liver injury and function, body parameters (weight, composition (MRI), gastrocnemius muscle weight/circumference and grip strength), HE (open field test for anxiety, rota-rod test for motor coordination and night-time activity) and brain edema (by gravimetric density method). Finally, we assessed susceptibility to develop severe cognitive impairment (mild-moderate lethargy, ataxia and loss of righting reflex) in male and female BDL rats after an ammonia challenge (6 mmoles/kg of ammonium acetate injected subcutaneously). Both plasma ammonia (Randox kit) and ROS (2',7'-dichlorodihydrofluorescein diacetate test (DCFDA)) were investigated. Results: Female BDL rats, similar to male BDL rats, had CLD, with impaired liver markers (ALP (p<0.001), AST (p<0.001), bilirubin (p<0.0001) and albumin (p<0.001)) compared to respective Sham controls. Male BDL rats experienced loss of lean mass (p<0.001), muscle weight (p<0.01) and strength (p<0.01) while similar differences were not found in female BDL vs respective Sham controls. Both female and male BDL developed HE (impaired motor-coordination (p<0.05) and reduced night activity (p<0.05)), compared to respective Shams. However, contrary to male BDLs, female BDLs did not develop brain edema compared to respective Sham controls. When compared to male BDL, baseline plasma ammonia levels did not differ to female BDL rats. However, plasma ROS levels were lower in females compared to males (p<0.05). Following ammonia challenge, male BDL rats had progressive worsening of mental status, reaching ataxia and loss of righting reflex while female BDL were protected, reaching only moderate lethargy (p<0.05). During the ammonia challenge, female BDL rats had similar ammonia levels but lower levels of ROS compared to male BDL rats (p<0.01). Discussion: We demonstrated BDL surgery in females leads to hepatic and neurological impairment comparable to male BDL rats. Contrary to males, female BDL did not develop loss of muscle mass nor brain edema compared to respective controls. However, sustained muscle mass in females did not lead reduced blood ammonia therefore the protection versus brain edema in females in due to protection against systemic oxidative stress. Female BDLs did not developed severe HE following ammonia challenge. Whether brain edema or systemic oxidative stress renders the females resistant to ammonia insults remains to be determined.
Manila Sophasath, Yvette Mukaneza, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Liver disease affects over 9 million Canadians and kills 2 million people annually worldwide. One of the most prevalent complications of chronic liver disease (cirrhosis) is malnutrition, which greatly affects the quality of life of patients and their caregivers. In this context, several nutritional guidelines for cirrhotic patients have been developed. However, the application of these guidelines seems to have important shortcomings, such as the difficulty of achieving certain nutritional goals for the majority of patients. The Nutrition in Cirrhosis guide was developed by a national team of hepatology and nutrition experts. Objectives: 1) To assess the impact in cirrhotic patients, in the short and long term, of the Guide on: i) nutritional status; ii) nutrition knowledge; iii) quality of life; iv) liver function; and, in the long term, v) complications; vi) number and duration of hospitalization. 2) Determine the impact on the quality of life and the perceived burden of caregivers. 3) Survey the appreciation of the Guide by patients and their caregivers. Method: A randomized controlled study targeting 100 cirrhotic patients at the Centre Hospitalier de l’Université de Montréal (CHUM) divided into 2 groups: Intervention (Guide) and Control (without Guide). Nutritional status (Liver Disease Undernutrition Screening Tool), nutrition knowledge (homemade questionnaire), quality of life (Chronic Liver Disease Questionnaire) and liver function (from medical charts) are assessed at t = 0, 3 and 6 months, and hospitalizations after 6 months. For the second objective, the quality of life (Short-Form 36 Questionnaire) and perceived burden (Zarit Burden Interview) of caregivers is evaluated at t = 0 and 6 months. Three focus groups of 10 patients and their caregiver, randomly chosen from the intervention group, will be created to assess their appreciation of the Guide. Preliminary results: 26 patients completed the 3-month pilot study (67% men, mean age = 60 years and etiologies: 30% non-alcoholic hepatic steatosis, 25% alcoholic, 12.5% hepatitis C, hepatitis B and mixed etiologies, and 4% other etiologies). The results show that the patients in the intervention group (n=20) have better knowledge of nutrition than the controls (n=5) (79.5 ± 7.7% vs 68.4 ± 7.9%; p=0.02) after 3 months. The control group also displayed an improvement in their quality of life after 3 months compared to t = 0 (5.10 ± 1.15 vs 5.61 ± 1.08, p<0.0005). Conclusion: The Guide seems to offer a beneficial effect on the quality of life and nutrition knowledge of cirrhotic patients after 3 months. The long-term impact of this resource still needs to be established in order to develop implementation vehicles and eventually include it in cirrhotic patient care.
The Nutrition in Cirrhosis Guide: Potential beneficial effects in cirrhotic patients and caregivers.
Manila Sophasath, Yvette Mukaneza, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Liver disease affects over 9 million Canadians and kills 2 million people annually worldwide. One of the most prevalent complica-tions of chronic liver disease (cirrhosis) is malnutrition, which greatly affects the quality of life of patients and their caregivers.The Nutrition in Cirrhosis guide was developed by a national team of hepatology and nutrition experts. Objectives: 1) To assess theimpact in cirrhotic patients, in the short and long term, of the Guide on: i) nutritional status; ii) nutrition knowledge; iii) qualityof life; iv) liver function; and, in the long term, v) complications; vi)number and duration of hospitalization. 2) Determine the impact on the quality of life and the perceived burden of caregivers. 3) Survey the appreciation of the Guide by patients and their caregivers. Method: A randomized controlled study targeting 100 cirrhoticpatients at the Centre Hospitalier de l’Université de Montréal (CHUM) divided into 2 groups: Intervention (Guide) and Control (without Guide). Nutritional status, nutrition knowledge, quality of life and liver function are assessed at t = 0, 3 and 6 months, and hospitalizations after 6 months. The quality of life and perceived burden of caregivers is evaluated at t=0 and 6 months.Threefocusgroups of 10 patients and their caregiver, randomly chosen from the intervention group, will be created to assess their appreciation of the Guide. Results (pilot study): 26 patients completed the 3-month pilot study (67% men, mean age = 60 years and etiologies: 30% non-alcoholic hepatic steatosis, 25% alcoholic, 12.5% hepatitis C, hepatitis B and mixed etiologies, and 4% other etiologies). The results show that the patients in the intervention group (n=20) have better knowledge ofnutritionthanthecontrols(n=5)(79.5 ±7.7% vs 68.4 ±7.9%; p=0.02) after 3 months. The control group also displayed an improvement in their quality of life after 3 months compared to t = 0 (5.10 ± 1.15 vs 5.61 ± 1.08, p<0.0005). Conclusion: The Guide seems to offer a beneficial effect on the quality of life and nutrition knowledge of cirrhotic patients after 3 months.
Manila Sophasath, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
L’une des complications les plus prévalentes de la maladie hépatique chronique (cirrhose) est la malnutrition, qui affecte grandement la qualité de vie. Le Guide de nutrition pour la cirrhose a été élaboré par une équipe nationale d’experts en hépatologie et en nutrition. Objectifs: 1)Évaluer l’impact chez les patients cirrhotiques, à court et long terme, du Guide sur : i) l’état nutritionnel; ii) les connaissances en nutrition; iii) la qualité de vie; iv) la fonction hépatique; et, à long terme uniquement, v) les complications; vi) le nombre et la durée d’hospitalisation. 2)Déterminer l’impact sur la qualité de vie et la perception de fardeau des proches aidants. 3)Sonder l’appréciation du Guide par les patients et leurs proches aidants. Méthode: Une étude randomisée contrôlée visant 100 patients cirrhotiques du CHUM divisés en 2 groupes : intervention (Guide) et contrôle (sans Guide). L’état nutritionnel, connaissances en nutrition, qualité de vie et fonction hépatique sont évalués à t=0, 3 et 6 mois, et les hospitalisations survenues après 6 mois. La qualité de vie et perception de fardeau des proches aidants sera évaluée à t=0 et 6 mois. Trois groupes de discussion de 10 patients et leur proche aidant, aléatoirement choisis dans le groupe intervention, seront créés afin d’évaluer leur appréciation du Guide. Résultats (pilote): 25 patients ont complété l’étude pilote de 3 mois (67% hommes, âge moyen = 60 ans et étiologies : 30% stéatose hépatique non-alcoolique, 25% alcoolique, 12,5% hépatite C, hépatite B et étiologies mixtes, et 4% autres étiologies). Les résultats montrent que les patients du groupe intervention (n=20) ont des connaissances en nutrition supérieures aux contrôles (n=5) (79,5±7,7% vs 68,4±7,9%; p=0,02) après 3 mois. Le groupe contrôle voit aussi une amélioration de leur qualité de vie après 3 mois en comparaison au t=0 (5,10±1,15 vs 5,61 ±1,08, p<0.0005). Conclusion: Le Guide semble démontrer un effet bénéfique sur la qualité de vie et les connaissances des patients cirrhotiques après 3 mois.
Rafael Ochoa‐Sanchez, Mariana M. Oliveira, Mélanie Tremblay, Grégory Petrazzo, Asha Pant, Cristina R. Bosoi, Mylene Perreault, William Querbes, Caroline B. Kurtz, Christopher F. Rose.
Les métaux lourds et l’encéphalopathie hépatique.
Mihai Alexandru Popovici, Mélanie Tremblay, Christopher F. Rose.
L’encéphalopathie hépatique est une complication neurologique d’une maladie du foie. Les mécanismes menant au développement de l’encéphalopathie sont complexes et mettraient en scène plusieurs facteurs d’origines différentes. L’ammoniac est considéré comme le facteur principal. Cette substance neurotoxique est présente en grande quantité chez les patients atteints d’encéphalopathie hépatique. D’autres facteurs comme la présence d’inflammation ou l’accumulation d’acides aminés aromatiques participent aussi au développement de la maladie. Beaucoup de travail reste à faire au sujet de la pathogenèse de l’encéphalopathie hépatique. Les métaux lourds représentent une piste éventuelle vers une meilleure compréhension de la pathologie. Parmi ceux-ci, le zinc et le manganèse retiennent particulièrement l’attention. Le zinc est l’un des éléments traces les plus importants pour le corps humain dont les niveaux dans l’organisme peuvent être affectés par une maladie hépatique. Les suppléments de zinc incorporés dans la diète ont été avancés comme une possible thérapie, mais sont loin d’être concluants. Établir l’efficacité d’un tel traitement est un des buts de ce papier. Des rats recevront une diète avec supplément de carbonate de zinc. Ils passeront plusieurs tests d’orientation et d’attention et leurs niveaux et activité d’ornithine transcarbamylase et de glutamine synthetase seront comparés avec ceux de rats témoins ayant reçu une diète normale. Le manganèse est un élément omniprésent dans les aliments. Comme le zinc, il entre dans la composition de nombreuses métalloenzymes. Le manganèse est excrété en très grande partie dans la bile. Une maladie hépatique jouant sur l’élimination ou la composition de la bile peut entrainer une augmentation des taux de manganèse dans le corps. De là, le manganèse s’accumule au niveau des ganglions de la base et peut causer une toxicité. L’évaluation des effets de l’accumulation de manganèse sur le cerveau des rats cirrhotiques avec épisodes d’encéphalopathie hépatique fut un autre but de ce projet. Les rats recevront tout d’abord une diète exempte de manganèse puis une injection d’acétate d’ammonium (3,5 mmoles/kg, ip). Ils passeront des tests tels le labyrinthe de Morris, le champ ouvert et le pont étroit. Il sera important de mesurer la formation et la présence de molécules affectées par le manganèse comme l’acétylcholine ou encore des neurostéroides. En s’intéressant davantage aux métaux lourds et à leur rôle dans l’encéphalopathie hépatique, il sera possible de mieux caractériser les facteurs à la genèse de la maladie. Les solutions pour pallier aux concentrations anormales de zinc et de manganèse offriront de futures thérapies potentielles à l’encéphalopathie hépatique.
Constantine J. Karvellas, Jaime L. Speiser, Mélanie Tremblay, William M. Lee, Christopher F. Rose, U. S. Acute Liver Failure Study Group.
Liver-type fatty acid binding protein (FABP1) has previously been demonstrated to improve prognostic discrimination in acetaminophen (APAP)-induced ALF but has not been investigated in other etiologies of ALF. To determine whether FABP1 levels (early: admission or late: days 3-5) are associated with 21-day transplant-free survival in non-APAP ALF. FABP1 was measured in serum samples from 384 ALF patients (n = 88 transplant-free survivors (TFS), n = 296 died/LT-NTFS) using solid-phase enzyme-linked immunosorbent assay and analyzed with US ALFSG registry data. Of 384 ALF patients (autoimmune hepatitis n = 125, drug-induced liver injury n = 141, Hepatitis B n = 118), 177 (46%) patients received LT. Early FABP1 levels were significantly higher in ALF patients requiring vasopressor support (203.4 vs. 76.3 ng/mL) and renal replacement therapy (203.4 vs. 78.8 ng/mL; p < 0.001 for both). Late FABP1 levels were significantly higher in patients requiring mechanical ventilation (77.5 vs. 53.3 ng/mL), vasopressor support (116.4 vs. 53.3 ng/mL) and in patients with grade 3/4 hepatic encephalopathy (71.4 vs. 51.4 ng/mL; p = 0.03 for all). Late FABP1 levels were significantly lower in TFS patients (TFS 54 vs. NTFS 66 ng/mL; p = 0.049) but not admission (TFS 96 vs. NTFS 87 ng/mL; p = 0.67). After adjusting for significant covariates, serum FABP1 did not discriminate significantly between TFS and patients who died/received LT at day 21 either on admission (p = 0.29) or late (days 3-5, p = 0.087) time points. In this first report of FABP1 in non-APAP ALF, FABP1 levels at late time points (days 3-5) were significantly lower in ALF patients who were alive without transplant at day 21 but not after adjusting for covariates reflecting severity of illness. Higher FABP1 levels were associated with the presence of increased organ failure.
Marc-André Clément, Cristina R. Bosoi, Mariana M. Oliveira, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Hepatic encephalopathy (HE) is a debilitating neurological complication of cirrhosis. By definition, HE is considered a reversible disorder, and therefore HE should resolve following liver transplantation (LT). However, persisting neurological complications are observed in as many as 47% of LT recipients. LT is an invasive surgical procedure accompanied with various perioperative factors such as blood loss and hypotension which could influence outcomes post-LT. We hypothesize that minimal HE (MHE) renders the brain frail and susceptible to hypotension-induced neuronal cell death. Six-week bile duct-ligated (BDL) rats with MHE and respective SHAM-controls were used. Several degrees of hypotension (mean arterial pressure of 30, 60 and 90mmHg) were induced via blood withdrawal from the femoral artery and maintained for 120 minutes. Brains were collected for neuronal cell count and apoptotic analysis. In a separate group, BDL rats were treated for MHE with the ammonia-lowering strategy ornithine phenylacetate (OP; MNK-6105), administered orally (1g/kg) for 3 weeks before induction of hypotension. Hypotension 30 and 60mmHg (not 90mmHg) significantly decreased neuronal marker expression (NeuN) and cresyl violet staining in the frontal cortex compared to respective hypotensive SHAM-operated controls as well as non-hypotensive BDL rats. Neuronal degeneration was associated with an increase in cleaved caspase-3, suggesting the mechanism of cell death was apoptotic. OP treatment attenuated hyperammonemia, improved anxiety and activity, and protected the brain against hypotension-induced neuronal cell death. Our findings demonstrate that rats with chronic liver disease and MHE are more susceptible to hypotension-induced neuronal cell degeneration. This highlights MHE at the time of LT is a risk factor for poor neurological outcome post-transplant and that treating for MHE pre-LT might reduce this risk.
Oliver Tavabie, Constantine J. Karvellas, SIamak Salehi, Jaime Speiser, Christopher F. Rose, Krishna Menon, Andreas Prachalias, Michael A. Heneghan, Kosh Agarwal, William M. Lee, Mark J. McPhail, Varuna Aluvihare, U. S. Acute Liver Failure Study Group.
Background: Acute liver failure (ALF) remains a rare but life‐threatening condition which requires early prognostication for transplantation (LTx). Existing models such as the King’s College Criteria (KCC) lack sensitivity. We have previously demonstrated the potential for regeneration linked miRNA to perform as biomarkers in acute and chronic liver disease. The aim of this study was to develop miRNA‐based prognostic models for acetaminophen (APAP) ALF 21‐day transplant‐free mortality. Methods: We assessed serum miRNA expression from 193 patients (94 survivors, 89 non‐survivors defined as mortality within 21 days) with APAP‐ALF at two time points (early; day 1, late; day 3‐5) from the US Acute Liver Failure Study Group registry (1998‐2014). Patients who underwent liver transplantation for APAP‐ALF were excluded from this study. A panel of 24 miRNA identified from our previous studies were analysed. Multiple logistic regression was used to create outcome prediction models at both early and late time points. Clinical data were incorporated to improve prognostication. The DeLong method was used to compare models. Correction for multiple comparisons was performed using the Benjamini‐Hochberg procedure with a false discovery rate set at 0.05. Primary outcome was prediction of 21‐day transplant‐free mortality. Results: At the early time point; up‐regulation of miR‐150, down‐regulation of ‐16‐2, the detection of miR‐20a and absence of miR‐149 were associated with mortality. At the late time point; up‐regulation of miR‐30a, down‐regulation of ‐122, 16‐2 and ‐21 and the detection of miR‐149, ‐17 and ‐191 were associated with mortality. MiRNA based prognostic models were made for both early and late time points (Table 1). The early time point model contained miRNA associated with regeneration (area under the receiver operator curve (AUC) 0.78, 95% CI 0.71‐0.84, p<0.01). The late time point model contained miRNA associated with cell death (AUC 0.83, 95% CI 0.76‐0.89, p<0.01). Both models were enhanced when combined with the Model for End‐Stage Liver Disease score (MELD) and vasopressor requirements (early time point; AUC 0.83, 95% CI 0.78‐0.90, p<0.01, late time point; AUC 0.91, 95% CI 0.86‐0.96, p<0.01). These enhanced models significantly outperformed the KCC (early time point; AUC 0.54, 95% CI 0.49‐0.59, late AUC 0.59, 95% CI 0.54‐0.53) and early time point MELD (early; AUC 0.72 95% CI 0.64‐0.79), and performed comparably to MELD at the late time point (late; AUC 0.87, 95% CI 0.81‐0.92) and the ALF Study Group Prognostic Index (early AUC 0.76, 95% CI 0.69‐0.83, late AUC 0.88, 95% CI 0.82‐0.95). Conclusion: We demonstrate that specific serum miRNA have prognostic value as biomarkers to discriminate outcome in ALF. Our early model utilised regeneration linked miRNA whereas our late model utilised cell‐death linked miRNA; this may signify mechanistic differences at early and late time points which determine patient mortality.
Hepatic encephalopathy: Novel insights into classification, pathophysiology and therapy.
Christopher F. Rose, Piero Amodio, Jasmohan S. Bajaj, Radha Krishan Dhiman, Sara Montagnese, Simon D. Taylor-Robinson, Hendrik Vilstrup, Rajiv Jalan.
Hepatic encephalopathy (HE) is a frequent and serious complication of both chronic liver disease and acute liver failure. HE manifests as a wide spectrum of neuropsychiatric abnormalities, from subclinical changes (mild cognitive impairment) to marked disorientation, confusion and coma. The clinical and economic burden of HE is considerable, and it contributes greatly to impaired quality of life, morbidity and mortality. This review will critically discuss the latest classification of HE, as well as the pathogenesis and pathophysiological pathways underlying the neurological decline in patients with end-stage liver disease. In addition, management strategies, diagnostic approaches, currently available therapeutic options and novel treatment strategies are discussed.
Stroop EncephalApp as a predictive test for overt Hepatic Encephalopathy in cirrhotic patients.
Silicia A. Très, Mélanie Tremblay, Yvette Mukaneza, Christopher F. Rose.
Introduction: Hepatic Encephalopathy (HE) is a severe neuropsychiatric syndrome associated with liver failure, ranging from covert HE, with subclinical impairments, to overt HE, with evident symptoms that require hospitalization. HE can be prevented, but prophylaxis would be justified and cost-effective only for patients at risk. Efficient and practical screening models are needed to detect these patients. EncephalApp, smartphone-based Stroop test is a valid method to screen for covert HE, one of the main risk factors. • Objective: Identify patients who are at high risk of developing overt hepatic encephalopathy within one year. • Hypothesis: The risk of developing an overt HE episode within 1 year and mortality will be higher in patients with covert HE and with a high risk of decompensation due to cirrhosis measured by ALBI-FIB4 score. Presence of ascites, high risk of gastrointestinal bleeding and high levels of inflammation biomarkers associated with covert HE at the screening are also expected to predict the outcomes. • Methods: For a multicentric longitudinal prospective study, 25 to 30 cirrhotic patients will be selected from 7 centers in Canada: Montreal (2 centers), Toronto, Ottawa, Calgary, Vancouver and Edmonton. Covert HE will be diagnosed with the Stroop test and blood samples will be collected at the screening and at the hospitalizations. Patients will be followed up for 1 year with phone calls every 3 months and medical records will be checked to identify HE- related hospitalizations and mortality. Statistical analysis will be performed to study the correlation between screening test results and patient outcomes. • Conclusion: The expected results can provide important information for identifying patients at high risk of developing overt HE within 1 year, by diagnosing covert HE and measuring biomarkers linked to the HE risk pathway. This data can help to justify prophylaxis, early treatment and prevention of episodes, which would have a great impact on the patient's quality of life, cost of health treatments and decrease in mortality.
Outcomes of sarcopenic obesity and metabolic syndrome in liver transplant patients.
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher Rose, Chantal Bémeur.
Background: Sarcopenia is associated with a worst prognosis in cirrhotic patients after liver transplantation (LT). As patients gain weight and sarcopenia remains after LT, sarcopenic obesity (SO) develops. Metabolic syndrome (MS), a cluster of factors that increase the risk of heart disease and diabetes, is caused by weight gain. There are limited data about the influence of SO and MS in LT recipients. Purpose: The goal of this study was to examine the impact of SO and MS on outcome after LT. Method: In total, 94 cirrhotic patients who underwent LT at the CHUM – Liver Unit were included. Sarcopenia was assessed at the third lumbar level vertebrae using a CT-scan. Obesity was determined using BMI whereas MS was diagnosed using the presence of ³3 modified NCEP ATP III criteria. The prognostic factors were collected 6 months before and during 1 year after LT through medical records and included number of complications, episodes of infections, length of stay, and frequency of readmissions. Result(s): Most of the patients ( 70%) were not obese before LT. Approximately 20% of the patients developed obesity after LT. Among patients who were obese before LT, 40% of the patients remained obese after LT. SO affected 10% and less of the patients before and after LT. Among patients with MS before LT (64%), 40% of them was still affected after LT. Among patients who were not affected by MS before LT, 38% developed MS after LT and one patient remained not affected after LT. Prognostic factors were worst in patients with SO and MS before and after LT. Conclusion(s): SO affected a small proportion of patients while MS was prevalent before and after LT. Nevertheless, these conditions were associated with worst prognosis. Strategies to manage SO and MS could help to improve recovery in patients who have undergone LT.
New insights on the impact of sex on chronic liver disease and hepatic encephalopathy.
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Background The impact of sex differences on chronic liver disease (CLD) and hepatic encephalopathy (HE) is unknown. The majority of animals used in research are male since the main difficulty with using female animals is the potential impact of the estrous cycle which increases intra-group variability. The bile duct ligated (BDL) rat is a well-characterized model of CLD and HE in males, which has not been investigated in females. Therefore, we aimed to characterize a female BDL model of CLD and HE and compare to male BDL rats. Material and methods Female rats underwent either BDL (n=8) or Sham (n=8) surgery. After 5 weeks, we assessed estrous cycle phase (by cellular cytology), anxiety (open field test), motor incoordination (rota-rod test) and night-time activity. We also assessed body weight, body composition (MRI), gastrocnemius muscle weight/circumference, grip strength, plasma ammonia and liver enzymes. Results from female BDL rats were compared to historical laboratory data from male BDL rats. Results Female BDL rats had increased markers of liver injury: ALP, AST and bilirubin (p<0.001) and impaired markers of liver function (increased ammonia and decreased albumin (p<0.001)) compared to female Shams. Furthermore, Female BDL rats did not differ in body weight, muscle circumference/weight and grip strength but had decreased fat mass (p<0.0001) and increased lean mass (p<0.005) compared to female shams. All results were comparable to male BDL rats except for plasma ammonia levels which were significantly lower in females (p<0.01). Moreover, male BDL rats had decreased fat mass, lean mass, muscle circumference/weight and grip strength. BDL surgery in female rats induced a dysregulated estrous cycle compared to sham (increased metestrus phase (p<0.01)). However, similar to male BDL rats, female BDL rats had increased anxiety (p<0.005), motor incoordination (p<0.05), and decreased night activity (p<0.05), independent of the estrous cycle phase. Discussion We demonstrated BDL surgery in females leads to hepatic and neurological impairment comparable to male BDL rats (similar intra-group variability). Interestingly, female BDL rats developed unique features. Contrary to male BDL vs. Shams, body weight and muscle mass did not differ between female BDL and Shams. Since muscle mass plays an important compensatory role in regulating ammonia levels, this could explain the reason why blood ammonia levels in female BDL rats are significantly lower compared to male BDL rats. We expect that this model will provide new insights on the effect of sex differences on the pathogenesis of CLD and HE and help to personalize HE treatment.
Diabetes is associated to the development of hepatic encephalopathy in cirrhotic patients.
Cristina R. Bosoi, Corina Cerlat, Mimosa Nguyen, Mélanie Tremblay, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Background: Non-alcoholic fatty liver disease (NAFLD) is associated with type II diabetes (T2D) and has become the main cause of cirrhosis. Both NAFLD and T2D are associated with cognitive and neurological impairments. T2D has been established as a risk factor for first-time development of overt hepatic encephalopathy (HE) in cirrhotic patients. The onset of HE in diabetic patients with cirrhosis develops earlier compared to cirrhosis patients without T2D. However it remains unclear whether NAFLD-induced cirrhosis increases the risk for HE. The present study aims to address the association between NAFLD, T2D and HE. Methods: Our retrospective study includes 102 cirrhotic patients on the liver transplant list at the Liver Unit of the Montreal University Hospital Center. Patients were classified by etiology of cirrhosis; 1) NAFLD and 2) non-NAFLD. Demographic data, blood biochemistry, clinical information on T2D-related comorbidities and cirrhosis complications (including number and severity of HE episodes) were collected. These factors were statistically associated with HE episodes. Results: Our cohort comprised 20 (19%) NAFLD and 82 (79%) non-NAFLD patients presenting similar MELD and Child-Pugh scores. The prevalence of T2D was higher in NAFLD vs non-NAFLD cirrhotics (15 (75%) vs 24 (29%) respectively) and was associated to co-morbidities such as cardiac disease, dyslipidemia, hypertension and obesity. Among non-NAFLD cirrhotics, 47 (57%) patients had a history of HE whereas 8 (40%) were found in the NAFLD cirrhotics (p>0.05). Since T2D is already known as a risk factor for HE, we subdivided both NAFLD and non-NAFLD groups into non-T2D and T2D subgroups. HE was significantly more prevalent in patients with T2D: in the NAFLD group, 5 (25%) T2D patients had developed an episode of HE compared to 3 (15%) patients without T2D (p<0.05); in the non-NAFLD group, 16 (67%) patients had T2D and HE compared to 31 (53%) HE patients without T2D (p<0.001). Fasting glycemia levels analysis in the 4 sub-groups of patients revealed increased levels in patients with history of HE and T2D, regardless of NAFLD etiology; in the NAFLD group 8.60 ± 0.84 mmol/l in patients with HE and T2D vs 6.00 ± 1.35 mmol/l in patients with HE without T2D (p<0.01); in the non-NAFLD group: 9.23 ± 0.93 mmol/l in patients with HE and T2D vs 5.82 ± 0.27 mmol/l in patients with HE without T2D (p<0.001). Conclusion: Our results sustain the association between T2D and HE and suggest high glucose might play a pathological role in the development of cognitive decline. NAFLD is not a risk factor for the development of HE. These interesting results provide new insights in the role of T2D in the development of HE and further studies are required to understand the underlying mechanisms. Furthermore, identifying patients who are at higher risk of developing HE is imperative to initiate early treatment strategies to protect neurological decline in patients with cirrhosis.
Diabetes is associated to the development of hepatic encephalopathy in cirrhotic patients.
Corina Cerlat, Cristina R. Bosoi, Mimosa Nguyen, Mélanie Tremblay, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Background: Non-alcoholic fatty liver disease (NAFLD) is associated with type II diabetes (T2D) and has become the main cause of cirrhosis. Both NAFLD and T2D are associated with cognitive and neurological impairments. T2D has been established as a risk factor for first-time development of overt hepatic encephalopathy (HE) in cirrhotic patients. The onset of HE in diabetic patients with cirrhosis develops earlier compared to cirrhosis patients without T2D. However it remains unclear whether NAFLD-induced cirrhosis increases the risk for HE. The present study aims to address the association between NAFLD, T2D and HE. Methods: Our retrospective study includes 102 cirrhotic patients on the liver transplant list at the Liver Unit of the Montreal University Hospital Center. Patients were classified by etiology of cirrhosis; 1) NAFLD and 2) non-NAFLD. Demographic data, blood biochemistry, clinical information on T2D-related comorbidities and cirrhosis complications (including number and severity of HE episodes) were collected. These factors were statistically associated with HE episodes. Results: Our cohort comprised 20 (19%) NAFLD and 82 (79%) non-NAFLD patients presenting similar MELD and Child-Pugh scores. The prevalence of T2D was higher in NAFLD vs non-NAFLD cirrhotics (15 (75%) vs 24 (29%) respectively) and was associated to co-morbidities such as cardiac disease, dyslipidemia, hypertension and obesity. Among non-NAFLD cirrhotics, 47 (57%) patients had a history of HE whereas 8 (40%) were found in the NAFLD cirrhotics (p>0.05). Since T2D is already known as a risk factor for HE, we subdivided both NAFLD and non-NAFLD groups into non-T2D and T2D subgroups. HE was significantly more prevalent in patients with T2D: in the NAFLD group, 5 (25%) T2D patients had developed an episode of HE compared to 3 (15%) patients without T2D (p<0.05); in the non-NAFLD group, 16 (67%) patients had T2D and HE compared to 31 (53%) HE patients without T2D (p<0.001). Fasting glycemia levels analysis in the 4 sub-groups of patients revealed increased levels in patients with history of HE and T2D, regardless of NAFLD etiology; in the NAFLD group 8.60 ± 0.84 mmol/l in patients with HE and T2D vs 6.00 ± 1.35 mmol/l in patients with HE without T2D (p<0.01); in the non-NAFLD group: 9.23 ± 0.93 mmol/l in patients with HE and T2D vs 5.82 ± 0.27 mmol/l in patients with HE without T2D (p<0.001). Conclusion: Our results sustain the association between T2D and HE and suggest high glucose might play a pathological role in the development of cognitive decline. NAFLD is not a risk factor for the development of HE. These interesting results provide new insights in the role of T2D in the development of HE and further studies are required to understand the underlying mechanisms. Furthermore, identifying patients who are at higher risk of developing HE is imperative to initiate early treatment strategies to protect neurological decline in patients with cirrhosis.
Frailty and Sarcopenia in Cirrhosis: The Basics, the Challenges, and the Future
Chantal Bémeur, Christopher F. Rose.
Hepatic encephalopathy (HE), sarcopenia, and frailty are serious complications of chronic liver disease that may negatively affect quality of life and survival. HE is a complex neuropsychiatric multifactorial syndrome for which ammonia is believed to play a key role. Sarcopenia, which is nearly universal in chronic liver disease, is a muscle disease characterized by low muscle strength, low muscle quantity or quality, and low physical performance. Physical frailty is described as a multidimensional syndrome of decreased reserve, functional impairment, and resistance to stressors, resulting from cumulative declines across multiple systems, whereas cognitive frailty is characterized by reduced neurophysiological reserve. Pathophysiology of sarcopenia and frailty in the setting of HE and chronic liver disease remains to be elucidated. Therapeutic strategies of HE aim at reducing blood ammonia concentrations. In that context, muscle may play a vital compensatory role in reducing ammonia levels since it contains an ammonia-removing enzyme. Assessing HE, sarcopenia, and frailty during chronic liver disease is the cornerstone of optimal intervention.
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
Introduction: Sarcopenia is associated with a worst prognosis in cirrhotic patients after liver transplantation (LT). As patients gain weight and sarcopenia remains after LT, sarcopenic obesity (SO) develops. Metabolic syndrome (MS), a cluster of factors that increase the risk of heart disease and diabetes, is caused by weight gain. There are limited data about the influence of SO and MS in LT recipients. The goal of this study was to examine the impact of SO and MS on outcomes after LT. Method: In total, 94 cirrhotic patients who underwent LT at the CHUM – Liver Unit were included. Sarcopenia was assessed at the third lumbar level vertebrae using a CT-scan. Obesity was determined using BMI whereas MS was diagnosed using the presence of 3 modified NCEP ATP III criteria. The prognostic factors were collected 6 months before and during 1 year after LT through medical records and included number of complications, episodes of infections, length of stay, and frequency of readmissions. Results: Most of the patients ( 70%) were not obese before LT. Approximately 20% of the patients developed obesity after LT. Among patients who were obese before LT, 40% of the patients remained obese after LT. SO affected 10% and less of the patients before and after LT. Among patients with MS before LT (64%), 40% of them was still affected after LT. Among patients who were not affected by MS before LT, 38% developed MS after LT and one patient remained not affected after LT. Prognostic factors were worst in patients with SO and MS before and after LT. Conclusion: SO affected a small proportion of patients while MS was prevalent before and after LT. Nevertheless, these conditions were associated with worst prognosis. Strategies to manage SO and MS could help to improve recovery in patients who have undergone LT.
An Investigation of PS‐b‐PEO Polymersomes for the Oral Treatment and Diagnosis of Hyperammonemia
Simon Matoori, Yinyin Bao, Aaron Schmidt, Eric J. Fischer, Rafael Ochoa-Sanchez, Mélanie Tremblay, Mariana M. Oliveira, Christopher F. Rose, Jean‐Christophe Leroux.
Ammonia-scavenging transmembrane pH-gradient poly(styrene)-b-poly(ethylene oxide) polymersomes are investigated for the oral treatment and diagnosis of hyperammonemia, a condition associated with serious neurologic complications in patients with liver disease as well as in infants with urea cycle disorders. While these polymersomes are highly stable in simulated intestinal fluids at extreme bile salt and osmolality conditions, they unexpectedly do not reduce plasmatic ammonia levels in cirrhotic rats after oral dosing. Incubation in dietary fiber hydrogels mimicking the colonic environment suggests that the vesicles are probably destabilized during the dehydration of the intestinal chyme. The findings question the relevance of commonly used simulated intestinal fluids for studying vesicular stability. With the encapsulation of a pH-sensitive dye in the polymersome core, the local pH increase upon ammonia influx could be exploited to assess the ammonia concentration in the plasma of healthy and cirrhotic rats as well as in other fluids. Due to its high sensitivity and selectivity, this polymersome-based assay could prove useful in the monitoring of hyperammonemic patients and in other applications such as drug screening tests.
Mathilde Mouchiroud, Étienne Camiré, Manal Aldow, Alexandre Caron, Éric Jubinville, Laurie Turcotte, Inès Kaci, Marie-Josée Beaulieu, Christian Roy, Sébastien M. Labbé, Thibault V. Varin, Yves Gélinas, Jennifer Lamothe, Jocelyn Trottier, Patricia L. Mitchell, Frédéric Guénard, William T. Festuccia, Philippe Joubert, Christopher F. Rose, Constantine J. Karvellas, Olivier Barbier, Mathieu C. Morissette, André Marette, Mathieu Laplante.
Objectives Hepatokines are proteins secreted by the liver that impact the functions of the liver and various tissues through autocrine, paracrine, and endocrine signaling. Recently, Tsukushi (TSK) was identified as a new hepatokine that is induced by obesity and cold exposure. It was proposed that TSK controls sympathetic innervation and thermogenesis in brown adipose tissue (BAT) and that loss of TSK protects against diet-induced obesity and improves glucose homeostasis. Here we report the impact of deleting and/or overexpressing TSK on BAT thermogenic capacity, body weight regulation, and glucose homeostasis. Methods We measured the expression of thermogenic genes and markers of BAT innervation and activation in TSK-null and TSK-overexpressing mice. Body weight, body temperature, and parameters of glucose homeostasis were also assessed in the context of TSK loss and overexpression. Results The loss of TSK did not affect the thermogenic activation of BAT. We found that TSK-null mice were not protected against the development of obesity and did not show improvement in glucose tolerance. The overexpression of TSK also failed to modulate thermogenesis, body weight gain, and glucose homeostasis in mice. Conclusions TSK is not a significant regulator of BAT thermogenesis and is unlikely to represent an effective target to prevent obesity and improve glucose homeostasis.
Victor Dong, Maxime Gosselin, Nishita Jagarlamudi, Beverley Kok, Mark G. Swain, Jasmohan S. Bajaj, Juan G. Abraldes, Vladimir Marquez, R. Todd Stravitz, Aldo J. Montano-Loza, Manuela Merli, Phil Wong, Amanda Brisebois, Punetta Tandon, Julia Wendon, Scott L. Nyberg, François M. Carrier, Michael R. Lucey, Florence Wong, Jordan J. Feld, Constantine J. Karvellas, Christopher F. Rose, Julien Bissonnette.
Rafael Ochoa-Sanchez, Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mélanie Tremblay, Mylene Perreault, Bill Querbes, Caroline Kurtz, Christopher F. Rose.
Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) production that contributes to systemic hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering systemic NH3. As a therapeutic strategy, Escherichia coli Nissle 1917 bacterium (EcN), a well characterized probiotic, was genetically modified to consume and convert NH3 to arginine (SYNARG), and its administration to thiaoacetamide-treated mice reduced NH3 levels. SYNARG was further modified to synthesize butyrate (SYNARG+BUT), a short-chain fatty acid with anti-inflammatory properties, and both strains were tested in an animal model of CLD and HE, the bile duct ligation (BDL). Methods: One week (wk) post surgery, BDL rats were gavaged with SYNARG, SYNARG+BUT (3x1011 CFU/day, BID) or vehicle until they were sacrificed at 3- or 5-weeks along with respective sham controls. Plasma NH3 and liver markers were measured at 3 and 5 wk. Recognition memory, motor coordination, muscle strength, locomotion and anxiety were assessed in the 5-week groups. Results: BDL increased NH3 over time, with levels of 109.1±9.2µM (Shams 56.7±3.5µM, p<0.001) and 150.2±25.6µM (Shams 58.3±3.0µM, p<0.001) at 3- and 5-wk, respectively. In addition, plasma liver markers ALT, AST, bilirubin, and GGT as well as liver fibrosis (hydroxyproline) were increased in BDL rats at both timepoints while albumin was lowered. As compared to BDL-Veh rats, NH3 was attenuated by SYNARG (103.9±12.3µM) and SYNARG+BUT (110.8±8.5µM) at 5, but not 3-wk post-BDL, while liver fibrosis was attenuated at 3, but not 5-wk post-BDL. None of the systemic liver markers were changed by the treatments at any timepoint. Motor coordination, muscle strength, locomotion and anxiety were affected in all BDL groups without protective effect of treatments. Short-term memory (STM) was impaired in BDL-Veh (p<0.001) and BDL-SYNARG (p<0.05) vs Shams, while STM was improved in BDL-SYNARG+BUT (p<0.05 vs BDL-Veh). Long-term memory was impaired in BDL-Veh vs Shams (p<0.05), but BDL-SYNARG and BDL-SYNARG+BUT were partially protected. Conclusion: EcN, engineered to consume NH3 in the gut, is an effective approach to lower plasma NH3 in a model of CLD and HE. Moreover, the attenuation of NH3 in BDL rats is related to a protective effect on memory in this model. The therapeutic potential of these strains should be further evaluated in patients with CLD and HE.
Mariana Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background The impact of sex differences on chronic liver disease (CLD) and hepatic encephalopathy (HE) is unknown. The majority of animals used in research are male since the main difficulty with using female animals is the potential impact of the estrous cycle, increasing intragroup variability. The bile duct ligated (BDL) rat is a well-characterized model of CLD and HE in males which has not been investigated in females. Therefore, we aimed to characterize a female BDL model of CLD and HE and compare to male BDL rats. Material and Methods We assessed BDL or Sham female rats for estrous cycle phase, behavior (anxiety, motor incoordination and activity), body parameters (weight and composition, muscle weight/circumference, grip strength), liver parameters (enzymes and ammonia). We than compared to historical laboratory data from male BDL rats. Results Female BDL rats had impaired liver markers (P<0.0001) and ammonia (p<0.001) compared to female Shams. These results were comparable to male BDL rats except ammonia which was lower in females (p< 0.01). Female BDL rats did not differ in body weight, muscle circumference/weight and grip strength and had increased lean mass (p<0.005) compared to female shams. Whereas, male BDL rats have decreased lean mass, muscle circumference/weight and grip strength. Similar to male BDL rats, female BDL rats had increased anxiety (p<0.005), motor incoordination (p<0.05), and decreased activity (p<0.05) independent of the estrous cycle phase. Discussion and Conclusion We demonstrated BDL surgery in females leads to hepatic and neurological impairment comparable to male BDL rats (similar intra-group variability). Interestingly, contrary to male BDL vs Shams, body weight and muscle mass does not differ between female BDL and Shams. Since Mmuscle plays an important compensatory role in clearing ammonia during CLD, maintenance of muscle mass in females which could explain the lower blood ammonia levels in female BDL rats compared to male BDL rats. We conclude that this model provides new insights on the impact of sex on the pathogenesis of CLD and HE.
Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mylene Perreault, Bill Querbes, Caroline B. Kurtz, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome observed in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Investigate the synergistic effect of obesity and CLD on the development of neurological impairment in a novel rat model of cirrhosis and obesity. M&M: Animal model of CLD and HE: 5-week bile-duct ligation (BDL) rats and Sham-operated controls, were used. Groups: Obese-BDL and Obese-Sham received high-fat diet (HFD) for 25-days pre-BDL and high-carbohydrate diet (HCD) for 5-weeks post-BDL; Lean-BDL and Lean-Sham received regular-diet (RD) pre-BDL and HCD post-BDL. Body-weight and fat-mass (EchoMRI) were monitored pre-BDL as well as 3- and 5-weeks post-BDL. Behavior: Motor-coordination, motor skill-learning, and muscular-strength were assessed at 3- and 5-weeks post-BDL. Locomotion and anxiety were measured at 5-weeks. Plasma ammonia, liver enzymes, and lipids were measured at 3- and 5-weeks. RESULTS: Before BDL surgery, body-weight and fat-mass of rats on HFD increased compared to rats on RD. 3-week post-BDL, body-weight and fat-mass decreased in Lean-BDL and Obese-BDL vs respective Shams, while at 5-weeks this was only found in Lean-BDL. These parameters were higher in Obese-BDL vs Lean-BDL at 3- and 5-weeks. Plasma ammonia, bilirubin, albumin, ALT, AST, and ALP were impaired in Obese- and Lean-BDL vs respective Shams at 3- and 5-weeks. AST and ALP increased in Obese-BDL vs Lean-BDL at 5-weeks. Elevated HDL-cholesterol and decreased LDL-cholesterol were detected in Obese-BDL and Lean-BDL vs respective Shams at 3- and 5-weeks, while LDL-cholesterol was higher in Obese-BDL vs Lean-BDL at 5-weeks. Total-cholesterol increased in Obese-BDL vs all groups at 5-weeks. At 3 weeks; motor-coordination was reduced in Obese-BDL, but not in Lean-BDL vs respective Shams, while at 5-weeks, motor-coordination decreased in both Lean-BDL and Obese-BDL vs respective Shams, with worse performance in Obese-BDL vs Lean-BDL. At 3-weeks, skill-learning improved in all Shams and Lean-BDL, but not in Obese-BDL; at 5-weeks contrary to Sham-groups, both BDL groups did not improve performance. Muscle-strength decreased in Lean-BDL and Obese-BDL vs respective Shams at 3- and 5-weeks. Hypolocomotion and anxiogenic effects were detected in Obese-BDL, but not in Lean-BDL vs Shams at 5-weeks. CONCLUSION: HFD induces obesity pre-BDL which is maintained post-BDL with a HCD-diet which was accompanied with increase fat-mass and hyperlipidemia. Neurological decline in obese-cirrhotic rats developed earlier and was more severe versus Lean-BDL rats. Besides, some neurological impairments developed in Obese-BDL but not in Lean-BDL. These results suggest a synergistic effect, which accelerates/worsens the disease-associated abnormalities in CLD and HE.
Rafael Ochoa-Sanchez, Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mélanie Tremblay, Mylene Perreault, Bill Querbes, Caroline B. Kurtz, Christopher F. Rose.
Background: Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) production that contributes to systemic hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering circulating NH3. As a therapeutic strategy, Escherichia coli Nissle 1917 bacterium (EcN), a well characterized probiotic, was genetically modified to consume and convert NH3 to arginine (SYNARG), and its administration to thioacetamide-treated mice resulted in a significant reduction of NH3 levels1. SYNARG was further modified to synthesize butyrate (SYNARG+BUT), a short-chain fatty acid with anti-inflammatory/anti-oxidant properties, and both strains were tested in an experimental model of cirrhosis and HE, the bile duct ligation (BDL). Methods: One week post surgery, BDL rats were gavaged with SYNARG, SYNARG+BUT (3x1011 CFU/day, BID) or vehicle until they were sacrificed at 3- or 5-weeks along with respective sham controls. Plasma NH3 and liver markers were measured at 3 and 5 weeks. Recognition-memory, motor-coordination, muscle-strength, locomotion and anxiety were assessed in the 5-week BDL groups. Results: BDL significantly increased NH3 over time, with levels of 109.1±9.2µM (Shams 56.7±3.5µM, p<0.001) and 150.2±25.6µM (Shams 58.3±3.0µM, p<0.001) at 3- and 5-weeks, respectively. In addition, plasma liver markers alanine-transaminase, aspartate-transaminase, bilirubin, and gamma-glutamyl transferase were significantly increased in BDL rats at both timepoints while albumin was significantly lowered. As compared to BDL-Veh rats, hyperammonemia was attenuated by SYNARG (103.9±12.3µM) and SYNARG+BUT (110.8±8.5µM) at 5, but not 3 weeks post-surgery, while liver fibrosis (hydroxyproline content) was attenuated at 3, but not 5 weeks post-surgery. None of the circulating liver markers were changed by the treatments at any timepoint. Motor-coordination, muscle-strength, locomotion and anxiety were affected in all BDL groups without protective effect of treatments. Short-term memory (STM) was impaired in BDL-Veh (p<0.001) and BDL-SYNARG (p<0.05) versus Shams, while STM was resolved in BDL-SYNARG+BUT (p<0.05 vs BDL-Veh). Long-term memory (LTM) was impaired in BDL-Veh vs Shams (p<0.05), but BDL-SYNARG and BDL-SYNARG+BUT were protected. Conclusion: EcN, engineered to consume NH3 in the gut and synthesize butyrate, is an effective approach to lower plasma NH3 in a model of cirrhosis and HE. Moreover, the attenuation of hyperammonemia in cirrhotic rats is associated with a protective effect on memory in this model. The therapeutic potential of these engineered EcN strains should be further evaluated in patients with CLD and HE.
Uncovering sex-based differences in a rat model of chronic liver disease and hepatic encephalopathy
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background The impact of sex differences on chronic liver disease (CLD) and hepatic encephalopathy (HE) is unknown. The majority of animals used in research are male since the main difficulty with using female animals is the potential impact of the estrous cycle, increasing intragroup variability. The bile duct ligated (BDL) rat is a well-characterized model of CLD and HE in males which has not been investigated in females. Therefore, we aimed to characterize a female BDL model of CLD and HE and compare to male BDL rats. Material and methods Female rats underwent either BDL (n=8) or Sham (n=8) surgery. After 5 weeks, we assessed estrous cycle phase (by cellular cytology), anxiety (open field test), motor incoordination (rota-rod test) and night-time activity. We also assessed body weight, body composition (MRI), gastrocnemius muscle weight/circumference, grip strength, and ammonia and liver enzymes in plasma. Results from female BDL rats were compared to historical laboratory data from male BDL rats. Results Female BDL rats had increased liver enzymes (ALP (P=0.001) and AST (P<0.0001) (but not ALT)), bilirubin (P<0.0001) and ammonia (p<0.001), and decreased albumin (P<0.0001) compared to female Shams. These results were comparable to male BDL rats except ALT and ammonia which were lower in females (p< 0.01). Female BDL rats did not differ in body weight, muscle circumference/weight and grip strength but had decreased fat mass (p<0.0001), increased lean mass (p<0.005) compared to female shams. Whereas, male BDL rats have decreased fat mass, muscle circumference/weight and grip strength. BDL in female rats induced a dysregulated estrous cycle compared to Sham (increased metestrus phase (p<0.01)). Similar to male BDL rats, female BDL rats had increased anxiety (p<0.005), motor incoordination (p<0.05), and decreased night activity (p<0.05) independent of the estrous cycle phase. Discussion We demonstrated BDL surgery in females leads to hepatic and neurological impairment comparable to male BDL rats (similar intra-group variability). Interestingly, female BDL rats developed unique features. Contrary to male BDL vs Shams, body weight and muscle mass does not differ between female BDL and Shams. Since muscle mass plays an important compensatory role in regulating ammonia levels, this could explain why the increase in blood ammonia levels in female BDL rats (vs. female Shams) was lower compared to male BDL. We expect that this model will provide new insights on the effect of sex differences on the pathogenesis of CLD and HE and help to personalize HE treatment.
Sarcopenia Pre- and Post-liver Transplantation Implication for Hepatic Encephalopathy.
Mimosa Nguyen, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
BACKGROUND: Muscle wasting (sarcopenia) and hepatic encephalopathy affect 30 to 70% of cirrhotic patients. The presence of sarcopenia may be associated with a worst prognosis and complications, including hepatic encephalopathy, in cirrhotic patients awaiting and after liver transplantation (LT). To this day, few studies have evaluated and followed muscle mass (in terms of quantity and quality) after LT. The goal of this study was to assess the association between the evolution of sarcopenia and the prognosis of cirrhotic patients, including hepatic encephalopathy and neurological complications, before and after LT. METHODS: In total, 94 cirrhotic patients who underwent LT at the Montreal University Hospital Center - Liver Unit were included. Sarcopenia was assessed at the third lumbar level vertebrae using a computed tomography scan (CT-scan). The diagnostic of sarcopenia was based on previously established sex-specific cut-off values of skeletal muscle index. Patients were classified into two groups: (1) persistent or newly developed sarcopenia after LT (Sarc+); (2) resolved sarcopenia or absence of sarcopenia before and after LT (Sarc-). Muscle quality (myosteatosis) was assessed by calculating intramuscular adipose tissue content. The prognostic factors were collected 6 months before and during 1 year after LT through medical records and included the number of complications, the presence of hepatic encephalopathy and the episodes of infections, the length of stay, and the frequency of readmissions. RESULTS: Sarcopenia persisted or was newly developed (Sarc+) in 62% of the patients (n = 58). It remained absent or was resolved after LT in 38% of the patients (n = 35). Muscle quality was significantly decreased post-LT (P = 0.034). The group Sarc+ experienced more complications pre-LT (P = 0.012) and post-LT (P < 0.001), infections post-LT (P = 0.006) and readmissions (P = 0.048) compared to the group Sarc-. The length of stay was longer for the group Sarc+ as opposed to the group Sarc- (P < 0.001). Hepatic encephalopathy was present in 83% of patients pre-LT whereas 17% experienced persistent neurological complications post-LT. CONCLUSIONS: Persistent and newly developed sarcopenia after LT appear to have negative outcomes on the prognosis of patients. Interventional strategies to optimize, increase or preserve muscle mass could help to improve post-operative recovery as well as the quality of life in patients who have undergone LT.
Developing a New Animal Model of Episodic Hepatic Encephalopathy.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). The primary cause of hospital admissions for cirrhotic patients is an overt episode of HE. Precipitating factors of HE frequently lead to an increase in blood ammonia. Patients who have experienced multiple episodes of HE are associated with persisting neurological complications post-liver transplantation. Currently, the impact of HE episodes on neurological integrity is unknown. We hypothesize that multiple episodes of HE will accelerate and/or intensify neurological deterioration. To date, an animal model of episodic HE is lacking. Therefore, our goal was to characterize an animal model of episodic HE (precipitated with ammonia) and to evaluate the impact of cumulative episodes on neurological status in cirrhotic rats. Material and Methods: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, and Sham-operated controls were used. BDL and Sham rats were divided in two groups, episodic and non-episodic. Injection (i.p) of ammonium acetate was used to induce episodes of overt HE (pre-coma; loss of righting reflex) every 4 days starting 3-weeks post-BDL surgery (total 5 episodes). Saline was injected as vehicle for non-episodic groups. Two days following the last HE episode, we assessed motor-coordination (RotaRod), anxiety (elevated plus maze, EPMT), as well as short-term and long-term memory (novel object recognition) in all groups. Upon sacrifice, plasma ammonia was measured. Results: The concentration of ammonia required to induce an episode of overt HE in BDL rats lessened with each subsequent episode, ranging from 7 to 4.5 mmol/kg. Short-term memory (p<0.05) and motor-coordination (p<0.05) were impaired in both non-episodic and episodic BDL groups compared to respective Sham-operated controls. Long-term memory impairment (p=0.06) and increased anxiety (+60.0%, p<0.05) were exclusively found in episodic BDL rats compared to non-episodic BDL rats. Moreover, there was an increase in blood ammonia (+30.4%, p=0.06) in episodic compared to non-episodic BDL rats, suggesting that although episodic-BDL rats recover from each HE episode, baseline (pre-episode) ammonia remain higher than non-episodic BDL rats. Conclusion: Cumulative HE episodes escalate neurological impairments in cirrhotic-BDL rats. Thus, this new episodic HE model represents a good approach to explore the pathological mechanism arising from multiple episodes, as well as further investigate whether higher hyperammonemia and/or increased brain sensitivity to ammonia is responsible for more complex neurological manifestations in episodic-BDL rats. Moreover, this model is an excellent platform to investigate novel therapies to prevent/treat episodic HE.
Characterization of a novel animal model of episodic hepatic encephalopathy.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). The primary cause of hospital admissions for cirrhotic patients is an overt episode of HE. Precipitating factors of HE are frequently due to increased blood ammonia. Patients with history of multiple episodes of HE experienced persisting neurological complications post-liver transplantation. Nevertheless, the impact of HE episodes on neurological integrity is unknown. We hypothesize that multiple episodes of HE will accelerate and/or intensify neurological deterioration. To date, an animal model of episodic HE is lacking. Therefore, our goal was to characterize an animal model of episodic HE (precipitated with ammonia) and to evaluate the impact of cumulative episodes on neurological status in cirrhotic rats. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, and Sham-operated controls were used. Ammonium acetate was used to induce HE episodes, every 4 days starting at 3-weeks post-surgery (total 5 episodes). After the last episode, we assessed motor-coordination (RotaRod), anxiety (elevated plus maze), as well as, short-term and long-term memory (novel object recognition). Rats were then sacrificed, and plasma ammonia measured. Results: Short-term memory (p<0.05) and motor-coordination (p<0.05) were reduced in both non-episodic and episodic BDL groups when compared Sham-operated controls. Long-term memory impairment (p=0.06) and increased anxiety (+60.0%, p<0.05) were found only in episodic vs non-episodic BDL rats. Moreover, there was an increase in blood ammonia (+30.4%, p=0.06) in episodic BDL vs non-episodic BDL rats. Conclusion: The induction of HE episodes escalates neurological impairments in cirrhotic rats. Thus, this new episodic HE model represents a good approach to explore the pathological mechanism arising from multiple episodes, as well as further investigate brain sensitivity to ammonia. Moreover, this model is an excellent platform to investigate novel therapies to prevent or treat episodic HE.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. Aim: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Model of CLD and HE: 6-week bile-duct ligation (BDL) rats and Sham-controls were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Obese-BDL received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; Lean-BDL received RD pre-/post-BDL; Lean-Sham received RD pre-/post-Sham. Recognition memory, motor-coordination, muscular-strength and body-composition (fat vs lean mass) were assessed before, 3 and 6 weeks post-surgery. Results: Before surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased vs rats on RD (Lean-BDL, Lean-Sham). 3 weeks post-surgery, BW, fat and lean mass were increased in Obese-BDL vs. Lean-BDL. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham. 6 weeks post-surgery, similar to Lean-BDL, Obese-BDL lost BW, fat and Lean mass vs. Lean-Sham. Motor-coordination, forelimb strength and long-term memory were impaired in Obese-BDL vs Lean-BDL or Lean-Sham, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL. Conclusion: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats, while others are exacerbated. A synergistic effect of obesity and CLD accelerates/worsens the disease-associated abnormalities observed in HE, suggesting more susceptibility to poor neurological performance in obese-induced cirrhotic patients.
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsychiatric disorder, a major complication of chronic liver disease (CLD). Hyperammonemia is central in the pathogenesis of HE as ammonia crosses the blood-brain barrier (BBB) causing toxicity. Glutamine synthetase (GS), an enzyme which removes ammonia during the amidation of glutamate to glutamine, is expressed in liver, muscle and brain. Since liver is responsible for regulating blood ammonia levels, extra-hepatic GS plays an important compensatory role during CLD. However, GS expression in endothelial cells (EC) of the BBB has never been explored. Methods: GS protein and activity was assessed in 1) rat brain microvascular EC (+/- ammonia exposure and plasma from rats with CLD) and 2) isolated cerebral microvessels (CMV) from naïve rats. Results: GS was co-localized with EC in brain of naïve rats. GS protein and activity was detected in CMV and in vitro, but with lower levels compared to brain (p<0.05). EC exposed to ammonia resulted in increased GS activity (p<0.05). However, ECs exposed to plasma from CLD rats resulted in lower GS activity and protein expression compared to controls (p<0.05). Conclusion: We demonstrate for the first time the presence of GS in EC in both in vitro and in vivo. Stimulated by ammonia, GS is however reduced following exposure to plasma from hyperammonemic CLD rats. This suggests other systemic factors such as oxidative stress (present in CLD; Bosoi et al., Free Radic Biol Med, 2012) could hinder GS activity. We speculate a downregulation of GS in the BBB during CLD leads to rapid entry of ammonia into the brain and the development of HE. Hence, upregulating GS in the BBB could become a new therapeutic target for HE.
The hepatokine Tsukushi is released in response to NAFLD and impacts cholesterol homeostasis.
Mathilde Mouchiroud, Étienne Camiré, Manal Aldow, Alexandre Caron, Éric Jubinville, Laurie Turcotte, Inès Kaci, Marie-Josée Beaulieu, Christian Roy, Sébastien M. Labbé, Thibault V. Varin, Yves Gélinas, Jennifer Lamothe, Jocelyn Trottier, Patricia L. Mitchell, Frédéric Guénard, William T. Festuccia, Philippe Joubert, Christopher F. Rose, Constantine J. Karvellas, Olivier Barbier, Mathieu C. Morissette, André Marette, Mathieu Laplante.
Nonalcoholic fatty liver disease (NAFLD) prevails in obesity and is linked to several health complications including dyslipidemia and atherosclerosis. How exactly NAFLD induces atherogenic dyslipidemia to promote cardiovascular diseases is still elusive. Here, we identify Tsukushi (TSK) as a hepatokine induced in response to NAFLD. We show that both endoplasmic reticulum stress and inflammation promote the expression and release of TSK in mice. In humans, hepatic TSK expression is also associated with steatosis, and its circulating levels are markedly increased in patients suffering from acetaminophen-induced acute liver failure (ALF), a condition linked to severe hepatic inflammation. In these patients, elevated blood TSK levels were associated with decreased transplant-free survival at hospital discharge, suggesting that TSK could have a prognostic significance. Gain- and loss-of-function studies in mice revealed that TSK impacts systemic cholesterol homeostasis. TSK reduces circulating HDL cholesterol, lowers cholesterol efflux capacity, and decreases cholesterol-to-bile acid conversion in the liver. Our data identify the hepatokine TSK as a blood biomarker of liver stress that could link NAFLD to the development of atherogenic dyslipidemia and atherosclerosis.
Malnutrition in chronic liver disease: Implications for quality of life and hepatic encephalopathy
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Vincent, Geneviève Huard, Christopher Rose, Chantal Bémeur.
BACKGROUND: Malnutrition is the most common complication in patients with chronic liver disease (CLD) and may increase the risk of developing hepatic encephalopathy (HE) as well as affect pa-tients’ functional status and quality of life (QOL). Management strategies focussing on nutritional status in relation to complications of CLD are an unmet clinical need. PURPOSE: The objectives are to: 1) Assess nutri-tional status and its relationship to QOL; 2) Ascer-tain the presence, severity and history of HE; 3) Inquire the relationship of HE and history of HE on the QOL of CLD subjects. METHOD: Observational and cross-sectional study involving 50 patients (hospitalized and outpatients) from the CHUM’s Liver Unit, Montreal, Canada) and 18 controls (non-cirrhotic). All subjects were assessed for: 1) Nutritional Status: Standardized Questionnaire Subjective Global Assessment (SGA); 2) QOL: General questionnaire SF-36 evalu-ating the QOL as perceived by the patient (8 scales: physical functioning (PF), role limitations due to physical health (RP), due to emotional problems (RE), social functioning (SF), bodily pain (BP), vitality (VT), mental health (MH) and general health (GH)); 3) HE: History and presence of covert HE using EncephalApp Stroop test or overt HE using the Clinical Hepatic Encephalopathy Staging Scale.RESULT(S): 50 CLD patients (72% men) of various etiologies (18% NASH, 12% alcoholic, 8% autoim-mune, 6% viral, 12% others and 44% mixed eti-ologies), Child–Pugh (15A, 7B, 18C and 10 N/A), aged 56±12 years and 18 non-cirrhotic patients (33% male, aged 42±15). SGA revealed that 34% of CLD subjects were malnourished. Among mal-nourished CLD patients, 18% were diagnosed with covert or overt HE. CLD malnourished patients had a lower perception of QOL than well-nour-ished CLD patients for all SF-36 scales (p < 0.01). History of HE was associated with poor QOL as CLD patients with a history of HE (36%) showed decreased PF (p = 0.024) and RP (p < 0.01). Com-pared to controls, CLD patients displayed a lower score in PF (p < 0.05) and GH (p < 0.05).CONCLUSION(S): Our data suggest that a subop-timal nutritional status based on SGA negatively affects 6 scales out of 8 of QOL (RP, RE, SF, VT, MH and GH) but is not associated with presence of HE. However, history of HE does impact two scales of QOL (PF and RP). Identifying malnourished CLD patients is of great importance to improve QOL. Interventions to prevent and treat malnutrition in CLD remain a primary need.
Sarcopenia in the context of liver transplantation: What is the prognosis?
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher Rose, Chantal Bémeur.
BACKGROUND: Muscle wasting (sarcopenia) af-fects 30 to 70% of cirrhotic patients. The presence of sarcopenia may be associated with a worst prog-nosis in cirrhotic patients awaiting and after liver transplantation (LT). To this day, few studies have evaluated and followed muscle mass (in terms of quantity and quality) after LT.PURPOSE: The goal of this study was to assess the association between the evolution of sarcopenia and the prognosis of cirrhotic patients before and after LT.METHOD: In total, 94 cirrhotic patients who un-derwent LT at the Montreal University Hospital Center – Liver Unit were included. Sarcopenia was assessed at the third lumbar level vertebrae using a computed tomography scan (CT-scan). The diag-nostic of sarcopenia was based on previously estab-lished sex-specific cut-off values of skeletal muscle index. Patients were classified into two groups: 1) persistent or newly developed sarcopenia after LT (Sarc+); 2) resolved sarcopenia or absence of sarcope-nia before and after LT (Sarc–). Muscle quality (myo-steatosis) was assessed by calculating intramuscular adipose tissue content. The prognostic factors were collected 6 months before and during 1 year after LT through medical records and included the num-ber of complications, the episodes of infections, the length of stay, and the frequency of readmissions.RESULT(S): Sarcopenia persisted or was newly de-veloped (Sarc+) in 62% of the patients (n = 58). It remained absence or was resolved after LT in 38% of the patients (n = 35). Muscle quality was significantly decreased post-LT (p = 0.034). The group Sarc+ ex-perienced more complications pre-LT (p = 0.012) and post-LT (p < 0 .001), infections post-LT (p = 0.006) and readmissions (p = 0.048) compared to the group Sarc−. The length of stay was longer for the group Sarc+ as opposed to the group Sarc− (p < 0 .001).CONCLUSION(S): Persistent and newly developed sarcopenia after LT appear to have negative out-comes on the prognosis of patients. Interventional strategies to optimize, increase or preserve muscle mass could help to improve post-operative recov-ery as well as the quality of life in patients who have undergone LT.
Uncovering the protective mechanism of the BBB against ammonia neurotoxicity.
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Background: The liver plays a major role in regulating ammonia levels in the blood. Therefore, in liver disease, the loss of hepatic function leads to hyperammonemia and consequently hepatic encephalopathy (HE). Ammonia-lowering strategies remain the mainstay therapeutic strategy for HE. Ammonia, both as an ion (NH4+) and gas (NH3), readily crosses all plasma membranes, including the blood-brain barrier (BBB): the interface between the blood and the brain. Since ammonia is a natural toxin, we hypothesize endothelial cells of the BBB have the capacity to metabolize ammonia to protect the brain from ammonia toxicity. Glutamine synthetase (GS), is an enzyme which in the process of amidating glutamate to glutamine removes ammonia is expressed in liver, muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. The aim of this study was to assess the presence of GS in endothelial cells of the BBB. Methods: Using brain primary microvascular endothelial cells (ECs) and isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats, we assessed GS’s activity and mRNA and protein expression of GS by rtPCR and western blot (brain used as positive control). We also assessed GS expression in brain slices of naïve rats by immunofluorescence (co-localized with endothelial cell marker caveolin-1). In addition, to understand the effect of liver disease on GS, we exposed ECs to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) and sham-operated controls , as well as 1mM of ammonia chloride and 10μM of hydrogen peroxide as oxidative stress. Results: In vitro ECs and in vivo CMVs both expressed GS mRNA, protein and activity. Immunofluorescence showed GS colocalized with caveolin-1, further proving the presence of GS in endothelial cells of the BBB. The treatment with ammonia increased the activity of GS, while treatment with oxidative stress reduced its activity (p<0.05). ECs exposed to plasma from BDL rats reduced GS activity and protein expression compared with plasma from sham controls (p<0.01). Conclusion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. Interestingly, ammonia stimulates GS in endothelial cells, whereas oxidative stress inhibits GS activity. Furthermore, GS activity is decreased in the presence of plasma from cirrhotic rats, possibly due to the presence of systemic oxidative stress in BDL rats. We speculate that downregulation of GS allows for faster and easier entry of ammonia into the brain and therefore may increase the risk of HE. We anticipate that upregulating GS in ECs of the BBB could become a new therapeutic target for HE.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, as well as Sham-operated controls, were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; 2. Lean-BDL rats received RD pre- and post-BDL; 3. Lean-Sham rats received RD pre- and post-Sham surgery. Behaviour: Recognition memory, motor coordination and muscular strength were assessed before surgery, as well as 3 and 6 weeks post-surgery using the novel object recognition, rotarod and grip-strength tests, respectively. Body-composition (echoMRI): Fat vs. lean mass and free water (ascites) were also monitored. RESULTS: Before the surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on RD (Lean-BDL and Lean-Sham). 3 weeks after surgery, BW, fat mass, lean mass and free water were increased in Obese-BDL rats vs. Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham rats. 6 weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost BW, fat and Lean mass, while free water increased vs. Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats, compared to Lean-Sham rats. CONCLUSION: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Obesity also accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats (long-term memory), while others are exacerbated (motor coordination, forelimb strength). This new animal model of CLD and obesity suggests a synergistic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. Thus, obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting more susceptibility to poor neurological performance.
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
Introduction : La sarcopénie affecte jusqu’à 70% des patients atteints d’une maladie hépatique chronique (cirrhose). Sa présence a été associée à une qualité de vie détériorée. Par ailleurs, elle pourrait influencer le pronostic des patients cirrhotiques avant et après la transplantation hépatique (TH), seul traitement curatif pour la cirrhose à ce jour. Peu d’études ont étudié l’évolution de la sarcopénie après la TH. Le but de cette étude était d’évaluer l’évolution de la sarcopénie et son association avec le pronostic des patients cirrhotiques pré- et post-TH. Méthodes : L’évaluation de la masse musculaire a été effectuée chez 94 patients du département d’hépatologie du CHUM ayant reçu la TH entre le 5 juin 2012 et le 30 avril 2017. Elle était évaluée en utilisant l’index musculaire squelettique lequel est basé sur l’analyse de l’examen radiologique de tomodensitométrie au niveau de la 3e vertèbre lombaire. Par la consultation des dossiers médicaux, les critères de pronostic étaient évalués durant l’année suivant la TH et comprenaient le nombre de complications, les épisodes d’infection, la durée de l’hospitalisation et la fréquence des réadmissions. Résultats : La sarcopénie a persisté ou est apparue (Sarc+) chez 62% des patients (n=58) alors qu’elle a régressé ou est demeurée absente (Sarc-) chez 38% des patients (n=35). Le groupe Sarc+ a eu davantage de complications avant la TH (p=0,012) et après la TH (p<0,001) et d’infections après la TH (p=0,006) par rapport au groupe Sarc-. La durée de l’hospitalisation était aussi prolongée chez le groupe Sarc+ comparativement au groupe Sarc- (p<0,001). Conclusion : La sarcopénie persistante ou nouvellement développée telle qu’évaluée jusqu’à 1 an après la TH est associée à un mauvais pronostic, particulièrement en période post-TH. La mise en place de stratégies d’intervention pour préserver ou même augmenter la masse musculaire pourrait aider à améliorer le pronostic des patients suite à la TH. Bourses octroyées par le Département de nutrition et la Faculté de médecine de l’Université de Montréal
Manila Sophasath, Mélanie Tremblay, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Problématique : Au Canada, la prévalence de la maladie hépatique connaît une croissance exponentielle. L’une des complications les plus prévalentes y étant associée est la malnutrition, qui affecte la qualité de vie des patients par l’augmentation des risques de nombreuses complications. Objectif : L’objectif général est d’évaluer l’impact bénéfique potentiel du Guide de nutrition pour la cirrhose, un document éducatif basé sur des données probantes et sur l’expérience de patient-partenaire, qui traduit les lignes directrices en matière de nutrition et cirrhose. Les objectifs spécifiques sont d’évaluer l’état nutritionnel, la qualité de vie reliée à la santé et la fonction hépatique en plus de déterminer les connaissances en nutrition des patients cirrhotiques après l’implantation du Guide. Méthodologie : Nous visons l’inclusion de 132 patients visitant la clinique externe d’hépatologie du CHUM. Les patients seront rencontrés par une nutritionniste, qui évaluera l’état nutritionnel, la qualité de vie, la fonction hépatique et leurs connaissances en nutrition. Le Guide leur sera ensuite enseigné et fourni pour 12 semaines, pendant lesquelles un suivi téléphonique sera réalisé à chaque deux semaines. Au terme des 12 semaines, les mêmes évaluations seront réalisées par la nutritionniste. Ces patients seront comparés à un groupe contrôle d’environ 40 sujets cirrhotiques recevant des conseils nutritionnels « habituels », mais n’ayant pas accès au Guide. Résultats attendus : Les patients ayant eu accès au Guide devraient voir une amélioration au niveau des paramètres mesurés. Discussion : Les résultats de cette étude devraient permettre d’optimiser la qualité des soins chez les gens souffrant de maladies chroniques du foie.
Constantine Karvellas, Jaime Speiser, William M. Lee, Mélanie Tremblay, Christopher F. Rose.
Background and aims:Acute liver failure (ALF) is associated withsignificant mortality. Failure to identify non-survivors results inpreventable deaths, while misclassifying prospective survivors maylead to unnecessary transplants (LT). Liver-type fatty acid bindingprotein (FABP1) is a 15 kDa protein expressed in hepatocytes. It haspreviously been demonstrated to improve prognostic discriminationin acetaminophen (APAP)-induced ALF but has not been investigatedin other etiologies of ALF. Our primaryaim was too determine if serialFABP1 levels (early; admission or late; day 3–5) are associated with21-day transplant-free survival in non-APAP ALF. Method:FABP1 was measured in serum samples from 384 ALFpatients (n = 88 transplant-free survivors (TFS), n = 296 died/LT∼NTFS) using solid-phase enzyme-linked immunosorbent assay(ELISA) and analysed with US ALFSG registry data.Results:Of 384 patients, etiologies of ALF included autoimmunehepatitis (AIH, n = 125), drug-induced liver injury (DILI, n = 141) andHepatitis B (n = 118). Overall, 177 (46%) ALF patients received LT.Transplant-free survivors (n = 88) were less likely to requiremechanical ventilation (21 vs. 66%), vasopressors (10% vs. 35%) andor have Grade III/IV hepatic encephalopathy (30% vs. 73%, p < 0.0001for all) compared with patients who died/required LT. FABP1 levelswere significantly lower in TFS patients at day 3–5 (TFS 54 vs. NTFS 66 ng/ml; p = 0.049; see Figure) but not admission (TFS 96 vs. NTFS87 ng/ml; p = 0.67). After adjusting for significant covariates,increased FABP1 levels at late time points (day 3–5) were significantlyassociated with worse outcomes (death/LT) in AIH patients (LogFABP1 Odds Ratio (OR) 3.93 (1.50–10.32), P = 0.0055; AUROC 0.89).Increased late FABP1 was associated with worse outcomes in HBV(Log FABP1 OR 1.85 (0.96–3.59), P = 0.068; AUROC 0.82) and DILI (LogFABP1 OR 1.36 (0.97–1.91), P = 0.079; AUROC 0.72), but these werenot statistically significant after adjusting for covariates. Similarly,FABP1 on admission was not significant (adjusted p > 0.15 for allsubgroups). Conclusion:This is the first report of FABP-1 in non-APAP-ALF. Usedin combination with existing prognostic scores, FABP1 may poten-tiallyhelp identify ALF patients with higher recovery potential at latertime points after a period of organ support.
Mimosa Nguyen, Mélanie Tremblay, An Tang, Christopher F. Rose, Chantal Bémeur.
Background and aims:Musclewasting (sarcopenia) affects 30 to 70%of cirrhotic patients. The presence of sarcopenia may be associatedwith a worst prognosis in cirrhotic patients awaiting and after livertransplantation (LT). To this day, few studies have evaluated andfollowed muscle mass (in terms of quantity and quality) after LT.The goal of this study was to assess the association between theevolution of sarcopenia and the prognosis of cirrhotic patients beforeand after LT.Method:In total, 94 cirrhotic patients who underwent LT at theMontreal University Hospital Center-Liver Unit were included.Sarcopenia was assessed at the third lumbar level vertebrae using acomputed tomography scan (CT-scan). The diagnostic of sarcopeniawas based on previously established sex-specific cut-off values ofskeletal muscle index. Patients were classified into two groups: (1)persistent or newly developed sarcopenia after LT (Sarc+); (2)resolved sarcopenia or absence of sarcopenia before and after LT(Sarc-). Muscle quality (myosteatosis) was assessed by calculatingintramuscular adipose tissue content. The prognostic factors werecollected 6 months before and during 1 year after LT through medicalrecords and included the number of complications, the episodes ofinfections, the length of stay, and the frequency of readmissions.Results:Sarcopenia persisted or was newly developed (Sarc+) in 62%of the patients (n = 58). It remained absence or was resolved after LTin 38% of the patients (n = 35). Muscle quality was significantlydecreased post-LT (p = 0.034). The group Sarc+ experienced morecomplications pre-LT (p = 0.012) and post-LT (p < 0.001), infectionspost-LT (p = 0.006) and readmissions (p = 0.048) compared to thegroup Sarc-. The length of stay was longer for the group Sarc+ asopposed to the group Sarc- (p < 0.001).Conclusion:Persistent and newly developed sarcopenia after LTappear to have negative outcomes on the prognosis of patients.Interventional strategies to optimize, increase or preserve musclemass could help to improve post-operative recovery as well as thequality of life in patients who have undergone LT.
The association between sarcopenia and the prognosis of cirrhotic patients in liver transplantation.
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
Introduction: Sarcopenia affects up to 70% of patients suffering from chronic liver disease (cirrhosis). The presence of sarcopenia may influence the prognosis of cirrhotic patients before and after liver transplantation (LT). Few studies have assessed the evolution of sarcopenia in LT. The goal of this study was to follow the evolution and assess the impact of sarcopenia on the prognosis of cirrhotic patients before and after LT. Methods: Skeletal muscle index (SMI) was calculated from cross‐sectional muscle area at the third lumbar level (L3) on computed tomography (CT). The following CT‐scans were analysed: before LT + before discharge and/or nearest 1‐year post‐LT. Sarcopenia was defined using previously published cutoffs based on gender. The association of sarcopenia with prognostic factors (mortality, hospital stay, infections, readmissions) was assessed in cirrhotic patients who underwent LT. Results: Thus far, the average SMI before LT of sarcopenic and non‐sarcopenic patients were 40.3±5.3 cm2/m2 and 58.7±13.7 cm2/m2, respectively. The correlation of SMI with length of hospital stay, infections, and readmissions were high (rspearman = −0.714 p = 0.071), moderate (rspearman = −0.598 p = 0.156), and low (rspearman = −0.386 p = 0.393). Conclusions: Preliminary results indicate that low muscle mass before LT tends to be associated with prolonged hospitalization. As we analyse the remaining data, the strength of the relationship between sarcopenia and the prognosis in LT will help better guide patient care.
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
Introduction : La sarcopénie affecte jusqu’à 70% des patients atteints d’une maladie hépatique chronique (cirrhose). Sa présence a été associée à une qualité de vie détériorée. Par ailleurs, elle pourrait influencer le pronostic des patients cirrhotiques avant et après la transplantation hépatique (TH), seul traitement curatif pour la cirrhose à ce jour. Peu d’études ont étudié l’évolution de la sarcopénie après la TH. Le but de cette étude était d’évaluer l’évolution de la sarcopénie et son association avec le pronostic des patients cirrhotiques pré- et post-TH. Méthodes : L’évaluation de la masse musculaire a été effectuée chez 94 patients du département d’hépatologie du CHUM ayant reçu la TH entre le 5 juin 2012 et le 30 avril 2017. Elle était évaluée en utilisant l’index musculaire squelettique lequel est basé sur l’analyse de l’examen radiologique de tomodensitométrie au niveau de la 3e vertèbre lombaire. Par la consultation des dossiers médicaux, les critères de pronostic étaient évalués durant l’année suivant la TH et comprenaient le nombre de complications, les épisodes d’infection, la durée de l’hospitalisation et la fréquence des réadmissions. Résultats : La sarcopénie a persisté ou est apparue (Sarc+) chez 62% des patients (n=58) alors qu’elle a régressé ou est demeurée absente (Sarc-) chez 38% des patients (n=35). Le groupe Sarc+ a eu davantage de complications avant la TH (p=0,012) et après la TH (p<0,001) et d’infections après la TH (p=0,006) par rapport au groupe Sarc-. La durée de l’hospitalisation était aussi prolongée chez le groupe Sarc+ comparativement au groupe Sarc- (p<0,001). Conclusion : La sarcopénie persistante ou nouvellement développée telle qu’évaluée jusqu’à 1 an après la TH est associée à un mauvais pronostic, particulièrement en période post-TH. La mise en place de stratégies d’intervention pour préserver ou même augmenter la masse musculaire pourrait aider à améliorer le pronostic des patients suite à la TH. Bourses octroyées par le Département de nutrition et la Faculté de médecine de l’Université de Montréal
Rafael Ochoa-Sanchez, Mariana M. Oliveira, Grégory Petrazzo, Yossi Dagon, Kip West, Lauren Renaud, Caroline Kurtz, Christopher F. Rose.
Hyperammonemia associated with liver cirrhosis plays a major role in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) that contributes to systemic hyperammonemia in HE. Probiotic bacteria have shown benefits in the treatment of HE although the underlying mechanism is not completely understood. We engineered Escherichia coli Nissle 1917 bacterium (EcN) to consume NH3 and convert it to arginine in the gut (SYNARG). To enhance the beneficial effect of NH3 consumption, we further engineered the EcN to synthesize the short chain fatty acid butyrate in the gut (SYNARG+BUT). Both strains were tested in two experimental models of cirrhosis and HE: thioacetamide (TAA) or bile duct ligation (BDL). Methods: Cirrhosis was induced in BALB/c mice by treatment with TAA for 4 weeks, and in SD rats by BDL for 5 weeks (W). TAA-treated mice were gavaged with a daily dose of 1x1010 colony forming units (CFU) of SYNARG. BDL rats received 1x1012 CFU with SYNARG or SYNARG+BUT for 4 W. Plasma NH3 (mmol/L) was measured in both models at baseline (BL) and after treatment, with an additional measurement at 3 W post-BDL. Results: TAA mice developed hyperammonemia (BL: 22.4±3.3 to 4 W: 82.4±8.7, p<0.05) which was attenuated after SYNARG treatment (45.5±4.7, p<0.05). Longitudinal analysis in Veh-BDL rats developed hyperammonemia at 3 W (BL: 68.8±5.7 to 121.6±9.8, p<0.01) which was further increased after 5 W (158.8±22.0, p<0.001 vs BL and 3 W). At 3 W, SYNARG+BUT prevented a significant increase in blood NH3 in BDL rats (99.6±8.7, p<0.05 vs BL). Both SYNARG+BUT (115.9±17.2) and SYNARG (127.9±15.5) were protective in preventing further increase in blood NH3 from 3 to 5 W, as observed in Veh-BDL rats. Moreover, the analysis between groups at 5 W showed that SYNARG+BUT reduces NH3 compared to Vehicle-BDL rats (p<0.05). Conclusion: EcN, engineered to consume NH3 in the gut, is an effective approach to lower plasma NH3 in models of cirrhosis. Thus, the therapeutic potential of these engineered EcN strains should be further evaluated in patients with cirrhosis and HE.
Mariana Oliveira, Mélanie Tremblay, Christopher Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of liver disease. The prevalence of the development of cirrhosis is similar in both males and females. The use of animal models of chronic liver disease contributes greatly to the understanding of the mechanisms of HE. To date, fundamental and pre-clinical research in HE has not been investigated in female animals. Since behavioral tests used to assess HE in rats are influenced by the female’s reproductive cycle (the estrous cycle), the aims of this pilot project are 1) to evaluate the estrous cycle, 2) assess degree of liver failure and 3) investigate the neuro phenotype of female bile duct ligated (BDL) rats, (a model of HE) compared to female SHAMs. Methods and results: 6 weeks bile duct ligated (BDL) female rats (N=6) had an increase in body weight, food intake and lean mass (a trend for lower fat mass (p=0.066)), with impaired liver function (p=0.009), dysregulated estrous cycle (measured by daily vaginal cytology), and impaired short-term memory (p=0.057) vs SHAM operated controls (N=4). Historical data from male BDLs showed similar liver and memory impairment, but a decrease in the body weight and food intake vs male SHAMs. Compared to male BDL, female BDL rats showed a similar degree of liver disease as well as short-term memory impairment, which was evident in the diestrous phase. However, a higher increase in body weight and food intake was found in female vs male BDL. A dysregulated estrous cycle was found in female BDL, however, all 4 phases of the cycle were completed. Discussion: These preliminary results demonstrate that there are potential differences between both male and female BDL rats and thus merit to be addressed.
Evaluate the efficiency of Rifaximin treatment in a bile-duct ligated model of cirrhosis.
Grégory Petrazzo, Mariana M. Oliveira, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a common and potentially severe complication of liver failure. HE is related to impaired quality of life. The pathogenesis of HE is linked with gut-derived ammonia (NH3) therefore treatment aim to reduce the blood level of NH3. The standard care of HE is Lactulose, a non-absorbable disaccharide. Nonetheless, patient observance is poor due to some uncomfortable side-effect. Among new therapies that have arisen this past decade, Rifaximin is a potent candidate. The aim of this study is to assess the efficiency of Rifaximin in reducing plasma NH3 and ameliorate the HE in a murine bile duct model. Three weeks after BDL surgery, BDL and SHAM-operated rats were sorted into five groups according to their treatments : SHAM-vehicle, BDL-vehicle, BDL-Lactulose, BDL-Rifaximin, BDL-Lactulose+Rifaximin. Treatment was given by gavage for 3 weeks. Survival, body-weight, food consumption and body composition were assess every week during the six weeks of the study. Behavioral analysis was conduct to assess the HE status. At sacrifice, brain water was measured to assess potential brain edema and plasma sample was taken to measure NH3 and hepatic biochemistry parameters. No difference was seen in the survival, growth pattern, food consumption or body composition between BDL-groups. No difference was seen in the performance during EPM, OFT, Rotarod, NOR or night activity. No difference was seen in brain water between SHAM or BDL groups. Surprisingly, NH3 level was increase in all but BDL-vehicle compare to SHAM. The biochemistry parameters confirm the establishment of cirrhosis in BDL groups compare to SHAM. Overall, this study didn’t present strong, reliable and enough data to conclude on the efficacy of Rifaximin as sole treatment in the context of HE. Some results fail to reach significance probably because of the low number of animals. Future direction on this project will aim to : 1) increase the antibiotic dose; 2) reduce the duration of the model to five weeks; 3) trigger an episode of HE by injecting a dose of NH3 before treating the rats.
Rafael Ochoa-Sanchez, Mariana Oliveira, Grégory Petrazzo, Yossi Dagon, Kip West, Lauren Renaud, Caroline Kurtz, Christopher F. Rose.
Background: Hyperammonemia associated with chronic liver disease (cirrhosis) plays a major role in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) that contributes to systemic hyperammonemia in HE. Probiotic bacteria have shown benefits in the treatment of HE although the underlying mechanism(s) are not completely understood. We engineered Escherichia coli Nissle 1917 bacterium (EcN) to consume NH3 and convert it to arginine in the gut. To enhance the beneficial effect of NH3 consumption, we further engineered the EcN to synthesize the short chain fatty acid butyrate in the gut. The resulting strains, SYNARG (arginine producing) and SYNARG+BUT (arginine and butyrate producing) were tested in two experimental models of cirrhosis and HE: thioacetamide (TAA) or bile duct ligation (BDL). Methods: Cirrhosis was induced in BALB/c mice by intraperitoneal treatment with TAA for 4 weeks, and in Sprague-Dawley rats by BDL for 5 weeks. TAA-treated mice were gavaged with a daily dose of 1 x 1010 colony forming units (CFU) of SYNARG. BDL rats were gavaged with 1 x 1012 CFU with SYNARG or SYNARG+BUT for 4 weeks. Plasma NH3 was measured in both models at baseline (BL) and after treatment, with an additional measurement at 2 weeks post treatment (3 weeks post-BDL). Results: TAA mice developed hyperammonemia (BL: 22.4 ± 3.3 umol/L to 4 weeks: 82.4 ± 8.7 umol/L, p<0.05) which was attenuated after SYNARG treatment (45.5 ± 4.7 umol/L, p<0.05). Longitudinal analysis in Vehicle-BDL rats developed hyperammonemia at 3 weeks (BL: 68.8 ± 5.7 to 121.6 ± 9.8 umol/L, p<0.01) which was further increased after 5 weeks (158.8 ± 22.0 umol/L, p<0.001 vs BL and 3 weeks). At 3 weeks, SYNARG+BUT prevented a significant increase in blood NH3 in BDL rats (99.6 ± 8.7 umol/L, p<0.05 vs BL). Both SYNARG+BUT (115.9 ± 17.2 umol/L) and SYNARG (127.9 ± 15.5 umol/L) were protective in preventing further increase in blood NH3 from 3 to 5 weeks, as observed in Vehicle-BDL rats. Moreover, the analysis between groups at 5 weeks showed that SYNARG+BUT reduces NH3 compared to Vehicle-BDL rats (p<0.05). Conclusion: Our data suggest that EcN, engineered to consume NH3 in the gut, is an effective approach to lower plasma NH3 in models of cirrhosis and hyperammonemia. Based on these results, the therapeutic potential of these engineered EcN strains should be further evaluated in patients with liver disease and HE.
Progressive resistance training prevents loss of muscle mass and strength in bile duct ligated rats.
Mariana M. Oliveira, Christopher F. Rose, Luise Aamann, Rafael Ochoa-Sanchez, Mariana Oliveira, Mélanie Tremblay, Chantal Bémeur, Gitte Dam, Hendrik Vilstrup, Niels Kristian Aagaard, Christopher Rose.
Loss of muscle mass and strength is common in cirrhosis and increases the risk of hyperammonemia and hepatic encephalopathy. Resistance training optimizes muscle mass and strength in several chronic diseases. However, the beneficial effects of resistance training in cirrhosis remains to be investigated. Bile duct ligated (BDL)-rats develop chronic liver disease, hyperammonemia, reduced muscle mass and strength. Our aim was to test the effects of resistance training on muscle mass, function and ammonia metabolism in BDL-rats. A group of BDL-rats underwent a progressive resistance training program and a group of non-exercise BDL-rats served as controls. Resistance training comprised of ladder climbing with a progressive increase in carrying weights attached to the tail. Training was performed five days a week during four weeks. Muscle strength and body composition were assessed using grip strength and EchoMRI. Weight and circumference of the gastrocnemius muscle (normalized to bodyweight), plasma ammonia and glutamine synthetase protein expression and activity were assessed. BDL+exercise rats had significantly larger gastrocnemius circumference compared to non-exercise BDL-rats: ratio 0.082 vs. 0.075 (p<0.05). Gastrocnemius muscle weight was higher in exercisers than controls: 0.006 vs. 0.005 (p<0.05). A tendency towards a lower plasma ammonia in the exercise group compared to controls was observed (p=0.10). There were no differences in lean body mass, GS protein expression and activity between the groups. Resistance training in rats with chronic liver disease beneficially effects muscle mass and strength. The effects were followed by non-significant reduction in blood ammonia, however a tendency was observed. This article is protected by copyright. All rights reserved.
Pathogenesis of Hepatic Encephalopathy in Chronic Liver Disease.
Rafael Ochoa-Sanchez, Christopher F. Rose.
Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that occurs during chronic liver disease (CLD). While ammonia and other precipitating factors in liver disease including inflammation, bile acids, oxidative stress, and lactate play a role in the pathogenesis of HE, the exact mechanism that leads to HE is not fully understood. Notably, accumulating evidence points toward a synergic effect rather than independent actions among precipitating factors that contributes to the development and severity of HE in CLD. Hence, this review is aimed to briefly discuss the single and synergic interplay of pathological factors in the progression and severity of HE.
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Introduction: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (CLD; cirrhosis) and may increase the risk of developing other complications including hepatic encephalopathy (HE). Malnutrition in cirrhosis may also affect patient’s functional status and health-related quality of life (HRQOL). Management strategies focussing on nutritional status in relation to complications of cirrhosis are an unmet clinical need. We hypothesize that sub-optimal nutritional status in cirrhotic patients increases the risk of developing HE and decreases HRQOL. Objectives: The primary goal is to evaluate the impact of nutritional status on health-related quality of life in cirrhotic patients. The secondary goal is to ascertain the presence of HE and examine its relationship with nutritional status and HRQOL. Methods: Hospitalized and outpatients with cirrhosis as well as non-cirrhotic (NC) patients were assessed for 1) Nutritional status (SGA); 2) HE; 3) HRQOL (SF-36). Results: 50 cirrhotic patients (72% men) of various etiologies as well as 18 NC patients (33% men) were included. SGA analysis revealed that 34% of cirrhotic patients were malnourished whereas 12% of cirrhotic patients were diagnosed with HE at time of recruitment and 37% had a history of HE. Among malnourished CLD patients, 18% were diagnosed with HE. CLD malnourished patients showed a decreased HRQOL compared to well-nourished CLD patients (p<0,01). Moreover, HE had an impact on HRQOL as cirrhotic patients with a history of HE episode(s) showed decreased physical functioning (p=0,024) and role limitations due to physical health (p=0,002). Interestingly, when compared to NC patients, CLD patients displayed a lower score in physical functioning (p<0,0001) and general health (p=0,027). Conclusion: Our data suggest that poor nutritional status and history of HE episode(s) do negatively influence HRQOL in cirrhotic patients. Significance: Our study provides important results in order to empower the field of dietitics to actively engage in malnutrition management in cirrhosis.
Ammonia: This is not the end but rather the end of the beginning.
Nicolas Weiss, Gitte Dam, Christopher F. Rose.
Myosteatosis and sarcopenia are associated with hepatic encephalopathy in patients with cirrhosis.
Rahima A. Bhanji, Carlos Moctezuma-Velazquez, Andres Duarte-Rojo, Maryam Ebadi, Sunita Ghosh, Christopher Rose, Aldo J. Montano-Loza.
Skeletal muscle is known to play a role in hepatic encephalopathy. Fatty infiltration of the muscle (myosteatosis) and muscle mass depletion (sarcopenia) are frequent complications of cirrhosis. The purposes of the study were to investigate if myosteatosis and sarcopenia are associated with overt hepatic encephalopathy in patients with cirrhosis and to evaluate their impact on mortality. A total of 675 cirrhotic patients were studied. Computed tomography scans were used to analyze the skeletal muscle. Hepatic encephalopathy was defined by either a hepatic encephalopathy-related hospitalization and/or taking ammonia-lowering treatment (i.e., lactulose, rifaximin). Myosteatosis was observed in 348 patients (52%), sarcopenia in 242 (36%), and hepatic encephalopathy in 128 (19%) patients. Both myosteatosis (70 vs. 45%, p < 0.001) and sarcopenia (53 vs. 32%, p < 0.001) were more frequent in patients with hepatic encephalopathy. By multivariable regression analysis, both myosteatosis and sarcopenia were associated with a higher risk of hepatic encephalopathy, independent of the MELD score (OR 2.25; 95% CI, 1.32-3.85, p = 0.003 and OR 2.42; 95% CI, 1.43-4.10, p = 0.001, respectively). In univariate Cox proportional hazards analysis, sarcopenia (csHR 2.02; 95% CI, 1.57-2.58, p < 0.001), myosteatosis (csHR 1.45; 95% CI, 1.16-2.91, p = 0.004), and hepatic encephalopathy (csHR 1.61; 95% CI, 1.20-2.18, p = 0.002) were associated with mortality, but only sarcopenia was significant in the multivariable analysis (csHR 2.15; 95% CI, 1.52-3.05, p < 0.001). Myosteatosis and sarcopenia are independently associated with overt hepatic encephalopathy in patients with cirrhosis. The association between hepatic encephalopathy and mortality may be explained at least partially by the presence of sarcopenia. These results identify the importance of muscle mass and suggest therapeutic strategies to attenuate muscle mass loss and improve muscle quality.
Diagnosis and Management of Hepatic Encephalopathy: A Case-based Guide
Julien Bissonnette, Christopher F. Rose.
In liver disease, a number of systemic pathogenic factors are responsible for the onset and progression of hepatic encephalopathy. A reduced capacity of the liver to clear gut-derived ammonia leads to an increase in blood ammonia and neurotoxicity. Portal-systemic shunting as well as a reduction in muscle quality and/or muscle mass loss further increase the risk of developing hyperammonemia. Systemic oxidative stress and inflammation together with hyperammonemia can trigger cognitive decline. These factors can cross the blood-brain barrier and affect the brain directly or alter the permeability and signaling across the blood-brain barrier. Long considered a reversible disorder, there is ample amount of evidence demonstrating that repeated episodes of overt HE can prompt neuronal cell injury/death leading to irreversibility. This chapter describes a patient who embarks on the continuum of HE, describing precipitating factors involved in the pathogenesis of cognitive decline.
Vincent Forster, Valentina Agostoni, Giovanna Giacalone, Soohyeon Lee, Meriam Kabbaj, Christina Bosoi, Mélanie Tremblay, Christopher Rose, Jean-Christophe Leroux.
Alcohol accelerates hepatic encephalopathy in a rat bile duct ligation model.
Xiaoru Chen, Mariana Oliveira, Grégory Petrazzo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher Rose.
Hepatic encephalopathy (HE) is a common and debilitating neuropsychiatric complication of liver disease characterized by a constellation of symptoms, including cognitive, psychiatric and motor disturbances. One of the causes of liver disease is alcoholic cirrhosis, which can induce acquired cerebellar degeneration syndrome, atrophy of the cerebellum producing symptoms of ataxia and motor difficulties. In the literature, very few experimental studies concern the role of alcohol on the development of HE. Here we examine the effects of ethanol on bile duct ligation (BDL) rats, a HE model, using a variety of behavioral tasks on motor coordination, open field behavior and memory. BDL rats were subjected to double ligation on the common bile duct with dissection between the ligatures, sham-operated rats underwent the same surgery except for ligation. We first effectuated a dose-response study (N=4-5) in BDL rats to determine the optimal dosage regimen of ethanol. 7 days after surgery, BDL rats were given ethanol by intragastric gavage with 1, 2, 4, 6 and 8 g/kg or saline over a 4-weeks period. Crews Scale and blood ethanol concentration were checked weekly. The accelerating rotarod was used to assess motor coordination. Then the dose regimen of intermittent exposure of 2X3g/kg 3h apart 5 days per week during 4 weeks was chosen to further elucidate the evolution of neurological deficits in both BDL rats and their sham operated controls. The behavioral assessments were performed at 7, 14, 21, 28 and 35 days, one day after blood ethanol concentration dropped to zero of each treatment cycle. Rats were assigned to the following groups (N=9-10): Sham + saline, Sham + ethanol, BDL + saline, BDL + ethanol. The baseline of rotarod performance and gait analysis parameters (at 7 days) showed there were no difference among groups before ethanol gavage. However, BDL + ethanol group rats had gradually impaired coordination performance and motor activity, contrary to those of Sham+ ethanol group who improved their performance gradually by learning. No significant differencet was observed in gait analysis. In addition, blood ethanol concentration over time showed a different metabolism mode in BDL compared with that of Sham + ethanol rats. The changes in coordination performance of BDL+ethanol rats might be associated with some biomolecular changes in the brain. The fact that the front cortex water content in these rats slightly increased as well as the weight of cerebellum reduced suggests that neuroinflammation and cerebellar atrophy might be involved in. Results of the current study indicate heavy alcohol ingestion impairs gradually motor coordination during cirrhosis. BDL rats treated with alcohol allowed perform studies on motor alterations in less than 5 weeks, will be an efficient animal model for the study of HE induced by ethanol and for the search of new treatment strategies.
Impact de la sarcopénie lors de la transplantation hépatique : une étude rétrospective en cours.
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
Problématique: La sarcopénie (perte de masse et de fonction musculaires) affecte 30 à 70% des patients atteints de maladie hépatique chronique (cirrhose). Un Canadien sur 4 souffrirait de maladie hépatique. Chez les patients cirrhotiques, la présence de la sarcopénie avant et après la transplantation hépatique (TH) serait associée à un mauvais pronostic. Objectif : Le but général de cette étude rétrospective en cours est de déterminer, chez les patients cirrhotiques, la prévalence de la sarcopénie avant la TH ainsi que son impact sur le pronostic après la TH. Méthodologie : Notre étude vise l’inclusion de 100 patients cirrhotiques de diverses étiologies. L’évaluation de la masse musculaire avant la TH s’effectue en utilisant l’index musculaire squelettique lequel est basé sur l’analyse de l’examen radiologique de tomodensitométrie (CT-scan) au niveau de la 3e vertèbre lombaire. La durée de l’hospitalisation, la fréquence d’infections et le nombre de réadmissions durant la première année post-TH sont également recueillis par la consultation des dossiers médicaux. Résultats attendus: Nous anticipons que la sarcopénie soit répandue chez les patients cirrhotiques à l’étude. Sa présence pourrait être associée à une durée d’hospitalisation prolongée, à de fréquentes infections et à des réadmissions récurrentes. Discussion : Une prise en charge de la sarcopénie pourrait s’avérer primordiale si sa présence influence le pronostic des patients suite à la TH.
Grégory Petrazzo, Mariana M. Oliveira, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a common and severe complication of liver failure. The pathogenesis of HE is linked with gut-derived ammonia (NH3). The standard care for patient experiencing episodes of HE is lactulose but observance is poor due to uncomfortable side-effects. Rifaximin is a potent candidate but there is no clinical study that assess its efficiency solely. This study aim to assess, in a bile-duct ligation (BDL) model of HE, the efficiency of rifaximin to reduce plasma NH3 and ameliorate HE status. 3 wks after BDL surgery, all animals were sorted in 5 grps: SHAM-Veh, BDL-Veh, BDL-Lac, BDL-Rif, BDL-Lac+Rif. Ttm was given by gavage for 3 wks with vehicle, lactulose, rifaximin and the combination of both ttm. Survival, body-weight, food consumption and body composition was assess every wk. During the ttm, behavioral analysis was done to assess the HE status, including OFT, EPM, Rotarod, NOR and night activity. At the end of the study, brain water was measured and plasma sample was taken. No difference was seen in the survival, growth, food consumption or body composition between BDL-grps. In the last wk the free water was increased in all BDL rats. No difference was seen in any grps regarding behavioral tests nor was any evidence of brain edema. NH3 level was increased in BDL treated with either lactulose or rifaximin compare to SHAM but surprisingly not with vehicle. The biochemistry parameters confirm the onset of cirrhosis in BDL grps. This study don’t present strong, reliable and sufficient data to conclude on the efficacy of rifaximin. Future direction will aim to increase the dose of drug; reduce the duration of the model; trigger an episode of HE before ttm.
Assessment of the spectrum of hepatic encephalopathy: A multicenter study.
Bradley Reuter, Kara Walter, Julien Bissonnette, Michael D. Leise, Jennifer Lai, Puneeta Tandon, Patrick S. Kamath, Scott W. Biggins, Christopher F. Rose, James B. Wade, Jasmohan S. Bajaj.
Hepatic encephalopathy (HE) is a major cause of morbidity in cirrhosis. However, its severity assessment is often subjective, which needs to be studied systematically. The aim was to determine how accurately trainee and nontrainee practitioners grade and manage HE patients throughout its severity. We performed a survey study using standardized simulated patient videos at 4 US and 3 Canadian centers. Participants were trainees (gastroenterology/hepatology fellows) and nontrainees (faculty, nurse practitioners, physician assistants). We determined the accuracy of HE severity identification and management options between grades <2 or ≥2 HE and trainees/nontrainees. In total, 108 respondents (62 trainees, 46 nontrainees) were included. For patients with grades <2 versus ≥2 HE, a higher percentage of respondents were better at correctly diagnosing grades ≥2 compared with grades <2 (91% versus 64% P < 0.001). Specialized cognitive testing was checked significantly more often in grades <2, whereas more aggressive investigation for precipitating factors was ordered in HE grades >2. Serum ammonia levels were ordered in almost a third of grade ≥2 patients. For trainees and nontrainees, HE grades were identified similarly between groups. Trainees were less likely to order serum ammonia and low-protein diets, more likely to order rifaximin, and more likely to perform a more thorough workup for precipitating factors compared with nontrainee respondents. There was excellent concordance in the classification of grade ≥2 HE between nontrainees versus trainees, but lower grades showed discordance. Important differences were seen regarding blood ammonia, specialized testing, and nutritional management between trainees and nontrainees. These results have important implications at the patient level, interpreting multicenter clinical trials, and in the education of practitioners. Liver Transplantation 24 587-594 2018 AASLD.
Impact de la sarcopénie lors de la transplantation hépatique : une étude rétrospective en cours.
Mimosa Nguyen, Mélanie Tremblay, Geneviève Huard, An Tang, Christopher F. Rose, Chantal Bémeur.
Problématique: La sarcopénie (perte de masse et de fonction musculaires) affecte 30 à 70% des patients atteints de maladie hépatique chronique (cirrhose). Un Canadien sur 4 souffrirait de maladie hépatique. Chez les patients cirrhotiques, la présence de la sarcopénie avant et après la transplantation hépatique (TH) serait associée à un mauvais pronostic. Objectif : Le but général de cette étude rétrospective en cours est de déterminer, chez les patients cirrhotiques, la prévalence de la sarcopénie avant la TH ainsi que son impact sur le pronostic après la TH. Méthodologie : Notre étude vise l’inclusion de 100 patients cirrhotiques de diverses étiologies. L’évaluation de la masse musculaire avant la TH s’effectue en utilisant l’index musculaire squelettique lequel est basé sur l’analyse de l’examen radiologique de tomodensitométrie (CT-scan) au niveau de la 3e vertèbre lombaire. La durée de l’hospitalisation, la fréquence d’infections et le nombre de réadmissions durant la première année post-TH sont également recueillis par la consultation des dossiers médicaux. Résultats attendus: Nous anticipons que la sarcopénie soit répandue chez les patients cirrhotiques à l’étude. Sa présence pourrait être associée à une durée d’hospitalisation prolongée, à de fréquentes infections et à des réadmissions récurrentes. Discussion : Une prise en charge de la sarcopénie pourrait s’avérer primordiale si sa présence influence le pronostic des patients suite à la TH.
Christine Brew, Steven Smith, Lorette Noiret, Eric Labonte, Christopher F. Rose, Stephen Harrison.
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
The liver plays a major role in regulating ammonia levels in the blood. Therefore, in liver disease the loss of hepatic function leads to hyperammonemia and increased brain ammonia and consequently hepatic encephalopathy (HE). Ammonia-lowering strategies remain the mainstay therapeutic strategy. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. Therefore, the aim is to evaluate GS expression in endothelial cells of the BBB. Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS expression by western blot using brain lysates as positive control and by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, to understand the effect of ammonia on GS, ECs were exposed to 1mM of ammonia chloride for 48h. Finally, ECs were subjected to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) or sham-operated controls. We have characterized this BDL model and found both systemic oxidative stress and inflammation, in addition to hyperammonemia. ECs expressed GS mRNA, protein and activity. However, expression of GS was lower compared to brain lysate control samples (p<0.05). GS expression in CMV showed similar results to brain but GS activity was less (p<0.05). Using immunohistochemistry, GS was detected in ECs and in vessels of brain from naïve rats. When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS was decreased when compared to sham-operated controls (p<0.01). These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme compared to that found in the brain, could explain why GS has never been reported in these cells. Interestingly, ammonia stimulates GS in endothelial cells, but its activity is decreased in the presence of other pathogenic factors in plasma from cirrhotic rats such as oxidative stress and inflammation. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may be implicated in the pathogenesis of HE. We anticipate increasing GS in ECs of the BBB could become a new therapeutic target for HE
Christopher F. *Rose, Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (CLD; cirrhosis) and may increase the risk of developing other complications including hepatic encephalopathy (HE). Malnutrition in cirrhosis may also affect patient’s functional status and health-related quality of life (HRQOL). Management strategies focussing on nutritional status in relation to complications of cirrhosis are an unmet clinical need. We hypothesize that sub-optimal nutritional status in cirrhotic patients increases the risk of developing HE and decreases HRQOL. Purpose: The primary purpose is to evaluate the impact of nutritional status on health-related quality of life in cirrhotic patients. The secondary purpose is to ascertain the presence of hepatic encephalopathy and examine its relationship with nutritional status and HRQOL. Method: Hospitalized and outpatients (CHUM’s Liver Unit, Montreal, Canada) with cirrhosis as well as non-cirrhotic (NC) patients were assessed for 1) Nutritional status (Subjective Global Assessment (SGA)); 2) HE (presence or history); 3) HRQOL (Short-Form-36 (SF-36) questionnaire). Results: 50 cirrhotic patients (72% men) of various etiologies, Child-Pugh (15A, 7B, 18C, 10 unknown), mean age 56±12 as well as 18 NC patients (33% men, mean age 42±15) were included. SGA analysis revealed that 34% of cirrhotic patients were malnourished whereas 12% of cirrhotic patients were diagnosed with HE at time of recruitment and 37% had a history of HE. Among malnourished CLD patients, 18% were diagnosed with HE. CLD malnourished patients showed a decreased HRQOL compared to well-nourished CLD patients (p<0,01). Moreover, HE had an impact on HRQOL as cirrhotic patients with a history of HE episode(s) showed decreased physical functioning (p=0,024) and role limitations due to physical health (p=0,002). Interestingly, when compared to NC patients, CLD patients displayed a lower score in physical functioning (p<0,0001) and general health (p=0,027). Conclusion: Our data suggest that poor nutritional status does negatively influence HRQOL in cirrhotic patients but is not associated with HE. However, history of HE episode(s) does impact on HRQOL among this population. Therefore, identifying malnourished patients is of great importance and interventions for treating malnutrition remains an unmet clinical need.
Christopher F. *Rose, Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, as well as Sham-operated controls, were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; 2. Lean-BDL rats received RD pre- and post-BDL; 3. Lean-Sham rats received RD pre- and post-Sham surgery. Behaviour: Recognition memory, motor coordination and muscular strength were assessed before surgery, as well as 3 and 6 weeks post-surgery using the novel object recognition, rotarod and grip-strength tests, respectively. Body-composition (echoMRI): Fat vs. lean mass and free water (ascites) were also monitored. RESULTS: Before the surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on RD (Lean-BDL and Lean-Sham). 3 weeks after surgery, BW, fat mass, lean mass and free water were increased in Obese-BDL rats vs. Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham rats. 6 weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost BW, fat and Lean mass, while free water increased vs. Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats, compared to Lean-Sham rats. CONCLUSION: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Obesity also accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats (long-term memory), while others are exacerbated (motor coordination, forelimb strength). This new animal model of CLD and obesity suggests a synergistic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. Thus, obesity-induced cirrhosis in patients may result in more complex neurological
Impact de l’exercice sur la protection du cerveau lors de maladie hépatique chronique expérimentale.
Charlène Blanchette, Luise Aamann, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
La perte de masse musculaire et la malnutrition sont les complications les plus fréquentes de la maladie hépatique chronique (cirrhose). L’encéphalopathie hépatique (EH; désordre neuropsychiatrique) est une autre complication sérieuse observée chez 80% des patients cirrhotiques, qui pourrait être une conséquence de la perte de masse musculaire. Les myokines, dont « Brain-derived neurotrophic factor » (BDNF), sont des cytokines libérées par le muscle lors de la contraction musculaire exerçant plusieurs fonctions physiologiques bénéfiques. Leurs sécrétions impliquent la production d’un co-activateur transcriptionnel, le « Peroxisome proliferator-activated receptor coactivator-1alpha » (PGC-1a). Notre travail s’est donc concentré sur l’étude de l’impact de l’exercice sur la voie de signalisation PGC-1a/BDNF dans la prévention de la perte de masse musculaire et l’EH chez le rat cirrhotique. Matériel et méthode : La cirrhose et l’EH ont été induites chez le rat 6 semaines après la ligature des voies biliaires (BDL). Quatre groupes expérimentaux ont été constitués : 1) Contrôles; 2) BDL; 3) Contrôles + Exercice 4) BDL+ Exercice. Les rats faisant partie du 3e et du 4e groupe sont soumis à un exercice de type aérobique. L’expression génique et protéique musculaire de PGC-1a et BDNF a été évaluée par immunobuvardage Western. Résultats et discussion :L’exercice augmente l’expression de BDNF mature chez les rats sains, ce qui prévient la fonte musculaire typique de la maladie hépatique et le développement de l’EH. Le rôle du PGC-1a reste à clarifier. Conclusion :Une meilleure compréhension de la voie PGC-1a-BDNF pourrait permettre de cibler le muscle comme thérapie effectrice dans le but d’améliorer le pronostic ainsi que la qualité de vie des patients cirrhotiques. Sincères remerciements à PREMIER-FPC-Département de nutrition et à la Fondation Canadienne du foie
Nouveau modèle d’encéphalopathie hépatique épisodique due à la cirrhose.
Kim Phat Pham, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction :L'encéphalopathie hépatique (EH) est une complication neuropsychiatrique majeure de la maladie du foie chronique, telle que la cirrhose du foie. L’une de ses formes est l’EH épisodique dont les symptômes surgissent épisodiquement. Notre hypothèse était que les épisodes d’EH aggraveraient les dommages neurologiques de l’EH et accentueraient ainsi les troubles neurologiques. Toutefois, il n’existe aucun modèle animal d’EH épisodique à ce jour. Nous avions pour but de caractériser un modèle d’EH épisodique due à la cirrhose et d’évaluer l’impact de multiples épisodes sur le dysfonctionnement neurologique. Matériel et méthodes :Une cirrhose hépatique a été provoquée par ligatures du conduit biliaire (BDL) et a été combinée à des injections d’acétate d’ammonium afin d’induire l’EH épisodique dans le rat. La coordination motrice et l’apprentissage moteur ont été évalués avec le test RotaRod et la locomotion avec l’analyse de l’activité nocturne. Résultats et discussion :les déficits de coordination et de locomotion détectés chez les rats BDL ne se sont pas aggravés après des épisodes d’EH. Cependant, les rats cirrhotiques ayant eu des épisodes d’EH présentaient des déficits de coordination motrice après un épisode, alors que les rats cirrhotiques sans épisode ne présentaient pas de déficit de coordination. Un effet synergique de la cirrhose et des épisodes d’EH pourrait causer ces déficits de coordination motrice déjà après un seul épisode. Conclusion :Ce nouveau modèle de l’EH épisodique permettra d’explorer son mécanisme pathologique et d’y tester des traitements.Organisme subventionnaire : PREMIER-Département de médecine
Raluca Ticala, Mélanie Tremblay, Christopher F. Rose, Consortium de l'acidose lactique, Chantal Bémeur.
Introduction : Le Syndrome de Leigh version canadienne française (LSFC) est une maladie rare dont l’incidence est élevée dans la région du Saguenay-Lac-Saint-Jean. Il est causé par une mutation du gène « leucine-rich pentatricopeptide repeat motif containing protein » (LRPPRC), entrainant une déficience tissu-spécifique en cytochrome c oxydase de la chaîne respiratoire mitochondriale. L’activité de l’enzyme est réduite jusqu’à 80% dans le foie et le cerveau. Les atteintes hépatiques semblent avoir des répercussions au niveau cérébral; le phénotype consiste en un retard de développement, de l’hypotonie et un dimorphisme facial. Les enfants atteints sont susceptibles de souffrir d’épisodes aiguës d’acidose métabolique (crises) menant à la mort de 80% d’entre eux avant l’âge de 4 ans. Ces crises sont souvent déclenchées par plusieurs changements, incluant une infection ou un apport nutritionnel riche en matières grasses et sucres. Aucune avenue thérapeutique n’existe. Plusieurs recherches portent sur les atteintes hépatiques de la maladie, mais aucune investigation ne s’est penchée sur la caractérisation cérébrale de la microglie. Matériel et méthodes :L’hypothèse est qu’une activation de la microglie, cellules immunitaires du cerveau, est présente dans un modèle expérimental de souris ayant la mutation du gène LRPPRC au niveau hépatique et l’objectif est de caractériser cette activation. L’expression de marqueurs de cellules microgliales est mesurée par immunobuvardage Western, avec l’utilisation d’anticorps primaires (CD11b, OX-42 et Iba-1) spécifiques aux protéines d’intérêt. Résultats et discussion :Aucun anticorps n’a démontré des résultats valides et plusieurs sources d’erreur peuvent expliquer ces résultats. Conclusion :Ce projet de recherche a tenté de caractériser pour la première fois l’activation de la microglie dans un modèle expérimental de la maladie. Considérant les résultats obtenus, il est nécessaire de reproduire cette étude.
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (CLD; cirrhosis) and may increase the risk of developing other complications including hepatic encephalopathy (HE). Malnutrition in cirrhosis may also affect patient’s functional status and health-related quality of life (HRQOL). Management strategies focussing on nutritional status in relation to complications of cirrhosis are an unmet clinical need. We hypothesize that sub-optimal nutritional status in cirrhotic patients increases the risk of developing HE and decreases HRQOL. Purpose: The primary purpose is to compare the nutritional status of adult patients with cirrhosis to non-cirrhotic patients. The secondary purposes is to ascertain hepatic encephalopathy (current and previous history) and quality of life among this population. Method: Hospitalized and outpatients (CHUM’s Liver Unit, Montreal, Canada) with cirrhosis as well as non-cirrhotic (NC) patients were assessed for 1) Nutritional status (Subjective Global Assessment (SGA)); 2) HE (presence or history); 3) HRQOL (Short-Form-36 (SF-36) questionnaire). Results: 50 cirrhotic patients (72% men) of various etiologies, Child-Pugh (15A, 7B, 18C, 10 unknown), mean age 56±12 as well as 18 NC patients (33% men, mean age 42±15) were included. SGA analysis revealed that 34% of cirrhotic patients were malnourished whereas 12% of cirrhotic patients were diagnosed with HE at time of recruitment and 37% had a history of HE. Among malnourished CLD patients, 18% were diagnosed with HE. CLD malnourished patients showed a decreased HRQOL compared to well-nourished CLD patients (p<0,01). Moreover, HE had an impact on HRQOL as cirrhotic patients with a history of HE episode(s) showed decreased physical functioning (p=0,024) and role limitations due to physical health (p=0,002). Interestingly, when compared to NC patients, CLD patients displayed a lower score in physical functioning (p<0,0001) and general health (p=0,027). Conclusion: Our data suggest that poor nutritional status does negatively influence HRQOL in cirrhotic patients but is not associated with HE. However, history of HE episode(s) does impact on HRQOL among this population. Therefore, identifying malnourished patients is of great importance and interventions for treating malnutrition remains an unmet clinical need.
Jasmohan S. Bajaj, Bradley Reuter, Kara Walter, Scott W. Biggins, Julien Bissonnette, Christopher F. Rose, Jennifer C. Lai, Michael D. Leise, Patrick S. Kamath, Puneeta Tandon, James Wade.
Reliability of HE characterization and evaluation is essential for accurate management and conduct of multi-center trials but a systematic intervention of trainees and non-trainees is lacking. METHODS: US & Canadian GI trainee/non-trainee practitioners were shown standard- ized simulated patient videos during the same sitting. The videos demonstrated cirrhotic pts with HE below stage 2 (normal/covert), and overt stages (2, 3, & 4). Therespondents were asked to identify the HE stage and answer questions about management after each video. RESULTS: 108 respondents (62 trainees, 46 non-trainees) from 7 centers(77 from four 4 US & 31 from 3 Canadian centers) were included. Trainees included 18 1st yr, 16 2 nd yr, 12 3 rd yr, 14 4 th yr fellows & 2 residents. Non-trainees included 41 consultants & 5 mid-level practitioners. HE stages (Table): A higher % of respondents were better at correctly diagnosing stages ≥2 compared to stages <2 (91 vs 64%, p<0.001). HE sub-stages were identified similarly between trainees and non-trainees with a trend towards better “normal” identification in non-trainees(P=0.13).Management: Most respondents would send pts with stages < 2 rather than ≥2 HE for cognitive testing (70 vs 20%, p<0.001). The reverse was seen for ordering ammonia levels, higher in stages ≥2 than stage<2 HE (36 vs 11%, p<0.001). A higher percent of non-trainees tested ammonia levels in stage <2 HE compared to trainees (16 vs 7%, p=0.03). A higher percent of non-trainees than trainees would prescribe low protein diets in stage ≥2 HE(18 vs 8%, p=0.007). Most respondents would treat pts with stages ≥2 with lactulose (58 vs 93%, p<0.001) & rifaximin (14 vs 50%, p<0.001). More trainees would use rifaximin for stages ≥2 than non-trainees (68 vs 30%, p<0.001). CONCLUSION: In this multi-center study, North American GI practitioners demonstrated poor reliability in lower stages but not stages ≥2 HE. There were significant variations in ordering MHE testing, & lactulose/rifaximin and relatively few respondents order ammonia levels or a low-protein diet. Overt HE ≥stage 2 remains a reliable benchmark of diagnosis across training levels but there is a need for more standardized algorithms for HE management.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, as well as Sham-operated controls, were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; 2. Lean-BDL rats received RD pre- and post-BDL; 3. Lean-Sham rats received RD pre- and post-Sham surgery. Behaviour: Recognition memory, motor coordination and muscular strength were assessed before surgery, as well as 3 and 6 weeks post-surgery using the novel object recognition, rotarod and grip strength tests, respectively. Body-composition (echoMRI): Fat vs. lean mass and free water (ascites) were also monitored. RESULTS: Before the surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on RD (Lean-BDL and Lean-Sham). 3 weeks after surgery, BW, fat mass, lean mass and free water were increased in Obese-BDL rats vs. Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham rats. 6 weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost BW, fat and Lean mass, while free water increased vs. Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats, compared to Lean-Sham rats. CONCLUSION: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Obesity also accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats (long-term memory), while others are exacerbated (motor coordination, forelimb strength). This new animal model of CLD and obesity suggests a synergistic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. Thus, obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting more susceptibility to poor neurological performance.
Mariana Macedo de Oliveira, Mélanie Tremblay, Christopher F. Rose.
The liver plays a major role in regulating ammonia levels in the blood . Therefore, in liver disease, the loss of hepatic function leads to hyperammonemia and consequently hepatic encephalopathy (HE) . Ammonia-lowering strategies remain the mainstay therapeutic strategy for HE . Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain . Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease . GS is expressed in liver, muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB . Methods: Using brain primary microvascular endothelial cells (ECs) and isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats, the presence of GS was assessed using rtPCR, western blot (brain used as positive control), immunohistochemistry (co-localized with endothelial cell marker caveolin-1) . GS activity was also assessed . In addition, to understand the effect of liver disease on GS, ECs were exposed to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) and sham-operated controls, as well as ammonia chloride (1mM) and oxidative stress (10μM of hydrogen peroxide) . Results: Immunohistochemically, GS was detected in ECs and in vessels of brain from naïve rats . Furthermore, ECs expressed GS mRNA, protein and activity . However, expression of GS was lower compared to brain lysate control samples (p<0 .05) . GS expression in CMV showed similar results to brain but GS activity was less (p<0 .05) . When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0 .05) . However, when exposed to plasma from BDL rats or oxidative stress, GS activity was significantly reduced when compared to controls (p<0 .01) . Conclusion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro . Interestingly, ammonia stimulates GS in endothelial cells, whereas oxidative stress inhibits GS activity . Furthermore, GS activity is decreased in the presence of plasma from cirrhotic rats, possibly due to the presence of systemic oxidative stress in BDL rats. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may increase the risk of HE . We anticipate that upregulating GS in ECs of the BBB could become a new therapeutic target for HE.
Constantine J. Karvellas, Jaime L. Speiser, Mélanie Tremblay, William M. Lee, Christopher F. Rose, U. S. Acute Liver Failure Study Group.
Acetaminophen (APAP)-induced acute liver failure (ALF) is associated with significant mortality due to intracranial hypertension (ICH), a result of cerebral edema (CE) and astrocyte swelling. Brain-type fatty acid-binding protein (FABP7) is a small (15 kDa) cytoplasmic protein abundantly expressed in astrocytes. The aim of this study was to determine whether serum FABP7 levels early (day 1) or late (days 3-5) level were associated with 21-day mortality and/or the presence of ICH/CE in APAP-ALF patients. Serum samples from 198 APAP-ALF patients (nested case-control study with 99 survivors and 99 non-survivors) were analyzed by ELISA methods and assessed with clinical data from the US Acute Liver Failure Study Group (ALFSG) Registry (1998-2014). APAP-ALF survivors had significantly lower serum FABP7 levels on admission (147.9 vs. 316.5 ng/ml, p = 0.0002) and late (87.3 vs. 286.2 ng/ml, p < 0.0001) compared with non-survivors. However, a significant association between 21-day mortality and increased serum FABP7 early [log FABP7 odds ratio (OR) 1.16, p = 0.32] and late (log FABP7 OR 1.34, p = 0.21) was not detected after adjusting for significant covariates (MELD, vasopressor use). Areas under the receiver-operating curve for early and late multivariable models were 0.760 and 0.892, respectively. In a second analysis, patients were grouped based on the presence (n = 46) or absence (n = 104) of ICH/CE. A significant difference in FABP7 levels between patients with or without ICH/CE at early (259.7 vs. 228.2 ng/ml, p = 0.61) and late (223.8 vs. 192.0 ng/ml, p = 0.19) time points was not identified. Serum FABP7 levels were significantly elevated at early and late time points in APAP-ALF non-survivors compared to survivors. However, significant differences in FABP7 levels by 21-day mortality were not ascertained after adjusting for significant covariates (reflecting severity of illness). Our study suggests that FABP7 may not discriminate between patients with or without intracranial complications.
Valentina Agostoni, G. Giacalone, Cristina R. Bosoi, Christopher F. Rose, Jean-Christophe Leroux.
Introduction: Liposome-supported peritoneal dialysis (LSPD) has recently emerged as a versatile biodetoxification platform for improving the peritoneal extraction of small ionizable molecules via the intraperitoneal instillation of transmembrane pH-gradient liposomes.1 In a proof-of-concept study, these scavenging vesicles efficiently trapped weak bases/acids (e.g. ammonia), and were subsequently withdrawn together with their toxic cargo at the end of the dialysis session.1 We investigated here the translational potential of LSPD for treating hyperammonemia-associated hepatic encephalopathy, a life-threatening condition arising from impaired liver functions. The initial steps towards the clinical development of this technology consisted in optimizing the manufacturing process, assessing efficacy and safety in appropriate animal models, investigating interferences with drugs commonly administered to liver impaired patients, and establishing the extraction fingerprint of LSPD fluids. Methods: LSPD fluids were prepared by an osmotic-shock based method, sequentially mixing liposome aqueous suspensions with a hypertonic citrate solution (acidic) and an isotonic xylitol-based solution (alkaline).2 Efficacy experiments were performed in a rat model of secondary biliary cirrhosis induced by bile duct ligation.2 After 4 consecutive 4-h dialysis sessions with LSPD or a control fluid, brain edema was measured by densitometry, and plasma and dialysate ammonia concentrations determined by enzymatic assay. The immunogenicity of LSPD fluids was assessed in a porcine model of complement activation-related pseudoallergy (CARPA).2 For the interaction study, nine drugs commonly taken by liver impaired patients (e.g. antibiotics, β-blockers, diuretics) were tested for their influence on the in vitro ammonia scavenging activity of LSPD fluids. The metabolites’ fingerprints of LSPD or conventional PD, prior and after dialysis in healthy rats, were established by mass spectrometry. Results: LSPD fluids were prepared via an innovative, osmotic shock-based method overcoming sterilization and long-term stability issues.2 The liposomal suspensions outperformed conventional PD fluids in lowering plasmatic ammonia levels, and attenuating brain edema in cirrhotic rats and did not trigger any hypersensitive reaction in pigs, a gold standard model of CARPA.2 In general, the in vitro ammonia uptake of LSPD fluids was barely affected by the presence of the selected drugs in the incubation medium. Only the β-blocker propranolol considerably lowered the liposome scavenging capability. Metabolomics studies revealed a similar extraction profile between conventional PD and LSPD, confirming that important endogenous metabolites other than ammonia were not preferentially removed by LSPD. Conclusion: LSPD appeared as a promising strategy for ammonia detoxification, a first valuable alternative to extracorporeal dialysis, when the latter is not available or too risky. Acknowledgements. This work was supported by Versantis AG, the Swiss National Science Foundation (31003A_124882), Swiss Commission for Technology and Innovation (17525.1 PFLS-LS), and the OPO Foundation. The authors thank Seroscience Ltd (Budapest, Hungary) for their involvement in the safety studies.
José I. Fortea, Alexander Zipprich, Carolina Fernandez-Mena, Marta Puerto, Cristina R. Bosoi, Jorge Almagro, Marcus Hollenbach, Juan Bañares, Belén Rodríguez-Sánchez, Emilia Cercenado, Marc-André Clément, Christopher F. Rose, Rafael Bañares, Javier Vaquero, Cristina Ripoll.
Recent studies suggest that heparins reduce liver fibrosis and the risk of decompensation of liver disease. Here, we evaluated the effects of enoxaparin in several experimental models of advanced cirrhosis. Cirrhosis was induced in male Sprague-Dawley (SD) rats by: i. Oral gavage with carbon tetrachloride (CCl4ORAL ), ii. Bile duct ligation (BDL), and iii. CCl4 inhalation (CCl4INH ). Rats received saline or enoxaparin s.c. (40 IU/Kg/d or 180 IU/Kg/d) following various protocols. Blood biochemical parameters, liver fibrosis, endothelium- and fibrosis-related genes, portal pressure, splenomegaly, bacterial translocation, systemic inflammation, and survival were evaluated. Endothelial dysfunction was assessed by in-situ bivascular liver perfusions. Enoxaparin did not ameliorate liver function, liver fibrosis, pro-fibrogenic gene expression, portal hypertension, splenomegaly, ascites development and infection, serum IL-6 levels or survival in rats with CCl4ORAL or BDL-induced cirrhosis. Contrarily, enoxaparin worsened portal pressure in BDL rats and decreased survival in CCl4ORAL rats. In CCl4INH rats, enoxaparin had no effects on hepatic endothelial dysfunction, except for correcting the hepatic arterial dysfunction when enoxaparin was started with the CCl4 exposure. In these rats, however, enoxaparin increased liver fibrosis and the absolute values of portal venous and sinusoidal resistance. Our results do not support a role of enoxaparin for improving liver fibrosis, portal hypertension or endothelial dysfunction in active disease at advanced stages of cirrhosis. These disease-related factors and the possibility of a limited therapeutic window should be considered in future studies evaluating the use of anticoagulants in cirrhosis. This article is protected by copyright. All rights reserved.
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsychiatric disorder, a major complication of chronic liver disease (CLD). Hyperammonemia is central in the pathogenesis of HE as ammonia easily crosses the blood brain barrier (BBB) causing toxicity. Glutamine synthetase (GS), an enzyme which removes ammonia, plays an important compensatory role during CLD and is known to be expressed in muscle and brain. However, its expression in endothelial cells (EC) of the BBB has never been explored. Methods: GS protein and activity was assessed in 1) rat brain microvascular EC (+/- ammonia exposure and conditioned media from rats with CLD) and 2) isolated cerebral microvessels (CMV) from naïve rats. Results: GS was co-localized with EC in brain of naïve rats. GS protein and activity was detected in CMV, with less activity compared to brain (p<0.05). In vitro, EC expressed GS protein with activity, but lower compared to brain (p<0.05). EC exposed to ammonia resulted in increased GS activity (p<0.05). However, ECs exposed to plasma from CLD rats had lower GS activity and protein expression compared to controls (p<0.05). Conclusion: We demonstrate for the first time the presence of GS in EC. Stimulated by ammonia, GS is however reduced with conditioned plasma from hyperammonemic CLD rats. This suggests other factors such as oxidative stress (present in CLD) could lead to GS inhibition. We speculate a downregulation of GS in the BBB during CLD leads to rapid entry of ammonia into the brain and HE. Therefore upregulating GS in the BBB could become a new therapeutic target for HE.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Christopher F. Rose.
BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: The 6-week bile-duct ligation (BDL) rats and sham-operated controls, were used. Obesity: To induce obesity, high-fat diet (HFD) was given for 3 weeks before BDL. Experimental groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and normal diet (ND) for 6 weeks post-BDL; 2. Lean-BDL rats received ND pre- and post-BDL; 3. Lean-Sham rats received ND pre- and post-sham surgery. Behaviour: Motor coordination, muscular strength, and recognition memory were assessed before surgery, as well as 3 and 6 weeks post-BDL or sham surgery using the RotaRod, grip-strength and object recognition tests, respectively. Body-composition (echoMRI): Fat vs lean mass, as well as free water were also monitored. RESULTS: Before the surgery, body weight and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on ND (Lean-BDL and Lean-Sham). Three weeks after surgery, body-weight, fat mass, lean mass and free water were increased in Obese-BDL rats vs Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs Lean-Sham rats. Six weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost body weight, fat and lean mass, and increased free water vs Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats vs Lean-Sham rats. SUMMARY: HFD induces obesity features in healthy non-cirrhotic rats, and such effects are maintained in cirrhotic-BDL rats. Obesity also worsens and accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats, such as long-term memory, motor coordination and forelimb strength deficits. This new animal model of CLD and obesity suggests a synergic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. CONCLUSION: Obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting that these patients might be more susceptible to neuronal dysfunction and poor neurological performance. Therefore, this model of CLD and obesity will provide important clues to the underlying mechanisms of HE associated with obesity-induced cirrhosis and provide new insights into novel therapeutic strategies.
Comparison of nutritional assessment tools in cirrhotic patients.
Annie Lamoussenerie, Cassandra Picinbono-Larose, Mélanie Tremblay, Thierry Cresson, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Vincent, Geneviève Huard, Christopher Rose, Chantal Bémeur.
Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (cirrhosis; CLD). Malnutrition may increase the risk of developing other complications including hepatic encephalopathy (HE). Malnutrition in cirrhosis may also affect patient’s functional status and health-related quality of life (HRQOL). Management strategies focussing on nutritional status in relation to complications of cirrhosis are an unmet clinical need. We hypothesize sub-optimal nutritional status in cirrhotic patients increases the risk of developing HE and decreases HRQOL. Hospitalized and outpatients (CHUM’s Liver Unit in Montreal, Canada) with liver cirrhosis of different etiologies and healthy controls were assessed for 1) Nutritional status (Subjective Global Assessment (SGA)); 2) HE (Clinical HE Staging Scale (CHESS)); 3) HRQOL (Short-Form-36 (SF-36) questionnaire). This on-going prospective study included 33 cirrhotic patients (58% men) of various etiologies (% : 30 alcohol, 33 virus, 27 NASH and 33 others), Child-Pugh (13A, 9B, 5C and 6N/A), mean age 55,7±12,9 as well as 13 healthy controls (46% men, mean age 49,4±14,9). SGA analysis revealed that 27% of cirrhotic patients were malnourished. Furthermore, cirrhotic malnourished patients show decreased HRQOL compared to well-nourished cirrhotic patients (p<0.01). Cirrhotic patients, when compared to controls, displayed a lower score in physical functioning (p=0.03) and general health (p=0.03). CHESS analysis revealed none of the cirrhotic patients had HE while 21% of them had an HE diagnosis in medical chart, suggesting CHESS would not be sensible enough to screen HE. Our preliminary results suggest that nutritional status does influence particular domains of HRQOL in cirrhotic patients irrespective of their etiology. Further patients are required to statistically confirm the impact of nutritional status on HE and HRQOL in cirrhotic patients. Identifying factors associated with nutritional status, HRQOL and HE in cirrhotic patients may help improve patient care and guide future research.
Mariana Oliveira, Mélanie Tremblay, Christopher Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsyc hiatric disorder, a major complication of chronic liver disease (CLD). Hyperammonemia is central in the pathogenesis of HE as ammonia easily crosses the blood brain barrier (BBB) causing toxicity. Glutamine synthetase (GS), an enzyme which removes ammonia, plays an important compensatory role during CLD and is known to be expressed in muscle and brain. However, its expression in endothelial cells (EC) of the BBB has never been explored. Methods: GS protein and activity was assessed in 1) rat brain microvascular EC (+/- ammonia exposure and plasma from rats with CLD) and 2) isolated cerebral microvessels (C MV) from naïve rats. Results: GS was co-localized with EC in brain of naïve rats. GS protein and activity was detected in CMV, with less activity compared to brain (p<0.05). In vitro, GS protein and activity was detected in EC, but with lower levels compared to brain (p<0.05). EC exposed to ammonia resulted in increased GS activity (p<0.05). However, ECs exposed to plasma fr om CLD rats resulted in lower GS activity and protein expression compared to controls (p<0.05). Conclusion: We demonstrate for the first time the presence of GS in EC in both in vitro and in vivo. Stimulated by ammonia, GS is however reduced following exposur e to plasma from hyperammonemic CLD rats. This suggests other systemic factors such as oxidative stress (present in CLD; Bosoi et al., Free Radic Biol Med, 2012) could negative affect GS activity. We speculate a downregulation of GS in the BBB during CLD leads to rapid entry of ammonia into t he brain and the development of HE. H ence, upregulating GS in the BBB could become a new therapeutic target for HE.
Nouveau modèle d’encéphalopathie hépartique épisodique due à la cirrhose.
Kim Phat Pham, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction : L'encéphalopathie hépatique (EH) est une complication neuropsychiatrique majeure de la maladie du foie chronique, telle que la cirrhose du foie. L’une de ses formes est l’EH épisodique dont les symptômes surgissent épisodiquement. Notre hypothèse était que les épisodes d’EH aggraveraient les dommages neurologiques de l’EH etaccentueraient ainsi les troubles neurologiques. Toutefois, il n’existe aucun modèle animal d’EH épisodique à ce jour. Nous avions pour but de caractériser un modèle d’EH épisodique due à la cirrhose et d’évaluer l’impact de multiples épisodes sur le disfonctionnement neurologique.Matériel et méthodes : Une cirrhose hépatique a été provoquée par la chirurgie du bile duct ligation (BDL) et a été combinée à des injections d’acétate d’ammonium afin d’induire l’EH épisodique dans le rat. Les niveaux d’ammoniaque ont été mesurés. Les mémoires à court et long terme ont été évaluées avec le Novel Object Recognition test ; l’anxiétéavec le Elevated Plus Maze test ; et la coordination motrice avec le RotaRod test. Résultats : Les déficits de mémoire à court terme et de coordination motrice chez les rats BDL avec épisodes d’EH n’étaient pas altérés comparés aux rats BDL sans épisodes. Toutefois, leur mémoire à long terme a été réduite, et leurs niveaux d’anxiété et d’ammoniaque plasmique ont été augmentés. Discussion et conclusion : Un effet synergique de la cirrhose, des épisodes d’EH et des niveaux élevés d’ammoniaquepourraient causer ces altérations. Ce nouveau modèle de l’EH épisodique permettra d’explorer son mécanisme pathologique et d’y tester des traitements. Projet subventionné par la COPSE et la CLF.
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Introduction: La malnutrition est un facteur pronostic important influençant l’issue clinique des patients souffrant de maladie hépatique chronique (MHC). La malnutrition accroît le risque de développer d'autres complications, incluant l'encéphalopathie hépatique (EH) et peut également affecter la qualité de vie (QV). Objectif: Comparer l’état nutritionnel des patients MHC aux sujets non-cirrhotiques (NC) et son impact sur l’EH et la QV. Méthodes: 33 patients MHC, étiologies et sévérité de maladie (Child-Pugh Score) diverses et 13 sujets NC ont été évalués pour: A) État nutritionnel (ESG); B) EH (CHESS); C) QV (SF-36). Résultats: Cette étude prospective en cours inclut 33 patients MHC (58% hommes) d’étiologies variées (%: 30 Alcool, 33 Virus, 27 NASH et 33 autres), Child-Pugh (13A, 9B, 5C et 6N/D), âgés de 55,7±12,9 ans et 13 sujets NC (46% hommes, 49,4±14,9 ans). L’ESG révèle que 27% des patients MHC souffrent de malnutrition et présentent un score moindre de fonctionnement physique (p = 0,03) et de santé générale (p=0,03), comparativement aux sujets NC. Aussi, les patients MHC malnutris présentent une diminution de la QV par rapport aux sujets MHC bien nourris (p<0,01). L'analyse de CHESS a révélé qu'aucun des patients MHC n'avaient d'EH alors que 21% d'entre eux avaient un diagnostic médical positif, ce qui suggère que le CHESS ne serait pas assez sensible pour dépister l'EH. Discussion : Ces résultats suggèrent que l'état nutritionnel influe sur la QV des sujets MHC sans égard à l’étiologie. D'autres patients sont nécessaires pour confirmer l'impact de l'état nutritionnel chez les patients MHC et ce, afin d’améliorer les soins et guider les recherches futures.
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Vincent, Geneviève Huard, Christopher Rose, Chantal Bémeur.
Problématique : La malnutrition est un facteur pronostic important influençant l’issue clinique des patients souffrant de maladie hépatique chronique (MHC). La malnutrition accroît le risque de développer d'autres complications, incluant l'encéphalopathie hépatique (EH) et peut également affecter la qualité de vie (QV). Les stratégies thérapeutiques axées sur l'état nutritionnel en relation avec les complications de la cirrhose représentent un besoin clinique urgent. Objectif : Comparer l’état nutritionnel des patients MHC aux contrôles sains et son impact sur l’EH et la QV. Méthodologie : 33 patients MHC d’étiologies et de sévérité de maladie (Child-Pugh Score) diverses et 13 contrôles sains ont été évalués pour : A) État nutritionnel (ESG); B) EH (CHESS); C) QV (SF-36). Résultats : Cette étude prospective en cours inclut 33 patients MHC (58% hommes) d’étiologies variées (% : 30 Alcool, 33 Virus, 27 NASH et 33 autres), Child-Pugh (13A, 9B, 5C et 6N/D), âgés de 55,7±12,9 ans et 13 témoins sains (46% hommes, 49,4±14,9 ans). L’ESG révèle que 27% des sujets MHC souffrent de malnutrition et présentent un score moindre de fonctionnement physique (p = 0,03) et de santé générale (p=0,03), comparativement aux contrôles sains. Aussi, les patients MHC malnutris présentent une diminution de la QV par rapport aux patients MHC bien nourris (p<0,01). Discussion : Les résultats préliminaires suggèrent que l'état nutritionnel influe sur la QV des sujets MHC sans égard à l’étiologie. D'autres patients sont nécessaires pour confirmer l'impact de l'état nutritionnel chez les patients MHC, afin d’améliorer les soins et guider les recherches futures.
Comparaison d’outils d’évaluation nutritionnelle lors de cirrhose.
Annie Lamoussenerie, Cassandra Picinbono-Larose, Mélanie Tremblay, Thierry Cresson, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Problématique : La malnutrition est un facteur pronostic important influençant l’issue clinique des patients souffrant de maladie hépatique chronique (MHC). La malnutrition accroît le risque de développer d'autres complications, incluant l'encéphalopathie hépatique (EH) et peut également affecter la qualité de vie (QV). Les stratégies thérapeutiques axées sur l'état nutritionnel en relation avec les complications de la cirrhose représentent un besoin clinique urgent. Objectif : Comparer l’état nutritionnel des patients MHC aux contrôles sains et son impact sur l’EH et la QV. Méthodologie : 33 patients MHC d’étiologies et de sévérité de maladie (Child-Pugh Score) diverses et 13 contrôles sains ont été évalués pour : A) État nutritionnel (ESG); B) EH (CHESS); C) QV (SF-36). Résultats : Cette étude prospective en cours inclut 33 patients MHC (58% hommes) d’étiologies variées (% : 30 Alcool, 33 Virus, 27 NASH et 33 autres), Child-Pugh (13A, 9B, 5C et 6N/D), âgés de 55,7±12,9 ans et 13 témoins sains (46% hommes, 49,4±14,9 ans). L’ESG révèle que 27% des sujets MHC souffrent de malnutrition et présentent un score moindre de fonctionnement physique (p = 0,03) et de santé générale (p=0,03), comparativement aux contrôles sains. Aussi, les patients MHC malnutris présentent une diminution de la QV par rapport aux patients MHC bien nourris (p<0,01). Discussion : Les résultats préliminaires suggèrent que l'état nutritionnel influe sur la QV des sujets MHC sans égard à l’étiologie. D'autres patients sont nécessaires pour confirmer l'impact de l'état nutritionnel chez les patients MHC, afin d’améliorer les soins et guider les recherches futures.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Marcos Ocana-Sanchez, Christopher F. Rose.
BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the impact of obesity on the development of neurological impairment in cirrhotic rats. M&M: Obesity: To induce obesity, high-fat diet (HFD) was given for 3 weeks before surgical intervention (bile-duct ligation (BDL) or Sham). Experimental groups: 1. Obese-BDL: rats received HFD for 3 weeks pre-BDL and normal diet (ND) for 6 weeks post-BDL; 2. Lean-BDL: rats received ND pre- and post-BDL; 3. Lean-Sham: rats received ND pre- and post-sham surgery. HE parameters: Motor coordination, muscular strength, and recognition memory were assessed pre-pre-, 3 and 6 weeks post-surgical intervention using RotaRod, grip- strength and object recognition test respectively. Body-composition (echoMRI): Fat, lean mass and free water were monitored. RESULTS: Three weeks HFD lead to an increase in fat mass in comparison to rats on ND (p<0.01). Three weeks after surgery, fat, and lean mass and free water were increased in Obese-BDL vs Lean-BDL rats (p<0.05). Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs Lean-Sham rats (p<0.05). Six weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost fat and lean mass, and increased free water vs Lean-Sham rats (p<0.5). Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL and Lean-Sham rats (p<0.01), whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats when compared to Lean-Sham rats (p<0.01). CONCLUSION: Obesity exacerbates and accelerates the accumulation of free water and motor coordination deficits in cirrhotic-BDL rats. Interestingly, long-term memory and forelimb strength deficits were impaired in Obese-BDL but not in Lean-BDL rats. This novel animal model of CLD and obesity suggests obesity accelerates CLD-induced neurological impairment. Therefore, this model of CLD and obesity will provide important clues to the underlying mechanisms of HE associated with obesity-induced cirrhosis and provide new insights into novel therapeutic strategies.
Glutamine synthetase in endothelial cells of the blood-brain barrier: less but not insignificant?
Christopher F. *Rose, Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Background: The liver plays a major role in regulating ammonia levels in the blood. Therefore, impaired hepatic function leads to hyperammonemia and consequently hepatic encephalopathy (HE). Ammonia easily crosses the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which during the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain but has never been thoroughly explored in the endothelial cells of the BBB. Methods: Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS activity and protein expression (brain lysate was used as positive control). GS was also evaluated by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, GS activity was assessed in ECs exposed to 1mM of ammonium chloride for 48h. Finally, GS activity and protein expression were evaluated in ECs exposed to plasma from 6-week bile-duct ligated (BDL) rats or sham-operated controls for 72h. Results: ECs expressed mRNA, protein and activity of GS. However, EC`s expression (normalized to g/protein) of GS was lower compared to brain lysate control samples (p<0.05). The quantity of GS protein in CMV was similar to that found in brain but the activity of GS was significantly less in CMV (p<0.05). Using immunohistochemistry, GS was detected in ECs and in CMV from naïve rats. When ECs were exposed to ammonia (1mM), an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS activity and protein expression were decreased when compared to sham-operated controls (p<0.05). Discussion: These results demonstrate for the first time that GS is present in ECs. Interestingly, ammonia stimulates GS activity in ECs, but is reduced following treatment with plasma from BDL rats. This suggests other factors such as oxidative stress and inflammation (present in BDL rats), could inhibit GS activity. We speculate that a downregulation of GS in the BBB allows for a faster entry of ammonia into the brain and therefore may play a significant role in the onset of HE. We anticipate upregulating GS in ECs of the BBB could become a new therapeutic target for HE
The bile duct ligated rat: A relevant model to study muscle mass loss in cirrhosis.
Cristina R. Bosoi, Mariana M. Oliveira, Rafael Ochoa-Sanchez, Mélanie Tremblay, Gabriella A. Ten Have, Nicolaas E. Deutz, Christopher F. Rose, Chantal Bémeur.
Muscle mass loss and hepatic encephalopathy (complex neuropsychiatric disorder) are serious complications of chronic liver disease (cirrhosis) which impact negatively on clinical outcome and quality of life and increase mortality. Liver disease leads to hyperammonemia and ammonia toxicity is believed to play a major role in the pathogenesis of hepatic encephalopathy. However, the effects of ammonia are not brain-specific and therefore may also affect other organs and tissues including muscle. The precise pathophysiological mechanisms underlying muscle wasting in chronic liver disease remains to be elucidated. In the present study, we characterized body composition as well as muscle protein synthesis in cirrhotic rats with hepatic encephalopathy using the 6-week bile duct ligation (BDL) model which recapitulates the main features of cirrhosis. Compared to sham-operated control animals, BDL rats display significant decreased gain in body weight, altered body composition, decreased gastrocnemius muscle mass and circumference as well as altered muscle morphology. Muscle protein synthesis was also significantly reduced in BDL rats compared to control animals. These findings demonstrate that the 6-week BDL experimental rat is a relevant model to study liver disease-induced muscle mass loss.
Ammonia toxicity: from head to toe?
Srinivasan Dasarathy, Rajeshwar P. Mookerjee, Veronika Rackayova, Vinita Rangroo Thrane, Balasubramaniyan Vairappan, Peter Ott, Christopher F. Rose.
Ammonia is diffused and transported across all plasma membranes. This entails that hyperammonemia leads to an increase in ammonia in all organs and tissues. It is known that the toxic ramifications of ammonia primarily touch the brain and cause neurological impairment. However, the deleterious effects of ammonia are not specific to the brain, as the direct effect of increased ammonia (change in pH, membrane potential, metabolism) can occur in any type of cell. Therefore, in the setting of chronic liver disease where multi-organ dysfunction is common, the role of ammonia, only as neurotoxin, is challenged. This review provides insights and evidence that increased ammonia can disturb many organ and cell types and hence lead to dysfunction.
U. S. Acute Liver Failure Study Group, Constantine J. Karvellas, Jaime L. Speiser, Mélanie Tremblay, William M. Lee, Christopher F. Rose, U. S. Acute Liver Failure Study Group.
Constantine Jason Karvellas, Jaime Lynn Speiser, Mélanie Tremblay, William M. Lee, Christopher F. Rose.
Background: Acetaminophen (APAP) - induced Acute Liver Failure (ALF) is associated with significant morbidity and mortality related to cerebral edema (CE) and intracranial hypertension (ICH). Brain type fatty acid binding protein (FABP7) is a small (15 kDa) cytoplasmic protein abundantly expressed in astrocytes where there is active fatty acid metabolism. FABP7 levels have not been previously reported in ALF patients at high risk of CE/ICH. Aims: One: To determine whether serum FABP7 early (day 1) or late (day 3-5) levels are associated with 21-day mortality in APAP-ALF patients in the absence of liver transplant. Two: To determine whether serum FABP7 levels at serial time points are associated with the presence of ICH/CE Methods: Nested case control study 198 APAP-ALF patients (99 survivors, 99 non-survivors) from the US Acute Liver Failure Study Group (ALFSG) registry. Patient samples were analyzed for FABP1 using solidphase enzyme-linked immunosorbent assay (ELISA) and assessed with clinical data. In a second analysis where data were available, patients were stratified based on the presence of ICH/CE (n=46) or absence (n=104) based on death summaries or clinical data (ICP monitor, computed tomography). Multivariable logistic regression was used to determine independent associations with 21-day mortality (n=198) and development of ICH/CE (n=150) Results: APAP-ALF survivors had significantly lower serum FABP7 levels on admission (147.9 vs. 316.5 ng/ml, P=0.0002) and late (87.3 vs. 286.2 ng/ml, P<0.0001) compared with nonsurvivors (Figure 1). However using multivariable logistic regression, a significant association between 21- day mortality and increased serum FABP7 early (Log FABP7 Odds Ratio (OR) 1.16, P=0.32) and late (Log FABP7 OR 1.34, P=0.21) was not detected after adjusting for significant covariates (MELD, vasopressor use). Area under the receiver operating curve for early and late multivariable models were 0.760 and 0.892 respectively. A significant difference in FABP7 levels between patients with or without ICH/CE at early (259.7 vs. 228.2 ng/ml, P=0.61) and late (223.8 vs. 192.0 ng/ml, P=0.19) time points was not identified. FABP7 was not significantly associated with the development of ICH/CE after adjusting for significant covariates (Early OR 1.0, p=0.65; Late OR 1.0, p=0.57 see Table 1). Summary: Brain type FABP (FABP7) levels were elevated in APAP-ALF patients with serum significantly higher serum levels at early and late time points in APAP-ALF non-survivors. However, significant differences in FABP7 levels by 21-day mortality were not ascertained after adjusting for significant covariates reflecting severity of illness (MELD, vasopressor dependence). FABP7 did not discriminate between the APAP-ALF patients with and without ICH/CE. Conclusions: This suggests that while all APAP-ALF patients have some evidence of astrocyte inflammation and increased permeability in the blood brain barrier, FABP7 appears to not discriminate between patients who did and did not have significant intracranial complications of APAP-ALF. Disclosure: The study was sponsored by NIH grant U-01 58369 (from NIDDK), TL1 001451 (from NCATS), and a grant from the University of Alberta Hospital Foundation (UHF).
Constantine Jason Karvellas, Jaime Lynn Speiser, Mélanie Tremblay, William M. Lee, Christopher F. Rose.
Background: Acetaminophen (APAP) - induced acute liver failure (ALF) is associated with significant mortality. To date, traditional prognostic scores lack discrimination in identifying patients with APAP-ALF who will die without liver transplant (LT), and those who will survive with medical management alone. Failure to identify those in need of LT results in a potentially preventable death while wrongly classifying prospective survivors as in need of LT subjects the patient to unnecessary LT. Liver-type fatty acid binding protein (FABP1) is a small (15 kDa) cytoplasmic protein abundantly expressed in hepatocytes. The prognostic value of serum FABP1 levels in APAP-ALF patients has not been reported. Aim: To determine whether serial samples (early; day 1 or late; day 3-5) of serum FABP1 levels are associated with 21-day mortality in the absence of LT. Methods: Nested case control study 198 APAP-ALF patients (99 survivors, 99 non-survivors) from the US Acute Liver Failure Study Group (ALFSG) Registry. Patient samples were analyzed for FABP1 using solid-phase enzyme-linked immunosorbent assay (ELISA) and assessed with clinical data. In addition, 51 healthy controls (University of Alberta) were included. Results: APAP-ALF survivors had significantly lower serum FABP1 levels on admission (238.6 vs. 690.8 ng/ml, P<0.0001) and late (148.4 vs. 612.3 ng/ml, P<0.0001) compared with non-survivors (Figure 1). Using multivariable logistic regression, increased serum FABP1 early (Log FABP1 Odds Ratio (OR) 1.31, P=0.03) and late (Log L-FABP OR 1.50, P=0.005) were associated with significantly increased 21-day mortality after adjusting for significant covariates (MELD, vasopressor use). Area under the receiver operating curve (AUROC) for early and late multivariable models were 0.778 and 0.907 respectively. The addition of FABP1 (Table 1) significantly improved the AUROC of the King’s College Criteria (KCC) (Early: 0.552 alone, 0.711 with L-FABP; Late: 0.604 alone, 0.797 with FABP1, respectively) as well as the ALFSG prognostic index (Early: 0.686 alone, 0.766 with FABP1; Late: 0.711 alone, 0.815 with FABP1, respectively). Summary: Serum levels of FABP1 in APAP-ALF patients were significantly associated with 21-day mortality measured at early and late time points after adjusting for significant covariates. FABP1 improved performance (AUROC) when combined with existing prognostic scores at serial time points. Conclusions: In patients with APAP-ALF, FABP1 showed good potential to discriminate survivors from non- survivors at multiple time-points and significantly improved models currently used in clinical practice. Validation of FABP1 as a clinical prediction tool in APAP-ALF merits further investigation. The study was sponsored by NIH grant U-01 58369 (from NIDDK) and a grant from the University of Alberta Hospital Foundation (UHF).
Effects of anaerobic exercise in muscle mass optimization in bile duct ligated rats.
Mariana Oliveira, Christopher Rose, Luise Aamann, Rafael Ochoa-Sanchez, Mariana M. Oliveira, Mélanie Tremblay, Chantal Bémeur, Gitte Dam, Hendrik Vilstrup, Niels Kristian Aagaard, Christopher F. Rose.
Background/Aims: Skeletal muscle abnormalities, such as sarcopenia and myosteatosis are frequent complications of cirrhosis and are associated with increased morbidity and mortality. Hyperammonemia plays a significant role in the pathogenesis of hepatic encephalopathy (HE). Skeletal muscle have a significant compensatory part in detoxifying ammonia during liver disease since it houses the enzyme glutamine synthetase, an important ammonia-removing pathway during the amination of glutamate to glutamine. Therefore, we aimed to investigate whether reduced quantity and quality of muscle is associated with hyperammonemia, HE and mortality in patients with cirrhosis. Methods: A total of 677 cirrhotic patients were evaluated. Computed tomography (CT) scans were used to analyze the skeletal muscle (transverse CT image at the level of the 3rd. lumbar vertebra (L3) was selected from each scan) and sarcopenia and myosteatosis was evaluated. Overt HE was assessed clinically and ammonia blood levels were performed at the time of the muscularity assessment. Results: Sarcopenia was noted in 292 patients (43%), and 352 patients had myosteatosis (52%). A total of 225 patients (33%) had history of overt HE and 221 (of 267) patients (83%) had hyperammonemia. The prevalence of overt HE was higher in patients with sarcopenia (42% vs. 27%, P<0.001), and myosteatosis (41% vs. 25%, P<0.001). By multivariable regression analysis, sarcopenia and myosteatosis were independently associated with higher risk of overt HE (HR 1.89, P=0.007, HR 1.68, P=0.03) and hyperammonemia (HR 1.71, P=0.006, HR 1.88, P=0.001, respectively). Median survival was worse in patients with overt HE and sarcopenia (18±4 vs. 42±7 months, P=0.01), hyperammonemia and sarcopenia (11±7 vs. 38±16 months, P<0.001), and myosteatosis and hyperammonemia (19±3 vs. 38±20 months, P=0.005) compared to patients without these factors. Conclusions: Cirrhotic patients with sarcopenia and myosteatosis have a higher risk of hyperammonemia and overt HE. Further, skeletal muscle abnormalities present concomitantly with HE and hyperammonemia increase the risk of mortality in these patients.
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Demers, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (cirrhosis; CLD). Malnutrition may increase the risk of developing other complications including hepatic encephalopathy (HE). Malnutrition in cirrhosis may also affect patient’s functional status and wellbeing or health-related quality of life (HRQOL). Management strategies focussing on nutritional status in relation to complications of cirrhosis are an unmet clinical need. We hypothesize sub-optimal nutritional status in cirrhotic patients increases the risk of developing HE and decreases HRQOL. Methods: 33 patients (outpatients and hospitalized; CHUM’s Liver Unit in Montreal, Canada) with liver cirrhosis of different etiologies and 13 healthy controls were assessed for 1) Nutritional status (Subjective Global Assessment (SGA)); 2) HE (Clinical HE Staging Scale (CHESS)); 3) HRQOL (Short-Form-36 (SF-36) questionnaire). Results: This on-going prospective study included 33 cirrhotic patients (58% men) of various etiologies (30% alcohol, 33 % virus, 27% NASH and 33% others), Child-Pugh A/B/C (13A, 9B, 5C and 6 N/A), mean age 55,7±12,9 as well as 13 healthy controls (46% men, mean age 49,4±14,9). SGA analysis revealed that 27% of cirrhotic patients were malnourished in which 22% of them malnourished showed signs of HE as assessed by CHESS. Cirrhotic patients, when compared to controls, displayed a lower score in physical functioning (p=0.03) and general health (p=0.03), both part of the physical health domain of SF-36. Furthermore, cirrhotic malnourished patients show decreased physical aspects of HRQOL compared to well-nourished cirrhotic patients (p<0.01). Conclusion: Our preliminary results suggest that nutritional status does influence particular domains of HRQOL in cirrhotic patients irrelevant of their etiology. Further patients are required to statistically confirm the impact of nutritional status on HE and HRQOL in cirrhotic patients. Identifying factors associated with nutritional status, HRQOL and HE in cirrhotic patients may help improve patient care and guide future research.
Rahima A. Bhanji, Andres Duarte-Rojo, Christopher Rose, Aldo J. Montano-Loza.
Background/Aims: Skeletal muscle abnormalities, such as sarcopenia and myosteatosis are frequent complications of cirrhosis and are associated with increased morbidity and mortality. Hyperammonemia plays a significant role in the pathogenesis of hepatic encephalopathy (HE). Skeletal muscle have a significant compensatory part in detoxifying ammonia during liver disease since it houses the enzyme glutamine synthetase, an important ammonia-removing pathway during the amination of glutamate to glutamine. Therefore, we aimed to investigate whether reduced quantity and quality of muscle is associated with hyperammonemia, HE and mortality in patients with cirrhosis. Methods: A total of 677 cirrhotic patients were evaluated. Computed tomography (CT) scans were used to analyze the skeletal muscle (transverse CT image at the level of the 3rd. lumbar vertebra (L3) was selected from each scan) and sarcopenia and myosteatosis was evaluated. Overt HE was assessed clinically and ammonia blood levels were performed at the time of the muscularity assessment. Results: Sarcopenia was noted in 292 patients (43%), and 352 patients had myosteatosis (52%). A total of 225 patients (33%) had history of overt HE and 221 (of 267) patients (83%) had hyperammonemia. The prevalence of overt HE was higher in patients with sarcopenia (42% vs. 27%, P<0.001), and myosteatosis (41% vs. 25%, P<0.001). By multivariable regression analysis, sarcopenia and myosteatosis were independently associated with higher risk of overt HE (HR 1.89, P=0.007, HR 1.68, P=0.03) and hyperammonemia (HR 1.71, P=0.006, HR 1.88, P=0.001, respectively). Median survival was worse in patients with overt HE and sarcopenia (18±4 vs. 42±7 months, P=0.01), hyperammonemia and sarcopenia (11±7 vs. 38±16 months, P<0.001), and myosteatosis and hyperammonemia (19±3 vs. 38±20 months, P=0.005) compared to patients without these factors. Conclusions: Cirrhotic patients with sarcopenia and myosteatosis have a higher risk of hyperammonemia and overt HE. Further, skeletal muscle abnormalities present concomitantly with HE and hyperammonemia increase the risk of mortality in these patients.
Marc-André Clément, Cristina R. Bosoi, Mariana Oliveira, Mélanie Tremblay, Gabriella A. Ten Have, Nicolaas E. Deutz, Christopher F. Rose.
Background and Aims: Chronic liver disease (CLD) induces numerous complications including muscle mass loss and hepatic encephalopathy (HE) which negatively impact clinical outcomes. Hyperammonemia is considered the central component in the pathogenesis of HE, however recent studies have suggested ammonia to be toxic to other organs/tissues aside the brain, such as the muscle. The aim of this study was to investigate the effect of lowering ammonia on muscle mass in cirrhotic rats treated with an oral formulation of ornithine phenylacetate (OP; OCR-002). Methods: Six-week bile-duct ligated (BDL) and sham rats were used. OP was administered orally by gavage (1g/kg) daily for 5 weeks starting 1 week after surgery. Locomotor activity (day/night) was assessed in infrared beam cages for 24 h. Body weight, fat and lean mass (EchoMRI) were measured. Stable isotope tracers were injected (ip) in order to assess fractional protein synthesis rate (FSR). Blood ammonia and cerebral edema was also evaluaeted. Results: BDL rats demonstrated a 4-fold increase in blood ammonia vs sham-operated controls. This increase was reduced by 40% in OP-treated BDL rats. BDL rats gained less body weight compared to sham-operated controls (body weight of 360.2g ± 13.6 vs 476.8g ± 10.38 p<0.001) which was accompanied with a lower gain of lean mass and a lower muscle FSR. OP-treated BDL rats showed a significant increase in body weight (p<0.001 vs BDL) with a significant higher lean mass (303.1g ± 10.7 in BDL+OP vs 264.4g ± 10.5 in BDL p<0.01). Fat mass remained unchanged between the treated and untreated BDL groups. OP treatment normalized brain water content in BDL rats. In contrast, OP-treatment reduced muscle FSR in SHAM animals, but not in BDL rats. Locomotor activity in BDL rats was reduced compared with sham-operated controls but no significant change was found between BDL+OP and SHAM+OP. Conclusions: This is the first study demonstrating the efficient ammonia-lowering effect of an oral formulation of OP. Long-term treatment with OP is a safe, effective, non-antibiotic alternative demonstrating a significant ammonia-lowering effect, as well as a protective effect on the development of brain edema and muscle mass loss in rats with CLD. Whether the beneficial effect of OP on muscle mass loss is a result of lowering blood ammonia or direct result of OP on muscle metabolism remains to be established.
Glutamine synthetase in endothelial cells of the blood-brain barrier: less but not insignificant ?
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Therefore, during liver disease, the loss of hepatic function leads to hyperammonemia and consequently increased brain ammonia and hepatic encephalopathy (HE). Reducing ammonia neurotoxicity through systemic ammonia-lowering strategies remains the mainstay therapeutic strategy for HE. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which during the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (astrocytes) but has never been thoroughly explored in the endothelial cells of the BBB. Methods: Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS activity and protein expression (brain lysate was used as positive control). GS was also evaluated by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, GS activity was assessed in ECs exposed to 1mM of ammonium chloride for 48h. Finally, GS activity and protein expression were evaluated in ECs submitted to plasma from 6-week bile-duct ligated (BDL) rats or sham-operated controls for 72h. Results: ECs expressed mRNA, protein and activity of GS. However, EC`s expression (normalized to g/protein) of GS was lower compared to brain lysate control samples (p<0.05). GS protein expression in CMV showed similar results to that of brain but GS activity was significantly less in CMV (p<0.05). Using immunohistochemistry, GS was detected in ECs and in CMV from naïve rats. When ECs were exposed to ammonia (1mM), an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS activity and protein expression were decreased when compared to sham-operated controls (p<0.05). Discussion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme in CMVs compared to that found in the brain, could reason why GS has never been reported in these cells. Interestingly, ammonia stimulates GS activity in ECs, but GS activity is decreased following treatment with plasma from BDL rats. This suggests other factors such as oxidative stress and inflammation, could inhibit GS activity. We speculate that a downregulation of GS in the BBB allows for a faster entry of ammonia into the brain and therefore may play a significant role in the onset of HE. We anticipate upregulating GS in ECs of the BBB could become a new therapeutic target for HE.
Nutritional status assessment in patients with chronic liver disease: a pilot study.
Annie Lamoussenerie, Cassandra Picinbono-Larose, Mélanie Tremblay, Catherine Demers, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is one of the most common complications in the increasing number of patients suffering from chronic liver disease (CLD) and is a leading cause of morbidity and mortality. Traditional tools used to evaluate nutritional status are not reliable in CLD due to limitations related to weight, which may be artificially increased by the presence of ascites, underestimating malnutrition. New strategies to assess nutritional status focusing on early malnutrition detection are an unmet clinical need. The aim of this pilot study is to describe the performance of different measures of nutrition including body mass index (BMI), handgrip strength (HGS), mid-arm muscle circumference (MAMC) in relation to subjective global assessment (SGA). Methods: In this ongoing prospective study, patients with and without CLD are recruited at Centre Hospitalier de l’Université de Montréal (St-Luc Hospital) in Canada. We assess nutritional status via: BMI, HGS as measured with a calibrated dynamometer, MAMC calculated with the mid-arm circumference and the tricipital skinfold and a modified version of the SGA for CLD patients, Spearman correlation coefficient is used to assess correlation between different tools. Results : We recruited 36 and 18 patients with and without CLD and 18 patients, respectively. Conclusions: Our preliminary results indicate that BMI and HGS, but not MAMC tend to decrease according to the nutritional status (no malnutrition, mild or moderate malnutrition) determined by SGA. Further patients are required to statistically identify a reliable tool and be able to evaluate nutritional status in cirrhosis as well as detect malnutrition in its early stage.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Marcos Ocana-Sanchez, Christopher F. Rose.
BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: The 6-week bile-duct ligation (BDL) rats, as well as sham-operated controls, were used. Obesity: To induce obesity, high-fat diet (HFD) was given for 3 weeks before BDL. Experimental groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and normal diet (ND) for 6 weeks post-BDL; 2. Lean-BDL rats received ND pre- and post-BDL; 3. Lean-Sham rats received ND pre- and post-sham surgery. Behaviour: Motor coordination, muscular strength, and recognition memory were assessed before surgery, as well as 3 and 6 weeks post-BDL or sham surgery using the RotaRod, grip-strength and object recognition tests, respectively. Body-composition (echoMRI): Fat vs lean mass, as well as free water (ascites, fluid retention), were also monitored. RESULTS: Before the surgery, body weight and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on ND (Lean-BDL and Lean-Sham). Three weeks after surgery, body-weight, fat mass, lean mass and free water were increased in Obese-BDL rats vs Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs Lean-Sham rats. Six weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost body weight, fat and lean mass, and increased free water vs Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats when compared to Lean-Sham rats. SUMMARY: HFD induces obesity features in healthy non-cirrhotic rats, and such effects are maintained in cirrhotic-BDL rats. Obesity also exacerbates and accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats, such as long-term memory, motor coordination and forelimb strength deficits. This new animal model of CLD and obesity suggests a synergic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. CONCLUSION: Obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting that these patients might be more susceptible to neuronal dysfunction and poor neurological performance. Therefore, this model of CLD and obesity will provide important clues to the underlying mechanisms of HE associated with obesity-induced cirrhosis and provide new insights into novel therapeutic strategies.
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
The liver plays a major role in regulating ammonia levels in the blood. Therefore, in liver disease the loss of hepatic function leads to hyperammonemia and increased brain ammonia and consequently hepatic encephalopathy (HE). Ammonia-lowering strategies remain the mainstay therapeutic strategy. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. Therefore, the aim is to evaluate GS expression in endothelial cells of the BBB. Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS expression by western blot using brain lysates as positive control and by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, to understand the effect of ammonia on GS, ECs were exposed to 1mM of ammonia chloride for 48h. Finally, ECs were subjected to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) or sham-operated controls. We have characterized this BDL model and found both systemic oxidative stress and inflammation, in addition to hyperammonemia. ECs expressed GS mRNA, protein and activity. However, expression of GS was lower compared to brain lysate control samples (p<0.05). GS expression in CMV showed similar results to brain but GS activity was less (p<0.05). Using immunohistochemistry, GS was detected in ECs and in vessels of brain from naïve rats. When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS was decreased when compared to sham-operated controls (p<0.01). These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme compared to that found in the brain, could explain why GS has never been reported in these cells. Interestingly, ammonia stimulates GS in endothelial cells, but its activity is decreased in the presence of other pathogenic factors in plasma from cirrhotic rats such as oxidative stress and inflammation. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may be implicated in the pathogenesis of HE. We anticipate increasing GS in ECs of the BBB could become a new therapeutic target for HE.
Constantine J. Karvellas, Jaime L. Speiser, Mélanie Tremblay, William M. Lee, Elaine M. Leslie, Christopher F. Rose.
The liver plays a major role in regulating ammonia levels in the blood. Therefore, in liver disease the loss of hepatic function leads to hyperammonemia and increased brain ammonia and consequently hepatic encephalopathy (HE). Ammonia-lowering strategies remain the mainstay therapeutic strategy. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. Therefore, the aim is to evaluate GS expression in endothelial cells of the BBB. Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS expression by western blot using brain lysates as positive control and by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, to understand the effect of ammonia on GS, ECs were exposed to 1mM of ammonia chloride for 48h. Finally, ECs were subjected to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) or sham-operated controls. We have characterized this BDL model and found both systemic oxidative stress and inflammation, in addition to hyperammonemia. ECs expressed GS mRNA, protein and activity. However, expression of GS was lower compared to brain lysate control samples (p<0.05). GS expression in CMV showed similar results to brain but GS activity was less (p<0.05). Using immunohistochemistry, GS was detected in ECs and in vessels of brain from naïve rats. When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS was decreased when compared to sham-operated controls (p<0.01). These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme compared to that found in the brain, could explain why GS has never been reported in these cells. Interestingly, ammonia stimulates GS in endothelial cells, but its activity is decreased in the presence of other pathogenic factors in plasma from cirrhotic rats such as oxidative stress and inflammation. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may be implicated in the pathogenesis of HE. We anticipate increasing GS in ECs of the BBB could become a new therapeutic target for HE.
Brain-FABP serum levels and clinical outcomes in acetaminophen-induced acute liver failure.
Constantine Karvellas, Jaime Speiser, Mélanie Tremblay, William Lee, Christopher Rose.
Learning Objectives: Acetaminophen (APAP) - induced Acute Liver Failure (ALF) is associated with significant mortality related to intracranial hypertension (ICH). Brain type fatty acid binding protein (B-FABP) is a small (15 kDa) cytoplasmic protein abundantly expressed in astrocytes where there is active fatty acid metabolism. B-FABP levels have not been previously reported in ALF patients at high risk of ICH. We aimed to determine whether serum B-FABP early (day 1) or late (day 3-5) levels were associated with 21-day mortality and/or the presence of ICH in APAP-ALF patients. Methods: Serum samples from 198 APAP-ALF patients (99 survivors, 99 non-survivors) were analyzed by ELIZA methods and assessed with clinical data from the US Acute Liver Failure Study Group (ALFSG) Registry (1998-2013). No patients received transplant in this analysis. Results: APAP-ALF survivors had significantly lower serum B-FABP levels on admission (147.9 vs. 316.5 ng/ml, P=0.0002) and late (87.3 vs. 286.2 ng/ml, P<0.0001) compared with non-survivors. However using multivariable logistic regression, a significant association between 21-day mortality and increased serum B-FABP early (Log B-FABP Odds Ratio (OR) 1.16, P=0.32) and late (Log L-FABP OR 1.34, P=0.21) was not detected after adjusting for significant covariates (MELD, vasopressor use). Area under the receiver operating curve for early and late multivariable models were 0.760 and 0.892 respectively. In a second analysis where data were available, patients were stratified based on the presence of ICH (n=46) or absence (n=104) based on death summaries or clinical data (ICP monitor, computed tomography). A significant difference in B-FABP levels between patients with or without ICH at early (259.7 vs. 228.2 ng/ml, P=0.61) and late (223.8 vs. 192.0 ng/ml, P=0.19) time points was not identified. Conclusions: Serum B-FABP levels were significantly higher in APAP-ALF non-survivors but not after adjusting for covariates. While elevated B-FABP levels likely reflect astrocyte inflammation, B-FABP did not differentiate clinically between APAP-ALF patients with and without ICH.
Constantine J. Karvellas, Jaime L. Speiser, Mélanie Tremblay, William M. Lee, Elaine M. Leslie, Christopher F. Rose.
Background: Acetaminophen (APAP)-induced acute liver failure (ALF) is associated with significant mortality. To date, prog- nostic scores lack discrimination in identifying patients who will die without liver transplant (LT), and those who will sur- vive with medical management. Liver-type fatty acid binding protein (L-FABP) is a 15 kDa cytoplasmic protein expressed in hepatocytes with potential prognostic value in APAP-ALF. Meth-ods: With serial serum samples (early; day 1 and late; day 3-5) from 198 APAP-ALF patients (99 survivors, 99 non-survi-vors), we examined whether L-FABP levels, measured using sol- id-phase enzyme-linked immunosorbent assay, were associated with 21-day mortality in the absence of LT. Results: APAP-ALF survivors had significantly lower L-FABP levels early (238.6 vs. 690.8 ng/ml, p<0.0001) and late (148.4 vs. 612.3 ng/ml, p<0.0001) compared with non-survivors (Fig 1). Using logistic regression, elevated L-FABP early (Log L-FABP Odds Ratio (OR) 1.31, p=0.027) and late (Log L-FABP OR 1.50, p=0.005) were associated with significantly increased 21-day mortality after adjusting for covariates (MELD, vasopressor use). Area under the receiver operating curve (AUROC) for early and late models were 0.778 and 0.907. The addition of L-FABP significantly improved (p<0.01) the AUROC of the King’s Col- lege Criteria (KCC) (Early: 0.552 alone, 0.711 with L-FABP; Late: 0.604 alone, 0.797 with L-FABP) as well as the ALFSG prognostic index (Early: 0.686 alone, 0.766 with L-FABP; Late: 0.711 alone, 0.815 with L-FABP). Conclusion: Serum L-FABP levels were significantly higher at serial time points in APAP-ALF non-survivors. After adjusting for covariates, serum L-FABP was significantly associated with 21-day mortality. L-FABP improved prognostic discrimination (AUROC) when combined with KCC and the ALFSG prognostic index which may potentially help identify APAP-ALF patients with higher potential of recovery.
Constantine J. Karvellas, Jaime L. Speiser, Mélanie Tremblay, William M. Lee, Gurnit Kaur, Elaine M. Leslie, Christopher Rose, US Acute Liver Failure Study Group.
Evaluation of nutritional status in patients with chronic liver disease: a pilot study.
Annie Lamoussenerie, Mélanie Tremblay, Thierry Cresson, An Tang, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Evaluation of nutritional status in patients with chronic liver disease: a pilot study.
Annie Lamoussenerie, Mélanie Tremblay, Thierry Cresson, An Tang, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is one of the most common complication in the increasing number of patients suffering from chronic liver disease (cirrhosis; CLD) and is a leading cause of morbidity and mortality. Traditional tools used to evaluate nutritional status are not reliable in chronic liver disease due to limitations related to weight, which may be artificially increased by the presence of ascites, underestimating malnutrition. New strategies to assess nutritional status focussing on early malnutrition detection are an unmet clinical need. The aim of this ongoing pilot study is to describe the performance of different measures of nutrition including handgrip strength (HGS), mid-arm circumference (MAC) and subjective global assessment (SGA) in corre- lation to skeletal muscle index (SMI), an objective measure of skeletal muscle mass, among cirrhotic patients. Methods: In this ongoing pro- spective study, patients with and without CLD are recruited at the Centre hospitalier de l’Université de Montréal (St-Luc Hospital) in Can- ada. We assess nutritional status via: HGS as measured in the dominant hand with a calibrated dynamometer, MAC, SGA and SMI, as measured by computed tomography scan at the level of the third lumbar vertebrae. We also assess recommended and achieved calorie and protein intake. Spearman correlation coefficient is used to assess correlation between different tools. Results: To date, we recruited 21 patients with and 6 patients without CLD. Preliminary results indicate that SMI tends to correlate with HGS in both groups. Our prelim- inary results also suggest that SMI varies according to sex and etiology of cirrhosis. Discussion: The impact of age and sex of SMI needs fur- ther assessment. Objective measures of nutrition assessment in cirrhotics have the potential to reduce the dependence on subjective measures and allow accurate risk assessment. This may in turn lead to a change in clinical practice toward ensuring nutritional optimization in this high-risk population and attenuate CLD-related complications.
Évaluation de l'état nutritionnel lors de cirrhose.
Annie Lamoussenerie, Mélanie Tremblay, Thierry Cresson, An Tang, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Mariana Oliveira, Cristina Bosoi, Marc-André Clément, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD, cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence on obesity affecting neurological function, we hypothesize that obesity increases the risk, severity and accelerates the progression of HE in non-alcoholic fatty liver disease (NAFLD)-related cirrhosis. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and obesity: The 6-week bile-duct ligation (BDL) rat is a surgical model in which obstruction of the bile duct leads to cirrhosis and HE. We induced obesity with a high-fat diet (HFD). Previously, HFD was given after BDL, but it was not well accepted, and the body weight did not increased. Now we are pre-feeding the rats with HFD to see if that is a better model. Thus, rats will be fed HFD for 3 weeks (pre-BDL) and 6 weeks post-BDL. Food consumption, weight gain, as well as lean vs fat mass will be monitored. Preliminary results: 3-week HFD increases body weight (12.1%) and fat (30.5 vs 44.1g) mass compared to rats fed normal diet. Food consumption was decreased (HFD 12g/day vs normal diet 27g/day), while the calorie intake was not affected. Discussion: obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting these patients might be more susceptible to neuronal loss and poor neurological performance. Thus, this animal model of CLD and obesity will give important clues about psychiatric diseases including, HE and how they might be treated. Funded: CIHR
Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.
Introduction: The liver plays a major role in regulating ammonia levels in the blood. During liver disease, loss of hepatic function leads to hyperammonemia and consequently neurotoxicity and hepatic encephalopathy. Ammonia (NH₄⁺ and NH₃) easily crosses cell membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which amidates glutamate to glutamine, removing ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and the brain (primarily in astrocytes) but ammonia metabolism in endothelial cells (EC) has never been thoroughly explored. Methods: Using primary rat brain microvascular endothelial cells, GS detection was performed by rtPCR, western blot, immunohistochemistry and activity assay . In addition we evaluated GS expression in cerebral microvessels (CMV) from frontal cortex of naive rats by immunohistochemistry and western blot. Results: ECs expressed GS mRNA, protein and activity, although less intense compared to muscle and liver (controls). In vivo results showed GS in vessels of brain slices (co-localized with caveolin ) and in CMV-enriched lysate. Discussion: We demonstrate the presence of GS in endothelial cells both in vivo and in vitro. We speculate GS in ECs could act as a first barrier against ammonia neurotoxicity and play a compensatory role in liver disease. However, the latter has yet to be investigated. We anticipate GS in EC’s has the potential to become a new therapeutic target for treating hyperammonemia in liver disease and other hyperammonemic syndromes. Project financed by CIHR MO financed by MITACS, Université de Montréal
Cristina R. Bosoi, Mariana Oliveira, Marc-André Clément, Mélanie Tremblay, Gabriella A. Ten Have, Nicolaas E. Deutz, Christopher F. *Rose.
Background and Aims: Chronic liver disease (CLD) induces numerous complications including muscle mass loss and hepatic encephalopathy (HE) which negatively impact the clinical outcome. Furthermore, muscle mass wasting and HE have been shown to lead to poor prognosis following liver transplantation. Hyperammonemia is considered the central component in the pathogenesis of HE, however recent studies have suggested ammonia to be toxic to other organs besides the brain, such as the muscle. The aim of this study was to investigate the effect of ammonia on muscle mass in rats treated with an oral formulation of ornithine phenylacetate (OP; OCR-002). Methods: Six-week bile-duct ligated (BDL) and sham rats were used. OP was administered orally by gavage (1g/kg) daily for 5 weeks starting 1 week after surgery. Locomotor activity (day/night) was assessed in infrared beam cages for 24 h. Body weight, fat and lean mass (EchoMRI) were measured, followed by i.p. injection of a stable isotopes tracers cocktail in order to asses fractional synthesis of protein (FSR). Samples for blood ammonia, cerebral edema and muscle FSR were collected. Results: BDL rats demonstrated a 4-fold increase in blood ammonia vs sham-operated controls. This increase was reduced by 40% in OP-treated BDL rats. BDL rats gained less body weight compared to sham-operated controls (body weight of 360.2g ± 13.6 vs 476.8g ± 10.38 p<0.001) which was accompanied with a lower gain of lean mass and a lower muscle FSR. OP-treated BDL rats showed a significant increase in body weight (429.6g ± 117.9 p<0.001 vs BDL) with a significant higher lean mass (303.1g ± 10.7 in BDL+OP vs 264.4g ± 10.5 in BDL p<0.01). Fat mass remained unchanged between the treated and untreated BDL groups. OP treatment normalized brain water content in BDL rats. In contrast, OP-treatment reduced muscle FSR in SHAM animals, but not in BDL rats. Locomotor activity in BDL rats was reduced compared with sham-operated controls but no significant change was found between BDL+OP and SHAM+OP. Conclusions: This is the first study demonstrating the efficient ammonia-lowering effect of an oral formulation of OP. Long-term treatment with OP is a safe, non-antibiotic alternative demonstrating a significant ammonia-lowering effect, as well as a protective effect on the development of brain edema and muscle mass loss in rats with CLD. Whether the effect of OP on muscle mass loss attenuation is a result of lowering blood ammonia or directly improves muscle metabolism remains to be established.
Évaluation de l'état nutritionnel lors de cirrhose.
Annie Lamoussenerie, Mélanie Tremblay, Thierry Cresson, An Tang, Catherine Vincent, Christopher F. Rose, Chantal Bémeur.
Cristina R. Bosoi, Mariana Oliveira, Marc-André Clément, Mélanie Tremblay, Gabrie Ten Have, Nicholaas Deutz, Christopher F. Rose.
Background: Chronic liver disease (CLD) induces numerous complications including muscle mass loss and hepatic encephalopathy (HE) which negatively impact the clinical outcome. Furthermore, muscle mass wasting and HE have been shown to lead to poor prognosis following liver transplantation. Hyperammonemia is considered the central component in the pathogenesis of HE, however recent studies have suggested ammonia to be toxic to other organs besides the brain, such as the muscle. Aims: The aim of this study was to investigate the effect of ammonia on muscle mass in rats treated with an oral formulation of ornithine phenylacetate (OP; OCR-002). Methods: Bile-duct ligated (BDL) rats were divided into 4 experimental groups; 1) Sham; 2) BDL; 3) Sham + OP; 4) BDL + OP. OP was administered orally by gavage (1g/kg) daily for 5 weeks starting 1 week after surgery. Two days before sacrifice, locomotor activity (day/night) was assessed in infrared beam cages for 24 h. The day of the sacrifice, body weight, fat and lean mass (EchoMRI) were measured, followed by i.p. injection of a stable isotopes tracers cocktail (Phe/Gly) in order to asses fractional synthesis of protein (FSR). At sacrifice, samples were collected to measure blood ammonia (commercial kit), cerebral edema (specific gravity method) and muscle FSR. Results : At 6-weeks, BDL rats demonstrated a 4-fold increase in blood ammonia vs Sham-operated controls. This increase was reduced by 40% in OP-treated BDL rats. Body weight decreased in BDL rats compared to sham-operated controls (360.2g±13.6 vs 476.8g±10.4; p<0.001) and significantly increased following OP-treatment (429.6g±117.9; p<0.001 vs BDL). This was due to a higher gain of lean mass in OP-treated BDL rats compared to BDL rats (303.1g±10.7 in BDL+OP vs 264.4g±10.5 in BDL, p<0.01). This was accompanied by increased muscle FSR in OP-treated BDL rats. Fat mass remained unchanged between treated and untreated BDL groups. OP treatment also normalized brain water content in BDL rats. Locomotor activity in BDL rats was reduced compared with sham-operated controls but no significant change was found between BDL+OP and SHAM+OP. Conclusions: This is the first study demonstrating the efficient ammonia-lowering effect of an oral formulation of OP. Moreover, OP long-term treatment is a safe, non-antibiotic alternative with protective effects on the development of cirrhosis complications such as HE and muscle mass loss in rats with CLD. Whether the effect of OP on muscle mass loss attenuation is a result of lowering blood ammonia or directly improves muscle metabolism remains to be established.
Aldo Montano-Loza, Andres Duarte-Rojo, Rahima Bhanji, Christopher F. Rose.
Background: Muscle mass functions as an alternative site of ammonia detoxification in patients with cirrhosis.Aims: In this study we aimed to investigate if sarcopenia, myosteatosis, and obesity are associated with hyperammonemia and hepatic encephalopathy (HE) in patients with cirrhosis. Methods: A total of 204 cirrhotic patients were studied. Muscularity assessment was analyzed using CT scans at the level of the 3rd lumbar vertebral body. Sarcopenia was defined using the skeletal muscle index and myosteatosis according to the muscle attenuation index. Overweight-obesity was defined as a body mass index ≥25 kg/m2. Sarcopenic-obesity was defined as concomitant sarcopenia and overweight-obesity. HE was evaluated clinically (West -Heaven criteria) and defined as absent in patients not using specific treatment (i.e. lactulose, rifaximin) and with no prior episodes of HE in the preceding year. Ammonia blood levels were also performed (nl. 0-35 μmol/L) at the time of the muscularity assessment. Results: Mean age was 56±1 years and 141 were males (69%). Sarcopenia was noted in 96 patients (47%), 137 had myosteatosis (67%), 136 were overweight-obese (67%), and 53 (28%) had sarcopenic-obesity. Patients with sarcopenia (87±6 vs. 61±4 μmol/L, P<0.001), and sarcopenic-obesity (95±9 vs. 40±3 μmol/L, P<0.001) had higher levels of ammonia (Figure 1). Levels of ammonia were not different among patients with myosteatosis (77±5 vs. 67±5 μmol/L, P=0.2), and overweight-obesity (76±5 vs. 67±5 μmol/L, P=0.2). Patients with sarcopenia (84 vs. 63%, P=0.001) and sarcopenic-obesity (93 vs. 65%, P<0.001) had higher frequency of hyperammonemia. Patients with myosteatosis had a trend (P=0.06), and overweight-obesity was not associated with hyperammonemia (P=0.1). Lastly, patients with sarcopenia (42 vs. 26%, P<0.001), and sarcopenic obesity (47 vs. 30%, P<0.001) had higher frequency of HE. Sarcopenia and sarcopenic-obesity increased the risk of hyperammonemia (OR 3.2, P=0.001, and OR 7.0, P<0.001). Also, sarcopenia increased the risk of HE (OR 2.0, P<0.001, and OR 2.1, P<0.001). Conclusions: Cirrhotic patients with sarcopenia and sarcopenic-obesity have higher ammonia levels and risk for HE. Muscle mass plays a protective role for hyperammonemia and therapeutic strategies to avoid muscle depletion might decrease the risk of HE in cirrhosis. Funding Agencies:This study has been funded with a Clinical Research Award from the American College of Gastroenterology Institute 2011
Estudio del efecto de la enoxaparina sobre la cirrosis e hipertensión portal experimental.
Jose Ignacio Fortea, Alexander Zipprich, Carolina Mena Fernandez, Christopher F. Rose, Juan Bañares, Marta Puerto, Cristina R. Bosoi, Jorge Almagro, Marcus Hollenbach, Marc-André Clément, Javier Vaquero, Rafael Bañares, Cristina Ripoll.
INTRODUCCIÓN Y OBJETIVOS: Recientes estudios clínicos y experimentales sugieren que la administración de anticoagulantes podría reducir la fibrosis hepática y prevenir el desarrollo de complicaciones de la cirrosis hepática. Nuestro OBJETIVO fue evaluar los efectos de la enoxaparina sobre la fibrosis y la hemodinámica hepática en varios modelos experimentales de cirrosis. MÉTODOS: Utilizamos tres protocolos de cirrosis experimental en ratas Sprague- Dawley macho: 1) CCl4 oral; 2) CCl4 inhalado; 3) Ligadura del colédoco (LC). Los grupos (n=8-15/grupo) recibieron tratamiento diario con suero salino o con diferentes protocolos de enoxaparina sc: a) 40 U/Kg de peso corporal desde la 8a semana de administración de CCl4, b) 180 U/Kg desde la 1a u 8a semana de CCl4, y c) 180 U/Kg desde la 2a semana tras la LC. Se realizaron las siguientes mediciones: 1) presión portal (vena ileocólica), 2) fibrosis hepática (tinción de rojo Sirio y expresión de los genes Col1a1, Acta2 y Timp1 en hígado), 3) infección de líquido ascítico (cultivo en tioglicolato), 4) respuesta inflamatoria sistémica (niveles circulantes de IL-6), y 5) disfunción endotelial (estudios de perfusión hepática ex-vivo en ratas con cirrosis inducida por inhalación de CCl4). RESULTADOS: En comparación con los grupos control, las ratas con cirrosis inducida por la administración orogástrica de CCl4 mostraron una tendencia a una menor supervivencia y menor ganancia de peso corporal, las cuales fueron agravadas por el tratamiento con enoxaparina a dosis de 180 UI/kg (p<0,01). La administración de CCl4 oral también resultó en la alteración de diversos parámetros analíticos (elevación de INR, AST, ALT, bilirrubina y disminución de albúmina, glucosa y plaquetas), la cual no fue prevenida por el tratamiento con enoxaparina. La administración de enoxaparina no atenuó el aumento de presión portal en las ratas con cirrosis inducida por la administración oral de CCl4 o por la LC respecto a sus correspondientes controles (p<0,001). El desarrollo de fibrosis, la infección del líquido ascítico y los niveles circulantes de IL-6 tampoco fueron influidos por la enoxaparina en ninguno de los modelos. No se observaron efectos de la enoxaparina sobre la reactividad vascular hepática salvo en aquellas ratas que recibieron enoxaparina a dosis de 180 UI/Kg desde el inicio de la administración de CCl4, las cuales presentaron valores más elevados de resistencia venosa hepática con la exposición a dosis crecientes de acetilcolina y de S-nitroso acetilpenicilamina (SNAP, ambos p<0,05), y un incremento de la resistencia sinusoidal tras la adición de SNAP (p<0,05). CONCLUSIONES: La administración crónica de enoxaparina no mejoró la fibrosis hepática, la hipertensión portal o la disfunción endotelial en diversos modelos experimentales de cirrosis hepática avanzada en rata.
Marc-André Clément, Cristina Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by liver disease characterized by cognitive and motor dysfunction. Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following liver transplantation (LT). However, persisting neurological complications remain a common problem affecting as many as 47% of LT recipients. LT is a major surgical procedure accompanied by intraoperative stress, including blood loss and hypotension. Aim : We hypothesize, in the setting of minimal HE (MHE), the compromised brain becomes susceptible to hypotensive insults, resulting in cell injury and death. Methods: Six-week bile-duct ligated (BDL) rats with MHE and respective controls (SHAM) were used. Blood is withdrawn from the femoral artery (inducing hypovolemia) until a mean arterial pressure of 30, 60 and 90 mmHg (hypotension) and maintained for 120 minutes. Cerebral blood flow (BCF) was assessed by injecting fluorescent microspheres through the brachial artery. Upon sacrifice, brains were extracted for apoptotic analysis (western blot) and neuronal cell count (immunohistochemistry). In a separate group, BDL rats were treated for MHE with ornithine phenylacetate (OP; OCR-002), administered orally (1g/kg) for 3 weeks. Results: Both BDL rats and SHAM-operated controls without hypotension did not display any cell injury or neuronal loss. However, BDL rats following hypotension (30 and 60mmHg) demonstrated a significant decrease in neuronal cell count in the frontal cortex (using NeuN+DAPI and Cresyl Violet) compared to hypotensive SHAM-operated controls. In addition, neuronal loss was associated with an increased in cleaved caspase-3, suggesting apoptotic cell death. CBF decreased in BDL rats compared to SHAM and correlated with degree of hypotension insult. BDL rats treated with OP resulted in a decrease in blood ammonia and improvement in behaviour and did not lead to neuronal cell death following hypotension. Discussion: These findings strongly suggest that cirrhotic patients with MHE are more susceptible to hypotension-induced neuronal cell loss. Moreover, these results suggest a patient with HE (even MHE), with a “frail brain”, will fare worse during liver transplantation and consequently result in poor neurological outcome. Combination of MHE and hypotension may account for the persisting neurological complications observed in a number of cirrhotic patients following LT. Therefore, MHE, should not to be ignored and merits to be treated in order to reduce the risk of neurological complications occurring post-LT.
Liposome-supported peritoneal dialysis for the treatment of hepatic encephalopathy.
Valentina Agostoni, Soo Hyeon Lee, Vincent Forster, Meriam Kabbaj, Cristina Bosoi, Mélanie Tremblay, Matthias Zadory, Christopher F. Rose, Jean-Christophe Leroux.
Hyperammonemia can lead to cerebral dysfunction, encephalopathy, coma, and death if not treated adequately. The poor prognosis associated with this condition reflects the unmet medical need for effective ammonia-lowering treatments. Here, the translational potential of liposome-supported peritoneal dialysis (LSPD), a recently-developed detoxification strategy for the removal of small ionizable molecules like ammonia, is described. Dialysis fluids supplemented with micrometer-sized, transmembrane pH-gradient liposomes are prepared via an innovative, osmotic shock-based method overcoming sterilization and long-term stability issues. LSPD is able to sequester ammonia in healthy rats in relation to the injected dose, buffering capacity of the liposomal core, and membrane composition. In a rat model of cirrhosis, LSPD outperforms conventional peritoneal dialysis in lowering plasmatic ammonia levels and attenuating brain edema. LSPD does not trigger any hypersensitive reaction in pigs, a side effect commonly observed upon the injection of colloids in this animal model and in humans. These findings support the development of LSPD for the treatment of hyperammonemia-induced encephalopathy.
Brain edema: a valid endpoint for measuring hepatic encephalopathy?
Chantal Bémeur, Cristina Cudalbu, Gitte Dam, Alexander S. Thrane, Arthur J. L. Cooper, Christopher F. Rose.
Hepatic encephalopathy (HE) is a major complication of liver failure/disease which frequently develops during the progression of end-stage liver disease. This metabolic neuropsychiatric syndrome involves a spectrum of symptoms, including cognition impairment, attention deficits and motor dysfunction which eventually can progress to coma and death. Pathologically, HE is characterized by swelling of the astrocytes which consequently leads to brain edema, a common feature found in patients with acute liver failure (ALF) as well as in cirrhotic patients suffering from HE. The pathogenic factors involved in the onset of astrocyte swelling and brain edema in HE are unresolved. However, the role of astrocyte swelling/brain edema in the development of HE remains ambiguous and therefore measuring brain edema as an endpoint to evaluate HE is questioned. The following review will determine the effect of astrocyte swelling and brain edema on neurological function, discuss the various possible techniques to measure brain edema and lastly to propose a number of neurobehavioral tests to evaluate HE.
Glycine and hyperammonemia: potential target for the treatment of hepatic encephalopathy.
Rune Gangsøy Kristiansen, Christopher F. Rose, Lars Marius Ytrebø.
Hepatic encephalopathy (HE) is a neuropsychiatric disorder caused by hepatic dysfunction. Numerous studies dictate that ammonia plays an important role in the pathogenesis of HE, and hyperammonemia can lead to alterations in amino acid homeostasis. Glutamine and glycine are both ammoniagenic amino acids that are increased in liver failure. Modulating the levels of glutamine and glycine has shown to reduce ammonia concentration in hyperammonemia. Ornithine Phenylacetate (OP) has consistently been shown to reduce arterial ammonia levels in liver failure by modulating glutamine levels. In addition to this, OP has also been found to modulate glycine concentration providing an additional ammonia removing effect. Data support that glycine also serves an important role in N-methyl D-aspartate (NMDA) receptor mediated neurotransmission in HE. This potential important role for glycine in the pathogenesis of HE merits further investigations.
Geir I. Nedredal, Kjetil Elvevold, Marcio F. Chedid, Lars M. Ytrebø, Christopher F. Rose, Sambit Sen, Bård Smedsrød, Rajiv Jalan, Arthur Revhaug.
Background. Pulmonary complications are common in acute liver failure (ALF). The role of the lungs in the uptake of harmful soluble endogenous macromolecules was evaluated in a porcine model of ALF induced by hepatic devascularization (n = 8) vs. controls (n = 8). In additional experiments, pulmonary uptake was investigated in healthy pigs. Fluorochrome-labeled modified albumin (MA) was applied to investigate the cellular uptake.As compared to controls, the ALF group displayed a 4-fold net increased lung uptake of hyaluronan, and 5-fold net increased uptake of both tissue plasminogen activator and lysosomal enzymes. Anatomical distribution experiments in healthy animals revealed that radiolabeled MA uptake (taken up by the same receptor as hyaluronan) was 53% by the liver, and 24% by the lungs. The lung uptake of LPS was 14% whereas 60% remained in the blood. Both fluorescence and electron microscopy revealed initial uptake of MA by pulmonary endothelial cells (PECs) with later translocation to pulmonary intravascular macrophages (PIMs). Moreover, the presence of PIMs was evident 10 min after injection. Systemic inflammatory markers such as leukopenia and increased serum TNF-α levels were evident after 20 min in the MA and LPS groups.Significant lung uptake of harmful soluble macromolecules compensated for the defect liver scavenger function in the ALF-group. Infusion of MA induced increased TNF-α serum levels and leukopenia, similar to the effect of the known inflammatory mediator LPS. These observations suggest a potential mechanism that may contribute to lung damage secondary to liver disease.
Mathematical models and hepatology; oil and vinegar?
Lorette Noiret, Christopher F. Rose.
Leucine et exercices : bénéfique lors d'encéphalopathie expérimentale.
Corine Fontaine, Marc-André Clément, Cristina R. Bosoi, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Introduction : L’encéphalopathie hépatique (EH) est une complication neuropsychiatrique sérieuse de la maladie hépatique chronique (cirrhose). La pathogénèse de l’EH serait attribuable, entre autres à l’ammoniac. L’accumulation de cette neurotoxine jouerait un rôle clé. De plus, la malnutrition est associée à un risque élevé de développer une perte sévère de masse musculaire et à l’EH; ces complications augmentent le risque de mortalité. La déficience en leucine, a été démontrée lors de cirrhose. La leucine sert de substrat énergétique et de précurseur pour d’autres acides aminés en plus de stimuler la synthèse protéique. De plus, l’expression de mammalian target of rapamycin (mTOR) et sa cible p70S6 kinase, deux protéines impliquées dans de nombreuses réactions en lien avec la survie cellulaire, serait altérée dans le muscle lors de maladie hépatique chronique. Une masse musculaire optimale lors d’EH contribuerait à réduire l’ammoniac via l’enzyme glutamine synthase (GS). L’hypothèse de recherche est que l’optimisation de la masse musculaire permet de prévenir/atténuer les épisodes d’EH. Matériel et méthode: Un modèle deligature des voies biliaires (BDL) chez le rat qui récapitule les caractéristiques de la cirrhose et de l’EH est utilisé. Cinq groupes sont évalués: 1) Contrôle avec simulation de la chirurgie (Sham); 2) BDL; 3) BDL+ Leucine; 4) BDL + Exercices; 5) BDL + Leucine + Exercices. Six semaines post-chirurgie, l’EH est vérifiée par des tests comportementaux et phénotypage neurologique. La masse musculaire est évaluée par imagerie par résonance magnétique. Les rats sont ensuite sacrifiés et les muscles sont prélevés. L’expression protéique de mTOR et de p70S6 kinase est mesurée par immunobuvardage. Résultats: Chez le groupe BDL, on remarque une baisse de la masse musculaire et de la synthèse protéique comparativement au groupe Sham. La supplémentation en leucine et l’exercice favorise une augmentation de la masse musculaire chez les rats BDL. La voie de signalisation via mTOR semble moins exprimée dans le muscle du groupe BDL versus Sham. Conclusion: Dans le but d’optimiser le statut nutritionnel et d’améliorer la qualité de vie des patients cirrhotiques atteints d’EH, des recherches plus approfondies devront être effectuées.
Neuroinflammation et activation microgliale chez des rats cirrhotiques soumis à une hypotension.
Maxime Hovington, Marc-André Clément, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Introduction: L’encéphalopathie hépatique est une complication neuropsychiatrique causée par les maladies du foie. Ce syndrome devrait être réglé par la transplantation hépatique, mais d es complications neurologiques persistent chez près de 47% des p atients. La transplantation est accompagnée de stress intraopératoires dont l’hypovolémie et l’hypotension. Nous avons démontré qu’un cerveau fragilisé par l’encéphalopathie hépatique minimale (EHM) chez un rat cirrhotique devient plus susceptible à des dommages cérébraux causés par une hypotension, ce qui résulte en un stress cellulaire ainsi que la mort cellulaire neuronale. Ces signaux de stress cellulaire peuvent induire l’activation de la microglie, les cellules immunitaires du système nerveux central. Matériel et méthodes : Une hypotension par hypovolémie de l’artère fémorale a été effectuée pour atteindre une pression artérielle de 30 et 60 mmHg durant 2 heures chez des rats après 6 semaines suite à une cirrhose induite par ligation de la voie biliaire (BDL) et leu rs contrôles respectifs (SHAM). Nous avons évalué la présence de microglie activée dans le cortex frontal en immunobuvardage, avec CD11b, OX-42, CMH-II et COX-2 et en immunofluorescence, avec Iba-1. Résultats et discussion:Il n’y a pas d’activation microgliale chez les modèles SHAM et BDL dans le cortex préfrontal, mais l’insulte hypotensive induite chez le modèle cirrhotiqu e BDL, avec EHM , cause une activation de la microglie. Conclusion: L’activation microgliale chez les patients cirrhotiques soumis à une hypotension donne des outils supplémentaires dans la compréhension des processus pathophysiologiques délétères subséquents à une transplantation hépatique et permettra de suggérer des interventions cliniques afin de prévenir une aggravation des dommages cérébraux. Remerciements auCOPSE pour la bourse du Département de nutrition
Minimal hepatic encephalopathy leads to hypotension-induced neuronal cell loss in BDL rats.
Marc-André Clément, Cristina Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by liver disease characterized by cognitive and motor dysfunction. The only curative treatment to date remains liver transplantation (LT). Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following LT. However, persisting neurological complications remain a common problem affecting as many as 47% of LT recipients. LT is a major surgical procedure accompanied by intraoperative stress and confounding factors, including blood loss and hypotension. We hypothesize, in the setting of minimal HE (MHE), the compromised brain becomes susceptible to hypotensive insults, resulting in cell injury and death. Methods: Six-week bile-duct ligated (BDL) rats with MHE and respective controls (SHAM) were used. Blood is withdrawn from the femoral artery (inducing hypovolemia) until an mean arterial pressure of 30 and 60 mmHg (hypotension) and maintained for 120 minutes. Cerebral blood flow (BCF) was assessed by injecting fluorescent microspheres (1x106 microspheres/ml) through the brachial artery. Upon sacrifice, brains were extracted for apoptotic analysis (western blot) and neuronal cell count (immunohistochemistry). In a separate group, BDL rats were treated for MHE with ornithine phenylacetate (OP; OCR-002) (1g/kg) for 3 weeks. Results: Both BDL rats and SHAM-operated controls without hypotension did not display any cell injury or neuronal loss. However, BDL rats following hypotension (30 and 60mmHg) demonstrated a significant decrease in neuronal cell count in the frontal cortex (using NeuN+DAPI and Cresyl Violet) compared to hypotensive SHAM-operated controls. In addition, neuronal loss was associated with an increased in cellular stress protein, hsp32, hsp70 and caspase-3, suggesting apoptotic cell death. CBF decreased in BDL rats compared to SHAM and correlated with degree of hypotension insult. BDL rats treated with OP did not lead to neuronal cell death following hypotension. Discussion: These findings strongly suggest that cirrhotic patients with MHE are more susceptible to hypotension-induced neuronal cell loss. Moreover, these results suggest a patient with HE (even MHE), with a “frail brain”, will fare worse during liver transplantation and consequently result in poor neurological outcome. Combination of MHE and hypotension may account for the persisting neurological complications observed in a number of cirrhotic patients following LT. Therefore, MHE, i) should not to be ignored and ii) deserves to be treated in order to reduce the risk of neurological complications occurring post-LT.
Cristina R. Bosoi, Mariana Oliveira, Marc-André Clément, Mélanie Tremblay, Gabrie Ten Have, Nicolaas E. P. Deutz, Christopher F. Rose.
Background: Chronic liver disease (cirrhosis; CLD) is characterized by numerous metabolic disturbances which lead to complications that impact the clinical outcome. Among these, loss of muscle, characterized by a deterioration of muscle quantity and quality, leads to a decrease in functional capacity, adversely affecting survival, quality of life and outcome following liver transplantation. Hyperammonemia is central in the development of hepatic encephalopathy, a major complication of cirrhosis. However, it is speculated the toxic effect of ammonia extends beyond the brain, possibly affecting muscle. Therefore, we hypothesized that lowering blood ammonia will attenuate muscle mass loss in cirrhotic rats. Ornithine phenylacetate (OP; OCR-002) was used to lower blood ammonia. Methods: We induced CLD in rats following 6-week bile-duct ligation (BDL). Four experimental groups were tested; 1) Sham; 2) BDL; 3) Sham + OP; 4) BDL + OP. One week following BDL, rats were orally administered (gavage) OP (1g/kg) daily for 5 weeks. Body weight, fat and lean mass (EchoMRI), blood ammonia, cerebral edema (specific gravity method), fractional synthesis of protein (FSR) in muscle (with D2O) and locomotor activity (day/night) were measured. Results: At the end of the 6-weeks experiment, BDL rats demonstrated a 4-fold increase in blood ammonia vs Sham-operated controls. This increase was reduced by 40% in OP-treated BDL rats. BDL rats gained less body weight compared to sham-operated controls (body weight of 360.2g 13.6 vs 476.8g 10.38; p<0.001) which was accompanied with a lower gain of lean mass and a lower muscle FSR. OP-treated BDL rats showed a significant increase in body weight (429.6g 117.9; p<0.001 vs BDL) with a significant higher lean mass (303.1g 10.7 in BDL+OP vs 264.4g 10.5 in BDL, p<0.01). Fat mass remained unchanged between the treated and untreated BDL groups. OP treatment normalized brain water content in BDL rats. In contrast, OP-treatment reduced muscle FSR in SHAM animals, but not in BDL rats. Locomotor activity in BDL rats was reduced compared with sham-operated controls but no significant change was found between BDL+OP and SHAM+OP. Conclusion: This is the first study demonstrating the efficient ammonia-lowering effect of an oral formulation of OP. Long-term treatment with OP is a safe, non-antibiotic alternative demonstrating a significant ammonia-lowering effect, as well as a protective effect on the development of brain edema and muscle mass loss in rats with CLD. Whether the effect of OP on muscle mass loss attenuation is a result of lowering blood ammonia or directly improves muscle metabolism remains to be established.
Aldo J. Montano-Loza, Andres Duarte-Rojo, Rahima A. Bhanji, Christopher F. Rose.
Background/Aims: Muscle mass functions as an alternative site of ammonia detoxification in patients with cirrhosis. In this study we aimed to investigate if sarcopenia, myosteatosis, and obesity are associated with hyperammonemia and hepatic encephalopathy (HE) in patients with cirrhosis. Methods: A total of 204 cirrhotic patients were studied. Muscularity assessment was analyzed using CT scans at the level of the 3rd lumbar vertebral body. Sarcopenia was defined using the skeletal muscle index and myosteatosis according to the muscle attenuation index. Overweight-obesity was defined as a body mass index ≥25 kg/m2. Sarcopenic-obesity was defined as concomitant sarcopenia and overweight-obesity. HE was evaluated clinically (West-Heaven criteria) and defined as absent in patients not using specific treatment (i.e. lactulose, rifaximin) and with no prior episodes of HE in the preceding year. Ammonia blood levels were also performed (nl. 0-35 mmol/L) at the time of the muscularity assessment. Results: Mean age was 56±1 years and 141 were males (69%). Cirrhosis was caused by HCV in 78 patients (38%), alcohol in 56 (28%), NASH in 34 (16%), and other etiology in 36 patients (18%). Sarcopenia was noted in 96 patients (47%), 137 had myosteatosis (67%), 136 were overweight-obese (67%), and 53 (28%) had sarcopenic-obesity. Patients with sarcopenia (87±6 vs. 61±4 mmol/L, P<0.001), and sarcopenic-obesity (95±9 vs. 40±3 mmol/L, P<0.001) had higher levels of ammonia. Levels of ammonia were not different among patients with myosteatosis (77±5 vs. 67±5 mmol/L, P=0.2), and overweight-obesity (76±5 vs. 67±5 mmol/L, P=0.2). Patients with sarcopenia (84 vs. 63%, P=0.001) and sarcopenic-obesity (93 vs. 65%, P<0.001) had higher frequency of hyperammonemia. Patients with myosteatosis had a trend (77 vs. 64%, P=0.06), and overweight-obesity was not associated with hyperammonemia (77 vs. 66%, P=0.1). Lastly, patients with sarcopenia (42 vs. 26%, P<0.001), and sarcopenic-obesity (47 vs. 30%, P<0.001) had higher frequency of HE. Sarcopenia and sarcopenic-obesity increased the risk of hyperammonemia (OR 3.2, 95% CI 1.66.2, P=0.001, and OR 7.0, 95% CI 2.4-21, P<0.001, respectively). Also, sarcopenia and sarcopenic-obesity increased the risk of HE (OR 2.0, 95% CI 1.5-2.8, P<0.001, and OR 2.1, 95% CI 1.4-3.0, P<0.001, respectively). Conclusions: Cirrhotic patients with sarcopenia and sarcopenic-obesity have higher ammonia levels and risk for HE. Muscle mass plays a protective role for hyperammonemia and therapeutic strategies to avoid muscle depletion might decrease the risk of HE in cirrhosis.
Jose Ignacio Fortea, Alexander Zipprich, Carolina-Mena Fernandez, Christopher F. Rose, Juan Bañares, Marta Puerto, Cristina R. Bosoi, Jorge Almagro, Marcus Hollenbach, Marc-André Clément, Javier Vaquero, Rafael Bañares, Cristina Ripoll.
Recent clinical and experimental studies suggest that the administration of low molecular weight heparin may reduce the risk of decompensation of liver disease and liver fibrosis. Our aim was to evaluate the effects of enoxaparin on liver fibrosis and hemodynamics in several experimental models of cirrhosis. Methods: Cirrhosis was induced in male SD rats using 3 protocols: 1) Oral gavage with carbon tetrachloride (CCl4) twice a week for 12 weeks, 2) Inhalation of CCl4 twice a week for 12 weeks, and 3) Bile duct ligation (BDL) surgery. Rats (n= 8-15/group) underwent daily sc treatment with saline or diverse protocols of enoxaparin: 40 U/kg bw from 8th week of CCl4, 180 U/kg bw from 1st week or 8th week of CCl4, and 180 U/kg from 2nd week of BDL. Control groups of rats gavaged with water or sham-operated rats followed the same protocols. Portal pressure was measured in the ileocolic vein, liver fibrosis was assessed in Sirius Red and Masson’s Trichrome stained liver sections. Ascitic fluid was evaluated for bacterial growth in thioglycolate medium. Ex-vivo liver perfusion experiments were performed for assessing endothelium-dependent and -independent reactivity. Results: Compared with controls, rats with oral CCl4 gavage tended to show decreased survival and body weight gain, both of which were further worsened by enoxaparin 180 U/kg bw (p< 0.01). Rats with CCL4-induced cirrhosis showed altered laboratory parameters (increased INR, AST, ALT, bilirubin / decreased albumin, total proteins, glucose and platelets) regardless of enoxaparin treatment. In all experimental models, cirrhotic rats receiving saline and those receiving enoxaparin showed similar increases in the area of liver fibrosis compared with controls (p< 0.001). Rats with cirrhosis induced by oral CCl4 gavage and by BDL surgery developed increases of portal pressure and spleen-to-bw ratios compared with control rats (p< 0.001), regardless of enoxaparin treatment. Among rats with ascites, a similar proportion presented positive bacterial cultures (CCl4+saline 2 of 7 vs. CCl4+enoxaparin 4 of 8, NS; BDL+saline 1 of 6 vs. BDL+enoxaparin 2 of 7, NS). Potential effects of enoxaparin on hepatic vascular reactivity were only observed in rats receiving enoxaparin 180 U/kg bw from the beginning of CCl4 administration, and consisted of increased portal venous resistance after addition of acetylcholine or S-nitroso acetyl-penicillamine (SNAP, both p< 0.05) and increased sinusoidal resistance after addition of SNAP (p< 0.05). Conclusion: Our experimental data do not support a role of long-term treatment with enoxaparin for improving liver fibrosis, portal hypertension or endothelialdysfunction in cirrhosis.
Impact d'un stress inflammatoire et nutritionnel dans un modèle cellulaire d'acidose lactique.
Rafaela Almeida, Mélanie Tremblay, Christopher Rose, Consortium de l'acidose lactique, Chantal Bémeur.
Problématique : L’acidose lactique est une maladie mitochondriale rare causée par la mutation du gène LRPPRC (leucine-rich pentatricopeptide repeat motif containing). Elle est caractérisée principalement par une déficience tissu-spécifique (cerveau et foie) en cytochrome c oxydase, 4e complexe de la chaîne respiratoire ainsi que par des crises fatales d'acidose lactique. Il a été rapporté que ces crises, qui entraînent la mort de 80% des enfants atteints avant l’âge de 4 ans, sont déclenchées, entre autres, par une infection/inflammation et une surcharge nutritionnelle. À ce jour, aucun traitement n’existe pour prévenir ces crises. Question de recherche/objectif/hypothèse : Vérifier si la mutation rend les fibroblastes plus susceptibles au stress inflammatoire et nutritionnel. Méthodologie : Évaluer la viabilité des fibroblastes de peau de sujets contrôles (EBS) et de patients (AL), par microscopie à fluorescence, sous stress inflammatoire (TNF-α;100 ng/ml) et nutritionnel (palmitate; 0,5 mM) pour 24h. Les marqueurs Hoechst, caspase-3 et iodure de propidium ont été utilisés pour détecter les cellules viables, apoptotiques et nécrotiques, respectivement. Résultats : Nos résultats préliminaires démontrent qu’en conditions de stress, les taux de mort cellulaire par apoptose et nécrose semblent plus élevés dans les fibroblastes AL que les EBS. Discussion/conclusion/impact potentiel pour la nutrition : La mutation semble rendre les fibroblastes de patients atteints d’acidose lactique plus susceptibles au stress inflammatoire et nutritionnel. À plus long terme, l’appréciation du bienfait potentiel des acides gras oméga 3 dans ce processus pourrait s’avérer intéressante. Financement du projet / étudiant: Le Grand défi Pierre Lavoie.
Aldo J. Montano-Loza, Andres Duarte-Rojo, Rahima A. Bhanji, Christopher F. Rose.
Introduction: Muscle mass functions as an alternative site of ammonia detoxification in patients with cirrhosis. In this study we aimed to investigate if sarcopenia, myosteatosis, and obesity are associated with hyperammonemia and hepatic encephalopathy (HE) in patients with cirrhosis. Methods: A total of 204 cirrhotic patients were studied. Muscularity assessment was analyzed using CT scans at the level of the 3rd lumbar vertebral body. Sarcopenia was defined using the skeletal muscle index and myosteatosis according to the muscle attenuation index. Overweight-obesity was defined as a body mass index ≥25 kg/m2. Sarcopenic-obesity was defined as concomitant sarcopenia and overweight-obesity. HE was evaluated clinically (West-Heaven criteria) and defined as absent in patients not using specific treatment (i.e. lactulose, rifaximin) and with no prior episodes of HE in the preceding year. Ammonia blood levels were also performed (nl. 0-35 μmol/L) at the time of the muscularity assessment. Results: Mean age was 56±1 years and 141 were males (69%). Sarcopenia was noted in 96 patients (47%), 137 had myosteatosis (67%), 136 were overweight-obese (67%), and 53 (28%) had sarcopenic-obesity. Patients with sarcopenia (87±6 vs. 61±4 μmol/L, P < 0.001), and sarcopenic-obesity (95±9 vs. 40±3 μmol/L, P < 0.001) had higher levels of ammonia. Levels of ammonia were not different among patients with myosteatosis (77±5 vs. 67±5 μmol/L, P=0.2), and overweight-obesity (76±5 vs. 67±5 μmol/L, P=0.2). Patients with sarcopenia (84 vs. 63%, P=0.001) and sarcopenic-obesity (93 vs. 65%, P < 0.001) had higher frequency of hyperammonemia. Patients with myosteatosis had a trend (P=0.06), and overweight-obesity was not associated with hyperammonemia (P=0.1). Lastly, patients with sarcopenia (42 vs. 26%, P < 0.001), and sarcopenic obesity (47 vs. 30%, P < 0.001) had higher frequency of HE. Sarcopenia and sarcopenic-obesity increased the risk of hyperammonemia (OR 3.2, P=0.001, and OR 7.0, P < 0.001). Also, sarcopenia and sarcopenic-obesity increased the risk of HE (OR 2.0, P < 0.001, and OR 2.1, P < 0.001). Conclusion: Cirrhotic patients with sarcopenia and sarcopenic-obesity have higher ammonia levels and risk for HE. Muscle mass plays a protective role for hyperammonemia and therapeutic strategies to avoid muscle depletion might decrease the risk of HE in cirrhosis.
Liposome-Supported Peritoneal Dialysis in the Treatment of Severe Hyperammonemia.
Valentina Agostoni, Vincent Forster, Cristina Bosoi, Mélanie Tremblay, Christopher Rose, Jean-Christophe Leroux.
Purpose: Hyperammonemia (HA) is a clinical condition characterized by toxic increases in systemic ammonia levels predominantly resulting from inherited or acquired impairments in hepatic detoxification (e.g. cirrhosis). If not treated, HA can lead to irreversible brain damages and death in severe cases. The poor prognosis associated to this condition reflects the still unmet clinical need for a safe, fast, and efficient ammonia-removal therapy. Hemodialysis, the current standard of care for treating severe HA, suffers indeed some major drawbacks as it is technically difficult to implement, not readily available in hospitals and highly risky for some patients (e.g., newborns and patients suffering from hemodynamic instability). Peritoneal dialysis (PD) may represent a valuable therapeutic alternative as it is a less invasive procedure since it uses the peritoneum as a semipermeable filter and the peritoneal cavity as a dialysis chamber. However, its clinical application is limited by its lower clearance capacity compared with hemodialysis. To address this problem, we report herein a novel dialysis fluid based on transmembrane pH-gradient liposomes (acidic core) that efficiently scavenge the excess of ammonia from the body. The ammonia-removal efficacy of this system was assessed in a rat model of cirrhosis induced by bile duct ligation. The cirrhotic rats develop hyperammonemia, and consequently brain edema. Methods: Liposome-supplemented peritoneal dialysis (LSPD) fluids (350 mOsm/L, pH 6.5), were prepared by sequentially mixing sterile liposomes aqueous suspensions, obtained by film hydration method, with a hypertonic acidic citrate buffer (pH 2) and an isotonic xylitol-based basic solution (pH 12.4). The first step allowed the encapsulation of an acidic medium in the vesicles core by means of an osmotic shock; the second established the transmembrane pH-gradient. Dose-response studies were first performed in healthy Sprague-Dawley rats by injecting LSPD fluids (60 mL/kg) with increasing liposome concentrations (4, 8 and 16 mM) in the peritoneal space, and evaluating their ammonia extraction efficiency over 4 h of dialysis. Efficacy studies were then performed in a rat model of secondary biliary cirrhosis induced by bile duct ligation. Control normal rats (SHAM) underwent a sham operation. Five weeks after the surgery, two different groups of cirrhotic rats (LSPD and PD) received 4 consecutive PDs performed every other day with LSPD or a conventional glucose-based PD solution (injected dose = 50 mL/kg). One group (positive control) did not receive any treatment. At the end of the study, the brain edema was assessed by measuring the brain water content in the frontal cortex by densitometry method. The ammonia concentration in dialysate and plasma samples was determined by enzymatic method using a commercial kit. Results: LSPD was shown to efficiently scavenge ammonia in the peritoneal space of healthy rats in a dose-dependent manner. The extracted ammonia levels progressively increased over time reaching a concentration of 0.48 ± 0.05 mM, 1.25 ± 0.2 mM and 1.72 ± 0.24 mM in fluids supplemented with liposomes 4, 8 and 16 mM, respectively. In light of these results, the LSPD fluid 8 mM, which exhibited a good ammonia uptake, was tested in a cirrhotic rat model. Compared to the control PD solution, the liposomal formulation showed an enhanced ammonia extraction capability. At the end of each dialysis session, the ammonia concentrations in the liposome-supplemented dialysate exceeded those obtained with the control solution by 7-fold. Importantly, this improved ammonia clearance led to a significant attenuation of the brain edema. Indeed after four consecutive LSPDs, the brain water content of cirrhotic rats was significantly decreased and reverted to normal levels. On the contrary, no significant reduction was observed with the control solution. Plasma ammonia levels were also found to significantly decrease after treatment with LSPD fluid but not with the control PD solution. Conclusion: These findings demonstrate that LSPD is a promising strategy to improve the clearance capacity of PD towards ammonia, and pave the way for a less invasive, safe and efficient approach for treating severe HA. Financial support from the Swiss Commission for Technology and Innovation (17525.1 PFLS-LS) is acknowledged.
Rafaela Almeida, Mélanie Tremblay, Christopher Rose, Consortium de l'acidose lactique, Chantal Bémeur.
L’acidose lactique est une maladie mitochondriale rare causée par la mutation du gène LRPPRC (leucine-rich pentatricopeptide repeat motif containing). Elle est caractérisée principalement par une déficience tissu-spécifique (cerveau et foie) en cytochrome c oxydase, 4e complexe de la chaîne respiratoire ainsi que par des crises fatales d'acidose lactique. Il a été rapporté que ces crises, qui entraînent la mort de 80% des enfants atteints avant l’âge de 4 ans, sont déclenchées, entre autres, par une infection/inflammation et une surcharge nutritionnelle. À ce jour, aucun traitement n’existe pour prévenir ces crises. Dans ce contexte, nous étudions l'impact d'un stress inflammatoire et nutritionnel en utilisant des fibroblastes de peau de sujets contrôles (EBS) et de patients atteints d’acidose lactique (AL). L’objectif général du projet est de vérifier si la mutation du gène LRPPRC rend les fibroblastes plus susceptibles dans des conditions de stress inflammatoire et nutritionnel. À cet effet, la viabilité des fibroblastes est évaluée par microscopie à fluorescence dans les conditions de stress inflammatoire et nutritionnel telles que « tumor necrosis factor alpha » (TNF-α) à 100ng/ml et palmitate à 1mM, et ce, à différentes périodes d’incubation. Les marqueurs Hoechst, caspase-3 et iodure de propidium ont été utilisés pour détecter les cellules viables, apoptotiques et nécrotiques, respectivement. Nos résultats préliminaires démontrent qu’en conditions de stress, les taux de mort cellulaire par apoptose et nécrose semblent plus élevés dans les fibroblastes AL que les EBS. La mutation semble rendre les fibroblastes de patients atteints d’acidose lactique plus susceptibles au stress inflammatoire et nutritionnel. À plus long terme, l’appréciation du bienfait potentiel des acides gras oméga 3 dans ce processus pourrait s’avérer intéressante.
Marc-André Clément, Cristina R. Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Introduction L’encéphalopathie hépatique (EH) est une complication neuropsychiatrique causée par les maladies du foie. Ce syndrome est considéré comme un désordre métabolique réversible, qui devrait être réglé par la transplantation hépatique (TH), cependant les complications neurologiques persistent chez près de 47% des patients. La TH est une procédure accompagnée de stress intra-opératoires dont l’hypovolémie et l’hypotension. Nous supposons que le cerveau fragilisé devient plus susceptible à une hypotension, résultant en un stress ainsi que la mort cellulaire. Méthodologies Une hypotension par hypovolémie de l’artère fémorale est effectuée pour atteindre une pression artérielle de 30mmHg durant 2 heures chez des rats de 6 semaines avec ligation de la voie biliaire (BDL) et leurs contrôles respectifs (SHAM). Les cerveaux sont prélevés pour immunobuvardage et immunohistochimie. Résultats Les rats BDL avec hypotension ont démontré une diminution du compte neuronal dans le cortex préfrontal en utilisant NeuN+DAPI et le Crésyl violet, comparativement aux contrôles SHAM hypotendus. De plus, la protéine de stress cellulaire HSP32 est augmentée chez les groupes BDL hypotendus, tout comme la caspase-3 clivée, suggérant une mort cellulaire par apoptose. Les groupes contrôles sans hypotension ne démontrent aucune perte neuronale avec les marqueurs précédents. Discussion Ces résultats démontrent que les patients atteints d’EH sont plus susceptibles à une insulte hypotensive induisant une mort neuronale, et peuvent expliquer pourquoi certains patients ayant reçu une TH expérimentent des complications neurologiques.
Impact d'un stress inflammatoire et nutritionnel dans un modèle cellulaire d'acidose lactique.
Rafaela Almeida, Mélanie Tremblay, Christopher Rose, Consortium de l'acidose lactique, Chantal Bémeur.
Problématique : L’acidose lactique est une maladie mitochondriale rare causée par la mutation du gène LRPPRC (leucine-rich pentatricopeptide repeat motif containing). Elle est caractérisée principalement par une déficience tissu-spécifique (cerveau et foie) en cytochrome c oxydase, 4e complexe de la chaîne respiratoire ainsi que par des crises fatales d'acidose lactique. Il a été rapporté que ces crises, qui entraînent la mort de 80% des enfants atteints avant l’âge de 4 ans, sont déclenchées, entre autres, par une infection/inflammation et une surcharge nutritionnelle. À ce jour, aucun traitement n’existe pour prévenir ces crises. Question de recherche/objectif/hypothèse : Vérifier si la mutation rend les fibroblastes plus susceptibles au stress inflammatoire et nutritionnel. Méthodologie : Évaluer la viabilité des fibroblastes de peau de sujets contrôles (EBS) et de patients (AL), par microscopie à fluorescence, sous stress inflammatoire (TNF-α;100 ng/ml) et nutritionnel (palmitate; 0,5 mM) pour 24h. Les marqueurs Hoechst, caspase-3 et iodure de propidium ont été utilisés pour détecter les cellules viables, apoptotiques et nécrotiques, respectivement. Résultats : Nos résultats préliminaires démontrent qu’en conditions de stress, les taux de mort cellulaire par apoptose et nécrose semblent plus élevés dans les fibroblastes AL que les EBS. Discussion/conclusion/impact potentiel pour la nutrition : La mutation semble rendre les fibroblastes de patients atteints d’acidose lactique plus susceptibles au stress inflammatoire et nutritionnel. À plus long terme, l’appréciation du bienfait potentiel des acides gras oméga 3 dans ce processus pourrait s’avérer intéressante. Financement du projet / étudiant: Le Grand défi Pierre Lavoie.
Marc-André Clément, Cristina R. Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by liver disease characterized by cognitive and motor dysfunction. The only curative treatment to date remains liver transplantation (LT). Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following LT. However, even following the implantation of a new liver, persisting neurological complications remain a common problem affecting as many as 47% (8 47%) of liver transplant recipients. LT is a major surgical procedure accompanied by intraoperative stress and confounding factors, including blood loss (hypovolumia) and hypotension. We hypothesize, in the setting of MHE, that the compromised brain becomes predisposed to what would normally be an innocuous hypotensive insult, resulting in cell injury and death. Methods: Six-week bile-duct ligated (BDL) rats with MHE and respective controls will be used. Blood is withdrawn from the femoral artery (inducing hypovolemia) until an arterial pressure of 30 mmHg (hypotension) and maintained for 150 minutes. Upon sacrifice, brains are perfused and extracted for apoptotic analysis (western blot) and neuronal cell count (immunohistochemistry). Results: Both BDL rats and SHAM-operated controls without hypotension do not display any neuronal loss. However, BDL rats following hypotension demonstrated a significant decrease in neuronal cell count in the frontal cortex using NeuN+DAPI and Cresyl Violet compared to hypotensive SHAM-operated controls. In addition, neuronal loss was associated with an increased in cellular stress protein, hsp32, hsp70 and caspase-3, suggesting apoptotic cell death. Discussion: These findings suggest that patients with MHE are more susceptible to hypotension-induced neuronal cell loss. Moreover, these results suggest a patient with HE (even MHE), with a “frail brain”, will fare worse during LT and consequently result in poor neurological outcome. The combination of MHE and hypotension may justify for the persisting neurological complications observed in a number of cirrhotic patients following LT. This implies the impact of MHE on outcome is undervalued. MHE should not to be ignored and patients with MHE merit to be treated pre-LT.
Sarcopenia and Myosteatosis Increase the Risk of Hepatic Encephalopathy in Cirrhotic Patients.
Aldo J. Montano-Loza, Andres Duarte-Rojo, Christopher F. Rose.
Background: Sarcopenia is one of the most common complications of cirrhosis and it is associated with increased mortality. Muscle depletion is generally characterized by both a reduction in muscle size and increased proportion of inter- and intra-muscular fat denominated ldquomyosteatosisrdquo. Skeletal muscle may serve as an alternative site of ammonia detoxification in patients with cirrhosis. Aims: In this study we aimed to investigate if sarcopenia and myosteatosis are associated with overt hepatic encephalopathy in patients with cirrhosis. Methods: A total of 678 cirrhotic patients undergoing assessment for liver transplantation were studied. Sarcopenia and myosteatosis (characterized as low muscle attenuation) were analyzed using computed tomography (CT) scans at the level of the 3rd lumbar vertebral body. The area of paraspinal skeletal muscle (L3 SMI) at this location, and the muscle attenuation index were calculated. Hepatic encephalopathy was assessed clinically by applying the West-Heaven criteria (grade 0-IV). Results: Of the 678 patients, 457 patients were males (67%). Cirrhosis was caused by HCV in 256 patients (38%), alcohol in 152 (22%), NASH/cryptogenic in 171 (25%), autoimmune liver disease in 53 (8%), HBV in 41 (6%), other etiology in five patients (1%); and 292 patients had concomitant HCC (43%). Sarcopenia was noted in 291 patients (43%), and 353 patients had myosteatosis (52%). A total of 216 patients (32%) had history of hepatic encephalopathy (162 grade I-II, 54 grade III-IV). The prevalence of hepatic encephalopathy was significantly higher in patients with sarcopenia (40 vs. 26%, P<0.001), and myosteatosis (39 vs. 24%). By multivariate regression analysis (adjusted to age, gender, and MELD score), both sarcopenia (OR 1.68 [95% CI 1.04 to 2.40]; P=0.03), and myosteatosis (OR 1.97 [95% CI 1.32 to 2.99]; P=0.001) were significantly associated with hepatic encephalopathy. Conclusions: Cirrhotic patients with sarcopenia and myosteatosis have a higher risk of overt hepatic encephalopathy. Skeletal muscle seems to play a protective role in the pathogenesis of hepatic encephalopathy in cirrhosis, and therapeutic strategies to improve the muscle mass and quality may improve hepatic encephalopathy in cirrhosis. Funding Agency: American College of Gastroenterology.
Muscle mass optimization prevents experimental hepatic encephalopathy.
Sara Ghezzal, Marc-André Clément, Cristina R. Bosoi, Roxanne Beauchamp, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (cirrhosis; CLD). Malnutrition exacerbates severe muscle loss and hepatic encephalopathy (HE) in cirrhotic patients. New management strategies focussing on improving nutritional status and attenuating CLD-related complications are an unmet clinical need. Aims: We hypothesize supplementation with branched-chain amino acid leucine (LEU) and exercise training (EX) could possibly attenuate muscle mass loss and prevent HE (characterized by brain edema as well as cognitive and psychomotor impairments) in CLD. Methods: CLD was induced in rats following 6-week bile-duct ligation (BDL). Five experimental groups were tested; 1) BDL; 2) BDL + LEU; 3) BDL + EX; 4) BDL + LEU + EX; 5) Sham-operated rats. One week following BDL, rats were submitted to 15 min EX (10 cm/s) every other day and BDL rats receiving LEU, were gavaged daily (1.35 mg/kg) for five weeks. Body weight, muscle (gastrocnemius) mass, metabolic state (calculation of energy expenditure independent of food intake and fecal mass), cerebral edema (specific gravity method) and cognitive/psychomotor function (open-field test; anxiety-like behavior assessment and novel object recognition test; memory testing) were measured in all groups. Results: BDL rats gained less body weight compared to sham-operated rats (125.0±24.9 g vs 226.0±38.5 g; P<0.05). LEU-treated BDL rats display an improvement in brain edema (78.50±0.03% vs 80.27±0.14%; P<0.05), muscle mass (5.48±0.90 g/kg vs 4.83±0.11 g/kg; P<0.05) and circumference (15.6±0.8 cm/kg vs 13.1±0.7 cm/kg ; P<0.05) and metabolic activity (27.48±1.15 vs 32.99±2.35; P<0.05), which was further ameliorated with EX, compared to BDL animals. In addition, BDL rats receiving LEU and EX exhibited less anxiety-like behavior (4.9±1.2 s vs 2.2±0.9 s passed in the center; P<0.01) as well as better novel object recognition memory (69.6±15.2% vs 25.4±9.6%; P<0.01), in comparison with BDL rats. Conclusions: Our results demonstrate that supplemental LEU along with EX reduces body weight and muscle mass loss, improves metabolic activity, attenuates brain edema and improve cognitive and psychomotor function. These findings suggest that strategies aiming at improving nutritional status will attenuate muscle mass loss, reduce the risk of developing HE and therefore improve quality of life and decrease mortality in CLD. LEU supplementation and EX could rapidly be translated into clinical practice. Funding Agency: CIHR.
Marc-André Clément, Cristina R. Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by liver disease characterized by cognitive and motor dysfunction. The only curative treatment to date remains liver transplantation (LT). Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following LT. However, even following the implantation of a new liver, persisting neurological complications remain a common problem affecting as many as 47% (8 47%) of liver transplant recipients. LT is a major surgical procedure accompanied by intraoperative stress and confounding factors, including blood loss (hypovolumia) and hypotension. We hypothesize, in the setting of MHE, that the compromised brain becomes predisposed to what would normally be an innocuous hypotensive insult, resulting in cell injury and death. Methods: Using 6-week bile-duct ligated rats and respective controls, blood is withdrawn from the femoral artery (inducing hypovolemia) until an arterial pressure of 30 mmHg (hypotension) and maintained for 150 minutes. Upon sacrifice, brains are perfused and extracted for western blotting and immunohistochemistry. Results: Both BDL rats and SHAM-operated controls without hypotension do not display any neuronal loss. However, BDL rats following hypotension demonstrated a significant decrease in neuronal cell count in the frontal cortex using NeuN+DAPI and Cresyl Violet compared to hypotensive SHAM-operated controls. In addition, neuronal loss was associated with an increased in cellular stress protein, hsp32 and caspase-3, suggesting apoptotic cell death. Discussion: These findings suggest that patients with HE are more susceptible to hypotension-induced neuronal cell loss and this may explain why transplanted patients are experiencing persisting neurological complications. Aside from cirrhotic patients having a stroke, these results also suggest a patient with HE (even MHE) with a “frail brain”, fare worse during transplantation leading to poor neurological outcome. This implies MHE should not be ignored and therefore treated pre-LT.
Pivotal preclinical trial of the spheroid reservoir bioartificial liver.
Jaime M. Glorioso, Shennen A. Mao, Brian Rodysill, Taufic Mounajjed, Walter K. Kremers, Faysal Elgilani, Raymond D. Hickey, Hakon Haugaa, Christopher F. Rose, Bruce Amiot, Scott L. Nyberg.
The neuroprotective effect of the spheroid reservoir bioartificial liver (SRBAL) was evaluated in a porcine model of drug-overdose acute liver failure (ALF).Healthy pigs were randomized into three groups (standard therapy (ST) alone, ST+No-cell device, ST+SRBAL device) before placement of an implantable intracranial pressure (ICP) monitor and a tunneled central venous catheter. One week later, pigs received bolus infusion of the hepatotoxin D-galactosamine and were followed for up to 90h.At 48h, all animals had developed encephalopathy and biochemical changes confirming ALF; extracorporeal treatment was initiated and pigs were observed up to 90h after drug infusion. Pigs treated with the SRBAL, loaded with porcine hepatocyte spheroids, had improved survival (83 n=6) compared to ST alone (0 n=6, p=0.003) and No-cell device therapy (17 n=6, p=0.02). Ammonia detoxification, peak levels of serum ammonia and peak ICP, and pig survival were influenced by hepatocyte cell dose, membrane pore size and duration of SRBAL treatment. Hepatocyte spheroids remained highly functional with no decline in mean oxygen consumption from initiation to completion of treatment.The SRBAL improved survival in an allogeneic model of drug-overdose ALF. Survival correlated with ammonia detoxification and ICP lowering indicating that hepatocyte spheroids prevented the cerebral manifestations of ALF (brain swelling, herniation, death). Further investigation of SRBAL therapy in a clinical setting is warranted.
Reply to: "Pivotal preclinical trial of the spheroid reservoir bioartificial liver".
Jaime M. Glorioso, Shennen A. Mao, Brian Rodysill, Taufic Mounajjed, Walter K. Kremers, Faysal Elgilani, Raymond D. Hickey, Hakon Haugaa, Christopher F. Rose, Bruce Amiot, Scott L. Nyberg.
Marc-André Clément, Cristina R. Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Introduction L’encéphalopathie hépatique (EH) est une complication neuropsychiatrique causée par les maladies du foie. Ce syndrome est considéré comme un désordre métabolique réversible, qui devrait être réglé par la transplantation hépatique (TH), cependant les complications neurologiques persistent chez près de 47% des patients. La TH est une procédure accompagnée de stress intra-opératoires dont l’hypovolémie et l’hypotension. Nous supposons que le cerveau fragilisé devient plus susceptible à une hypotension, résultant en un stress ainsi que la mort cellulaire. Méthodologies Une hypotension par hypovolémie de l’artère fémorale est effectuée pour atteindre une pression artérielle de 30 mmHg durant 2 heures chez des rats de 6 semaines avec ligation de la voie biliaire (BDL) et leurs contrôles respectifs (SHAM). Les cerveaux sont prélevés pour immunobuvardage et immunohistochimie. Résultats Les rats BDL avec hypotension ont démontré une diminution du compte neuronal dans le cortex préfrontal en utilisant NeuN+DAPI et le Crésyl violet, comparativement aux contrôles SHAM hypotendus. De plus, la protéine de stress cellulaire HSP32 était augmentée chez les groupes BDL hypotendus, tout comme la caspase-3 clivée, suggérant une mort cellulaire par apoptose. Les groupes contrôles SHAM et BDL sans hypotension ne démontrent aucune perte neuronale avec les marqueurs précédents.
Rafaela Almeida, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur, Consortium de l'acidose lactique.
Introduction: Le Syndrome de Leigh version canadienne française (LSFC) ou «acidose lactique» est une maladie mitochondriale rare causée par la mutation du gène LRPPRC (leucine-rich pentatricopeptide repeat motif containing). Elle est caractérisée principalement par une déficience tissu-spécifique (cerveau et foie) en cytochrome c oxydase de la chaîne respiratoire ainsi que par des crises fatales d'acidose lactique. Celles-ci sont déclenchées, entre autres, par l'infection/inflammation et la surcharge nutritionnelle. À ce jour, aucun traitement n'existe pour les prévenir et l'identification d'un modèle expérimental de stress inflammatoire et nutritionnel est essentiel afin d'élucider les mécanismes par lesquels la mutation affecte les cellules LSFC. Méthodes: Nous avons évalué la viabilité des fibroblastes de peau de sujets contrôles et de patients atteints d'acidose lactique par Resazurin dans différentes conditions de stress inflammatoire et nutritionnel, telles que LPS (2 μg/ml), TNF-α (100 ng/ml) et palmitate (1 mM), et ce, à différentes périodes d'incubation (0, 24, 48 et 72h). Résultats: Une diminution significative (p<0,001) de la viabilité cellulaire a été observée 48h après l'exposition de palmitate ou en combinaison avec TNF-α. De plus, une tendance à une susceptibilité accrue au stress est notée dans les fibroblastes de patients LSFC. Discussion/Conclusion: Le stress inflammatoire et nutritionnel semble influencer la viabilité des fibroblastes de patients LSFC. À plus long terme, la modulation de la réponse cellulaire par des agents «thérapeutiques» incluant les acides gras oméga 3, pourrait s'avérer intéressante.
Sarcopenia and Myosteatosis Increase the Risk of Hepatic Encephalopathy in Cirrhotic Patients.
Aldo J. Montano-Loza, Andres Duarte-Rojo, Christopher F. Rose.
Background: Sarcopenia is one of the most common complications of cirrhosis and it is associated with increased mortality. Muscle depletion is generally characterized by both a reduction in muscle size and increased proportion of inter- and intra-muscular fat denominated “myosteatosis”. Skeletal muscle may serve as an alternative site of ammonia detoxification in patients with cirrhosis. Aims: In this study we aimed to investigate if sarcopenia and myosteatosis are associated with overt hepatic encephalopathy in patients with cirrhosis. Methods: A total of 678 cirrhotic patients undergoing assessment for liver transplantation were studied. Sarcopenia and myosteatosis (characterized as low muscle attenuation) were analyzed using computed tomography (CT) scans at the level of the 3rd lumbar vertebral body. The area of paraspinal skeletal muscle (L3 SMI) at this location, and the muscle attenuation index were calculated (Figure 1 & 2). Hepatic encephalopathy was assessed clinically by applying the West-Heaven criteria (grade 0-IV). Results: Of the 678 patients, 457 patients were males (67%). Cirrhosis was caused by HCV in 256 patients (38%), alcohol in 152 (22%), NASH/cryptogenic in 171 (25%), autoimmune liver disease in 53 (8%), HBV in 41 (6%), other etiology in 5 patients (1%); and 292 patients had concomitant HCC (43%). Sarcopenia was noted in 291 patients (43%), and 353 patients had myosteatosis (52%). A total of 216 patients (32%) had history of hepatic encephalopathy (162 grade I-II, 54 grade III-IV). The prevalence of hepatic encephalopathy was significantly higher in patients with sarcopenia (40 vs. 26%, P<0.001), and myosteatosis (39 vs. 24%). By multivariate regression analysis (adjusted to age, gender, and MELD score), both sarcopenia (OR 1.68, (95% CI 1.04-2.40, P=0.03), and myosteatosis (OR 1.97, 95% CI 1.32-2.99, P=0.001) were significantly associated with hepatic encephalopathy. Conclusions: Cir-rhotic patients with sarcopenia and myosteatosis have a higher risk of overt hepatic encephalopathy. Skeletal muscle seems to play a protective role in the pathogenesis of hepatic encephalopathy in cirrhosis, and therapeutic strategies to improve the muscle mass and quality may improve hepatic encephalopathy in cirrhosis.
Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Brain edema is a serious complication associated with hepatic encephalopathy (HE) due to chronic liver disease (CLD). An increase in blood brain barrier (BBB) ion permeability can occur across an intact BBB through alterations in transport mechanisms. NH4+ has very similar ionic properties to K+ and can be transported through K+ channels and cotransporters, implying that hyperammonemia could result in BBB hyperpermeability. An increase in BBB permeability via transport Na+-K+-2Cl- (NKCC1) has shown to promote brain edema and astrocyte swelling under pathophysiological conditions such as ischemia. Aim: To study the BBB integrity (vasogenic vs cytotoxic) and the role of NKCC1 in the pathogenesis of brain edema in cirrhotic rats. Methods: Two distinct animal models of HE are used in the present study; 1) biliary cirrhosis model (6 weeks bile duct ligation (BDL)). 2) portacaval shunt model (4 weeks portacaval anastomosis (PCA)). Both models develop hyperammonemia however brain edema is only observed in BDL. BBB breakdown was assessed by measuring brain extravasation of Evans blue and sodium fluorescein (injected i.v). Expression of BBB tight junction proteins (occludin, claudin-5, ZO-1 and ZO-2) were assessed by Western blot. Bumetanide was administered (i.p) for 10 days in BDL and SHAM animals. Brain water content was measured in the frontal cortex using the specific gravimetric method. Levels of brain NKCC1 mRNA were evaluated by RT-PCR in cerebral microvessels. Results: Extravasation of Evans blue and sodium fluorescein was not detected and no significant change in all tight junction protein was observed in both BDL and PCA models. Brain water content was reduced in bumetanide-treated BDL rats compared to control (77.66±0.15% vs 78.12±0.21%). In brain microvessels, NKCC mRNA increased in BDL rats compared to BDL SHAM (0.78±0.09 vs. 1.92±0.42) whereas no change was found in PCA compared to PCA SHAM (1.72±0.52 vs. 1.53±0.23). Conclusions: BDL rats did not demonstrate a change in BBB integrity or expression of tight junction proteins concluding brain edema in BDL is not of vasogenic origin. Furthermore, since brain edema was only observed in BDL rats (vs PCA), this implies additional factors aside ammonia, are involved in the pathogenesis of brain edema. Moreover, an increase of NKCC1 mRNA and an attenuation of brain edema following bumetanide treatment were demonstrated in BDL rats suggesting NKCC1 plays a role in the development of brain edema in CLD. These results demonstrate the potential therapeutic use of bumetanide for the treatment of HE.
Optimizing muscle mass: therapeutic target to prevent experimental hepatic encephalopathy.
Chantal *Bémeur, Sara Ghezzal, Marc-André Clément, Cristina R. Bosoi, Roxanne Beauchamp, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (cirrhosis; CLD). Malnutrition, considered a consequence of metabolic disturbances (hypermetabolism), exacerbates severe muscle loss and hepatic encephalopathy (HE) (complex neuropsychiatric disorder) in cirrhotic patients. New management strategies focussing on improving nutritional status and attenuating CLD-related complications are an unmet clinical need. We hypothesize supplementation with branched-chain amino acid leucine (LEU) and exercise training (EX) could possibly attenuate muscle mass loss and prevent HE (characterized by brain edema as well as cognitive and psychomotor impairments) in CLD. Methods: CLD was induced in rats following 6-week bile-duct ligation (BDL). Five experimental groups were tested; 1) BDL; 2) BDL + LEU; 3) BDL + EX; 4) BDL + LEU + EX; 5) Sham-operated rats. One week following BDL, rats were gavaged with LEU (1.35 mg/kg) daily and submitted to 15 min EX (10 cm/s) every other day for 5 weeks. Body weight, muscle (gastrocnemius) mass, metabolic state (calculation of energy expenditure independent of food intake and fecal mass), cerebral edema (specific gravity method) and cognitive/psychomotor function (open-field test; anxiety-like behavior assessment and novel object recognition test; memory testing) were measured. Results: BDL rats gained less body weight compared to sham-operated rats (125.0g ± 24.9 vs 226.0g ± 38.5; p<0.05). LEU-treated BDL rats display an improvement in brain edema (78.50% ± 0.03 vs 80.27% ± 0.14; p<0.05), muscle mass (5.48g/kg ± 0.90 vs 4.83g/kg ± 0.11; p<0.05) and circumference (15.6cm/kg ± 0.8 vs 13.1cm/kg ± 0.7; p<0.05) and metabolic activity (27.48 ± 1.15 vs 32.99 ± 2.35; p<0.05), which was further ameliorated with EX, compared to BDL animals. In addition, BDL rats receiving LEU and EX exhibited less anxiety-like behavior (4.9s ± 1.2 vs 2.2s ± 0.9 passed in the center; p<0.01) as well as better novel object recognition memory (69.6 ± 15.2% vs 25.4 ± 9.6%; p<0.01), in comparison with BDL rats. Conclusion: Our results demonstrate that supplemental LEU along with EX recovers body weight loss, increases muscle mass, improves metabolic activity, attenuates brain edema and improves cognitive and psychomotor function. These findings suggest that strategies aiming at improving nutritional status will attenuate muscle mass loss and reduce the risk of developing HE. This in turn will improve quality of life, decrease mortality and enhance outcome post-liver transplantation. LEU supplementation and EX could rapidly be translated into clinical practice.
Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Brain edema is a serious complication associated with hepatic encephalopathy (HE) due to chronic liver disease. An increase in BBB ion permeability (increase uptake of ions and accompanied by water fluxes towards the brain) can occur across an intact BBB through alterations in transport mechanisms. Endothelial cells that comprise the BBB secrete up to 40% of brain interstitial fluid as they transport Na+, K+ and 2 Cl- across the BBB. NH4+ has very similar ionic properties to K+ (similar ionic radius and diffusion coefficient) and can be transported through K+ channels and cotransporters, implying that hyperammonemia could result in BBB hyperpermeability. An increase in BBB permeability via transport Na+-K+-2Cl- (NKCC1) has shown to promote brain edema and astrocyte swelling under pathophysiological conditions such as ischemia. Aim: To study the BBB integrity (vasogenic vs cytotoxic) and the role of NKCC1 in the pathogenesis of brain edema in cirrhotic rats. Methods: Two distinct animal models of chronic liver failure and HE are used in the present study; 1) biliary cirrhosis model (6 weeks bile duct ligation (BDL)). 2) portacaval shunt model (4 weeks portacaval anastomosis (PCA)). Both models develop hyperammonemia however brain edema is only observed in BDL. BBB breakdown was assessed by measuring brain extravasation of Evans blue and sodium fluorescein (injected i.v). Expression of BBB tight junction proteins (occludin, claudin-5, ZO-1 and ZO-2) were assessed by Western blot. Bumetanide was administered (i.p) for 10 days in BDL and BDL SHAM. Brain water content was measured in the frontal cortex using the specific gravimetric method. Levels of brain NKCC1 mRNA were evaluated by RT-PCR in cerebral microvessels. Results: Extravasation of Evans blue and sodium fluorescein was not detected and there was no significant change in all tight junction protein levels measured in both BDL and PCA models. Brain water content was reduced in bumetanide-treated BDL rats compared to control (77.66±0.15% vs 78.12±0.21%). In brain microvessels, NKCC mRNA increased in BDL rats compared to BDL SHAM (0.78±0.09 vs. 1.92±0.42) whereas no change was found in PCA compared to PCA SHAM (1.72±0.52 vs. 1.53±0.23). Conclusions: BDL rats did not demonstrate a change in BBB integrity or in expression of BBB tight junction proteins. This suggests brain edema in BDL is not of vasogenic origin. Furthermore, since brain edema was only observed in BDL rats (vs PCA), this implies additional factors aside ammonia, are involved in the pathogenesis of brain edema. Moreover, an increase of NKCC1 mRNA and an attenuation of brain edema following bumetanide treatment were demonstrated in BDL rats suggesting NKCC1 plays a role in the development of brain edema in chronic liver disease. Furthermore, these results demonstrate the potential therapeutic use of bumetanide for the treatment of HE.
Chantal *Bémeur, Sara Ghezzal, Marc-André Clément, Cristina R. Bosoi, Roxanne Beauchamp, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (cirrhosis; CLD). Malnutrition, considered a consequence of metabolic disturbances (hypermetabolism), exacerbates severe muscle loss and hepatic encephalopathy (complex neuropsychiatric disorder) in cirrhotic patients. New management strategies focussing on improving nutritional status and attenuating CLD-related complications are an unmet clinical need. We hypothesize supplementation with branched-chain amino acid leucine (LEU) and exercise training (EX) could possibly attenuate muscle mass loss and prevent hepatic encephalopathy (characterized by brain edema as well as cognitive and psychomotor impairments) in CLD. Methods: CLD was induced in rats following 6-week bile-duct ligation (BDL). Five experimental groups were tested; 1) BDL; 2) BDL + LEU; 3) BDL + EX; 4) BDL + LEU + EX; 5) Sham-operated rats. One week following BDL, rats were submitted to 15 min EX (10 cm/s) every other day and BDL rats receiving LEU, were gavaged daily (1.35 mg/kg) for 5 weeks. Body weight, muscle (gastrocnemius) mass, metabolic state (calculation of energy expenditure independent of food intake and fecal mass), cerebral edema (specific gravity method) and cognitive/psychomotor function (open-field test; anxiety-like behavior assessment and novel object recognition test; memory testing) were measured in all groups. Results: BDL rats gained less body weight and muscle mass compared to sham-operated rats. LEU-treated BDL rats display an improvement in brain edema, muscle mass and circumference and metabolic activity, which was further ameliorated with EX. In addition, BDL rats receiving LEU and EX exhibited less anxiety-like behavior as well as better novel object recognition memory. Conclusion: Our results demonstrate that supplemental LEU along with EX reduces body weight and muscle mass loss, improves metabolic activity, attenuates brain edema and improve cognitive and psychomotor function. These findings suggest that strategies aiming at improving nutritional status will attenuate muscle mass loss, reduce the risk of developing hepatic encephalopathy and therefore improve quality of life and decrease mortality in CLD. LEU supplementation and EX could rapidly be translated into clinical practice.
Marc-André Clément, Cristina Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by liver disease characterized by cognitive and motor dysfunction. The only curative treatment to date remains liver transplantation (LT). Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following LT. However, even following the implantation of a new liver, persisting neurological complications remain a common problem affecting as many as 47% (8 47%) of liver transplant recipients. LT is a major surgical procedure accompanied by intraoperative stress and confounding factors, including blood loss (hypovolumia) and hypotension. We hypothesize, in the setting of MHE, that the compromised brain becomes predisposed to what would normally be an innocuous hypotensive insult, resulting in cell injury and death. Methods: Using 6-week bile-duct ligated rats and respective controls, blood is withdrawn from the femoral artery (inducing hypovolemia) until an arterial pressure of 30 mmHg (hypotension) and maintained for 150 minutes. Upon sacrifice, brains are perfused and extracted for western blotting and immunohistochemistry. Results: Both BDL rats and SHAM-operated controls without hypotension do not display any neuronal loss. However, BDL rats following hypotension demonstrated a significant decrease in neuronal cell count in the frontal cortex using NeuN+DAPI and Cresyl Violet compared to hypotensive SHAM-operated controls. In addition, neuronal loss was associated with an increased in cellular stress protein, hsp32, hsp70 and caspase-3, suggesting apoptotic cell death. Discussion: These findings suggest that patients with HE are more susceptible to hypotension-induced neuronal cell loss and this may explain why transplanted patients are experiencing persisting neurological complications. Aside from cirrhotic patients having a stroke, these results also suggest a patient with HE (even MHE) with a “frail brain”, fare worse during transplantation leading to poor neurological outcome. This implies MHE should not be ignored and therefore treated pre-LT.
Cristina R. Bosoi, Marc-André Clément, Mélanie Tremblay, Christopher F. Rose.
Background: Ammonia plays a major role in the pathogenesis of hepatic encephalopathy (HE) and therefore ammonia-lowering treatments remain a primary therapeutic strategy. Glutamine deamidation by the mitochondrial enzyme glutaminase (GLS) is believed to a major source of ammonia production in cirrhotic patients and increased intestinal GLS activity has been shown to be linked to minimal HE and to an increased risk of developing overt HE. CB-839 is a potent, selective and orally bioavailable GLS inhibitor (Gross et al., Mol Cancer Ther 13:890) that is currently in Phase 1 clinical trials for the treatment of cancer (clinicaltrials.gov). Aim: To evaluate the effect of CB-839, a GLS inhibitor, in preventing the onset of hyperammonemia following an oral glutamine challenge (OGC) in rats with portacaval anastomosis (PCA). Methods: Four week PCA rats received a single dose of CB-839 (gavage, 200 mg/kg diluted in 5ml/kg of vehicle). Control PCA rats received equivalent volumes of vehicle. Four hours after CB-839 or vehicle administration, PCA rats received an oral glutamine challenge (gavage, 100 mg/kg). Repeated aortic blood samples were obtained at baseline, 0.5h, 1h, 1.5h, 2.5h, 2.5h, 3h and 4h following OGC. Glutamine and ammonia were measured using commercial available kits. Results: Baseline ammonia levels were similar in both PCA groups. Following OGC, blood ammonia increased in vehicle-treated PCA rats with a peak at 2h (2.3-fold increase vs baseline, p<0.05). In CB-839 treated-PCA rats, ammonia levels did not change compared to the baseline value and were significantly decreased compared to non-treated PCA rats (p<0.05). At 4h, ammonia levels returned to baseline values in both groups. Baseline glutamine levels were not significantly different between treated and non-treated PCA rats. Following OGC, no significant difference between glutamine levels was observed in non-treated PCA rats compared to baseline values. However, in PCA rats treated with CB-839 glutamine levels significantly increased compared to non-treated PCA controls attaining a peak at 2.5 h (1.59 ± 0.40 mM vs 0.60 ± 0.15 mM, p<0.05). At 4h, glutamine levels remained significantly increased. Conclusions: CB-839 treatment inhibited glutamine induced hyperammonemia in PCA rats. These preliminary results strongly suggest CB-839 is an effective agent to attenuate GLS-induced ammonia production. Further studies are warranted to evaluate CB-839 as a novel agent for the treatment of HE.
Rune Gangsøy Kristiansen, Christopher Rose, Ole Martin Fuskevåg, Hanne Mæhre, Arthur Revhaug, Rajiv Jalan, Lars Marius Ytrebø.
Background: Glycine is an important ammoniagenic amino acid, which is increased in acute liver failure (ALF). We have previously shown that ornithine phenylacetate (OP) attenuates ammonia rise and intracranial pressure in pigs suffering from ALF, but failed to demonstrate a stoichiometric relationship between change in plasma ammonia levels and excretion of phenylacetylglutamine in urine. The aim was to investigate the impact of OP treatment on the phenylacetylglycine pathway as an additional ammonia- lowering pathway. Methods: A well-validated and characterized large porcine model of ALF (portacaval anastomosis followed by hepatic artery ligation), which recapitulates the cardinal features of human ALF was used. Twenty-four female pigs were randomised into three groups: (i) Sham operated + vehicle, (ii) ALF + vehicle, and (iii) ALF + OP. Results: There was a significant increase in arterial glycine concentration in ALF (p< 0.001 compared with sham), with a 3-fold increase in glycine release into the systemic circulation from the kidney compared to the sham group. This increase was attenuated in both blood and the brain of the OP treated animals (p<0.001 and p<0.05, respectively), and the attenuation was associated with renal removal of glycine through excretion of the conjugation product phenylacetylglycine in urine (ALF+vehicle: 1060 ± 106 μmol/L, ALF+OP: 27625 ± 2670 μmol/L, P<0.003). Conclusion: Data from this study provides solid evidence for the existence of a novel, and additional pathway for ammonia removal in ALF, that involves glycine production and removal, which is targeted by OP.
Systemic oxidative stress induction leads to brain edema in hyperammonemic portacaval-shunted rats.
Cristina Bosoi, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy is a neuropsychiatric disorder; a major complication of liver disease. Impairment in liver function leads to a reduced capacity to clear ammonia (via urea cycle) and subsequently hyperammonemia arises. The consequent neurotoxic levels of ammonia are considered to play a major role in the pathogenesis of hepatic encephalopathy. However, a correlation between ammonia and severity of neurological impairment is poor. Oxidative stress is another factor believed to play a role in the pathogenesis of this syndrome as it has demonstrated to exacerbate the neuropsychological effects of hyperammonemia. In the setting of liver disease, oxidative stress represents a systemic phenomenon induced by several mechanisms: decreased antioxidant synthesis, increased systemic release of oxidant enzymes, generation of reactive oxygen species and impaired neutrophil function. Furthermore, it has been demonstrated that high ammonia concentrations can induce oxidative stress. However, significantly lower degrees of hyperammonemia (<500 µM) are observed in patients with end-stage liver disease (cirrhosis), concentrations which do not induce cerebral nor systemic oxidative stress. Therefore defining ammonia and oxidative stress as independent factors. Data from both animal and human studies strongly suggest there is a synergistic effect between systemic oxidative stress and ammonia in the pathogenesis of hepatic encephalopathy and that induction of cerebral oxidative stress may be associated with severe neurological symptoms.
Sara Ghezzal, Marc-André Clément, Cristina R. Bosoi, Roxanne Beauchamp, Mélanie Tremblay, Christopher F. Rose, Chantal Bémeur.
Aims: The pathogenesis of hepatic encephalopathy (HE) is multifactorial. Even though ammonia is the central component in the pathogenesis of HE, oxidative stress is believed to play a role in exacerbating the neuropsychological effects of ammonia in patients with liver disease. With new, highly sensitive imaging techniques, brain edema is observed in HE patients. We previously demonstrated that portacaval shunted hyperammonemic rats do not develop oxidative stress or brain edema. In order to define a synergistic effect between hyperammonemia and systemic oxidative stress, the present study investigates the role of oxidative stress in the pathogenesis of brain edema in PCA rats following glutathione depletion by diethyl maleate (DEM). Methods: In the first set of experiments, we evaluated the effect of DEM in PCA and SHAM-operated control rats by injecting DEM at a dose of 0.4 and 1 mg/kg/day intraperitoneally for 10 days starting at day 18 after surgery. Rats were sacrificed at day 28 and oxidative stress was evaluated by arterial malon-dialdehyde (MDA, commercial kit). In the second set of experiments, 1 mg/kg/day DEM was used to induce oxidative stress. Ammonia (commercial kit) as well as other different oxidative stress markers: reactive oxygen species (DCFDA fluorescence technique), and 4-hydroxy-2-nonenal (HNE, Western blot) were assessed in arterial plasma and frontal cortex tissue. Brain water content was measured in the frontal cortex using a specific gravimetric technique. Results: DEM at 1 mg/kg/day (not 0.4 mg/kg/day) induced a significant increase in MDA levels in PCA rats. No increase in MDA was detected following either dose of DEM in SHAM-operated controls. Ammonia levels in both DEM-treated and non-treated PCA rats were significantly increased vs respective sham-operated controls (p<0.001) and remained unchanged between non-treated and DEM-treated PCA groups (p>0.05). An increase in brain water content was observed in DEM-treated PCA rats vs non-treated PCA rats (PCA+DEM: 78.45 ± 0.13% vs PCA: 77.38 ± 0.11, p< 0.001). Although no significant changes in reactive oxygen species were observed, there was an increase in plasma levels of HNE in DEM-treated PCA rats compared to non-treated PCA rats. No significant changes in any oxidative stress markers were observed in the frontal cortex. Conclusions: DEM treatment in PCA rats induced systemic oxidative stress but not central oxidative stress. This, imposed on hyperammonemia, was accompanied by the onset of brain edema in rats with PCA. Oxidative stress and brain edema were not detected in SHAM-operated rats, which were not hyperammonemic. Our findings suggest a synergistic effect between hyperammonemia and systemic oxidative stress is implicated in the pathogenesis of brain edema in hepatic encephalopathy.
Implication de l'hypotension dans la pathogenèse de l'encéphalopathie hépatique.
Marc-André Clément, Mélanie Tremblay, Christopher F. Rose.
L’encéphalopathie hépatique (EH) est une complication majeure des maladies du foie caractérisée par des troubles cognitifs, moteurs et psychiatrique qui peuvent mener jusqu’au coma et la mort. La transplantation du foie reste présentement la seule option curative de la maladie. Des études récentes ont démontré que la présence d’EH avant la transplantation est reliée aux complications neurologiques survenant après l’intervention. Le cerveau demandant plus de 20% de la demande énergétique du corps, l’hypotension et la perte sanguine associée à l’opération pourraient être responsables de ces dommages neurologiques, puisque l’apport sanguin au cerveau est réduit, particulièrement chez les cerveaux déjà fragilisés par l’EH. L’objectif principal de mon projet de maîtrise est de caractériser l’impact de l’EH sur la mort neuronale causée par une hypotension, induite par hypovolémie, chez des rats dont la cirrhose est causée par la ligation chirurgicale de la voie biliaire (BDL). Les animaux développent entre autres, une jaunisse, une hyperammonémie, des difficultés motrices, de mémoire et un œdème cérébral, étant des symptômes typiques de la cirrhose