Mariana Oliveira

Multiple ammonia-induced episodes of hepatic encephalopathy provoke neuronal cell loss in bile-duct ligated rats.

Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mariana M. Oliveira, Carina Lima, Maggy Lépine, Karine Dubois, Cristina Bosoi, Mélanie Tremblay, Lekha Sleno, Christopher F. Rose.

Neurological dysfunction is improved after faecal microbiota transplantation in rats with bile duct ligated-induced chronic liver disease.

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.

Constant alcohol consumption exacerbates hepatic encephalopathy and induces neuronal cell loss in rats with chronic liver disease.

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.

La transplantation de microbiote fécal améliore les troubles neurologiques chez les rats avec une maladie hépatique chronique induite par la ligature du canal biliaire.

Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Kraine Dubois, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.

L’acide urique comme facteur de risque pour les complications cognitives de la cirrhose du foie : développement et validation d’un nouveau modèle murin.

Sydnée L’Écuyer, Alexandra Matesan, Farzaneh Tamnanloo, Mariana Oliveira, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.

Oral

La transplantation de microbiote fécal améliore les troubles neurologiques chez les rats avec une maladie hépatique chronique induite par la ligature du canal biliaire.

Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Kraine Dubois, Mélanie Tremblay, Chantal Bémeur, F. Rose Christopher.

Oral

L’acide urique comme facteur de risque pour les complications cognitives de la cirrhose du foie : développement et validation d’un nouveau modèle murin.

Sydnée L’Écuyer, Alexandra Matesan, Farzaneh Tamnanloo, Mariana Oliveira, Mélanie Tremblay, Emmanuel Charbonney, Christopher F. Rose.

Oral

Hyperuricemia as an implicated factor in cognitive impairments in chronic liver disease: developing a new murine model.

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.

Neurological impairment in rats with bile duct ligated-induced chronic liver disease are improved after faecal microbiota transplantation.

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.

Oral

La transplantation de microbiote fécal améliore l’atteinte neurologique chez les rats ayant subi une la ligature du canal biliaire induisant une maladie hépatique chronique.

Alexandre Bourgeois, Felix Veillette, Mariana Oliveira, Karine Dubois, Mélanie Tremblay, Chantal Bémeur, F. Rose Christopher.

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.

Oral

La transplantation de microbiote fécal améliore la déficience neurologique chez les rats atteints d'une maladie hépatique chronique induite par la ligature du canal biliaire.

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.

Neurological dysfunction is improved after faecal microbiota transplantation in rats with bile duct ligated-induced chronic liver disease.

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.

Neurological impairment in rats with bile duct ligated-induced chronic liver disease are improved after faecal microbiota transplantation.

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.

Hyperuricemia as a risk factor for cognitive impairments in chronic liver disease: developing a new murine model.

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.

La transplantation de microbiote fécal améliore l’atteinte neurologique chez les rats avec une maladie hépatique chronique induite par la ligature du canal biliaire.

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.

Faecal microbiota transplantation improves neurological impairment in rats with 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 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.

Oral

La transplantation de microbiote fécal améliore la déficience neurologique chez les rats atteints d'une maladie hépatique chronique induite par la ligature du canal biliaire.

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.

OpenAccess

Sex is associated with differences in oxidative stress and susceptibility to severe hepatic encephalopathy in bile-duct ligated rats.

Mariana Macedo de Oliveira, Alexis Monnet-Aimard, Cristina R. Bosoi, Mélanie Tremblay, Christopher F. Rose.

Oral

La transplantation de microbiote fécal améliore la déficience neurologique chez les rats atteints d'une maladie hépatique chronique induite par la ligature du canal biliaire.

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.

Targeting glutamine synthetase in endothelial cells of the blood-brain barrier to treat hyperammonemia and hepatic encephalopathy.

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.

Faecal microbiota transplantation improves neurological impairment in rats with bile-duct ligated induced 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 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.

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.

Oral

La transplantation de microbiote fécal améliore la déficience neurologique chez les rats atteints d'une maladie hépatique chronique induite par la ligature du canal biliaire.

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.

Impact of sex on development of hepatic encephalopathy, sarcopenia and pathogenic factors 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) 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.

Oral

Sex disparity on the development of HE: impact of ammonia and ROS in the development of brain edema and hepatic encephalopathy severity in BDL rats

Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.

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.

Sex impacts muscle loss and ammonia metabolism in rats with chronic liver disease.

Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.

Effects of sex on liver disease: impact on sarcopenia and ammonia metabolism in bile-duct ligated rats.

Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.

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.

OpenAccess

Genetically engineered E. coli Nissle attenuates hyperammonemia and prevents memory impairment in bile‐duct ligated rats

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.

OpenAccess

Bile-duct ligation renders the brain susceptible to hypotension induced neuronal degeneration: implications of ammonia.

Marc-André Clément, Cristina R. Bosoi, Mariana M. Oliveira, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.

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.

OpenAccess

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.

The attenuation of hyperammonemia by genetically engineered E. coli Nissle counters memory deficits in an experimental model of cirrhosis and hepatic encephalopathy.

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.

Uncovering the impact of sex-based differences in a rat model of chronic liver disease and hepatic encephalopathy.

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.

Obesity accelerates and exacerbates neurological impairments associated to hepatic encephalopathy in chronic liver disease.

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.

Genetically Engineered E. coli Nissle attenuates hyperammonemia and improves memory in an experimental model of cirrhosis and hepatic encephalopathy.

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.

Oral

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.

Glutamine synthetase in the blood-brain barrier: role in hindering hyperammonemia-induced neurotoxicity?

Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.

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.

Genetically Engineered E. coli nissle Attenuates Hyperammonemia in Two Experimental Models of Hepatic Encephalopathy.

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.

Impact of gender on development of chronic liver disease and hepatic encephalopathy following bile duct ligation.

Mariana Oliveira, Mélanie Tremblay, Christopher Rose.

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.

Genetically Engineered E. coli nissle Attenuates Hyperammonemia in Two Experimental Models of Hepatic Encephalopathy.

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.

OpenAccess

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.

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.

Oral

Evaluate the efficiency of rifaximin treatment in a bile-duct ligated model of hepatic encephalopathy.

Grégory Petrazzo, Mariana M. Oliveira, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.

Oral

Glutamine synthetase in the blood-brain barrier: targeting endothelial cells for the treatment of hepatic encephalopathy?

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

Glutamine synthetase in endothelial cells of the blood-brain barrier: role in protecting the brain from hyperammonemia-induced neurotoxicity?

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.

Glutamine synthetase in endothelial cells of the blood-brain barrier: role in preventing protecting the brain in hepatic encephalopathy?

Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.

Glutamine synthetase in endothelial cells of the blood-brain barrier: role in preventing protecting the brain in hepatic encephalopathy?

Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.

Glutamine synthetase in endothelial cells of the blood-brain barrier: new target for the treatment of hepatic encephalopathy?

Mariana Oliveira, Mélanie Tremblay, Christopher Rose.

OpenAccess

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.

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

Oral

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.

Oral

Oral Ornithine Phenylacetate Attenuates Muscle Mass Loss and Prevents Hepatic Encephalopathy in BDL Rats.

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.

Oral

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.

Glutamine synthetase in endothelial cells of the blood-brain barrier: new target for the treatment of hepatic encephalopathy?

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.

Developing a new animal model of obesity and chronic liver disease for the study of hepatic encephalopathy.

Rafael Ochoa-Sanchez, Mélanie Tremblay, Mariana Oliveira, Cristina Bosoi, Marc-André Clément, Christopher F. Rose.

The role of the blood-brain barrier in ammonia homeostasis: targeting endothelial cells for the treatment of hepatic encephalopathy.

Mariana Oliveira, Mélanie Tremblay, Christopher F. Rose.

Oral

Beneficial effects of ornithine phenylacetate to attenuate muscle mass loss and to prevent hepatic encephalopathy in experimental cirrhosis.

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.

Oral

Ornithine phenylacetate attenuates loss of muscle mass and improves hepatic encephalopathy in bile-duct ligated rats.

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.

Long term oral treatment of ornithine phenylacetate increases lean mass and attenuates brain edema in bile-duct ligated rats.

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.

Mariana Oliveira, M.Sc.

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