The 17th ISHEN conference will be held from March 9th to 11th, at Gurgaon, India.
This week, our students Mariana, Rafael, Cassandra and Marc-André, and Dr. Rose will fly to India to attend ISHEN, the conference bringing together all experts in hepatic encephalopathy. They will present their results, whether in oral or poster. We wish them a good conference!
EDIT : Mariana awards the "best oral presentation", congrats!
Related Publications
Progressive resistance training prevents loss of muscle mass and strength in bile duct ligated rats.
Mariana M. Oliveira, Christopher F. Rose, Luise Aamann, Rafael Ochoa-Sanchez, Mariana Oliveira, Mélanie Tremblay, Chantal Bémeur, Gitte Dam, Hendrik Vilstrup, Niels Kristian Aagaard, Christopher Rose.
Loss of muscle mass and strength is common in cirrhosis and increases the risk of hyperammonemia and hepatic encephalopathy. Resistance training optimizes muscle mass and strength in several chronic diseases. However, the beneficial effects of resistance training in cirrhosis remains to be investigated. Bile duct ligated (BDL)-rats develop chronic liver disease, hyperammonemia, reduced muscle mass and strength. Our aim was to test the effects of resistance training on muscle mass, function and ammonia metabolism in BDL-rats. A group of BDL-rats underwent a progressive resistance training program and a group of non-exercise BDL-rats served as controls. Resistance training comprised of ladder climbing with a progressive increase in carrying weights attached to the tail. Training was performed five days a week during four weeks. Muscle strength and body composition were assessed using grip strength and EchoMRI. Weight and circumference of the gastrocnemius muscle (normalized to bodyweight), plasma ammonia and glutamine synthetase protein expression and activity were assessed. BDL+exercise rats had significantly larger gastrocnemius circumference compared to non-exercise BDL-rats: ratio 0.082 vs. 0.075 (p<0.05). Gastrocnemius muscle weight was higher in exercisers than controls: 0.006 vs. 0.005 (p<0.05). A tendency towards a lower plasma ammonia in the exercise group compared to controls was observed (p=0.10). There were no differences in lean body mass, GS protein expression and activity between the groups. Resistance training in rats with chronic liver disease beneficially effects muscle mass and strength. The effects were followed by non-significant reduction in blood ammonia, however a tendency was observed. This article is protected by copyright. All rights reserved.
Rahima A. Bhanji, Andres Duarte-Rojo, Christopher Rose, Aldo J. Montano-Loza.
Background/Aims: Skeletal muscle abnormalities, such as sarcopenia and myosteatosis are frequent complications of cirrhosis and are associated with increased morbidity and mortality. Hyperammonemia plays a significant role in the pathogenesis of hepatic encephalopathy (HE). Skeletal muscle have a significant compensatory part in detoxifying ammonia during liver disease since it houses the enzyme glutamine synthetase, an important ammonia-removing pathway during the amination of glutamate to glutamine. Therefore, we aimed to investigate whether reduced quantity and quality of muscle is associated with hyperammonemia, HE and mortality in patients with cirrhosis. Methods: A total of 677 cirrhotic patients were evaluated. Computed tomography (CT) scans were used to analyze the skeletal muscle (transverse CT image at the level of the 3rd. lumbar vertebra (L3) was selected from each scan) and sarcopenia and myosteatosis was evaluated. Overt HE was assessed clinically and ammonia blood levels were performed at the time of the muscularity assessment. Results: Sarcopenia was noted in 292 patients (43%), and 352 patients had myosteatosis (52%). A total of 225 patients (33%) had history of overt HE and 221 (of 267) patients (83%) had hyperammonemia. The prevalence of overt HE was higher in patients with sarcopenia (42% vs. 27%, P<0.001), and myosteatosis (41% vs. 25%, P<0.001). By multivariable regression analysis, sarcopenia and myosteatosis were independently associated with higher risk of overt HE (HR 1.89, P=0.007, HR 1.68, P=0.03) and hyperammonemia (HR 1.71, P=0.006, HR 1.88, P=0.001, respectively). Median survival was worse in patients with overt HE and sarcopenia (18±4 vs. 42±7 months, P=0.01), hyperammonemia and sarcopenia (11±7 vs. 38±16 months, P<0.001), and myosteatosis and hyperammonemia (19±3 vs. 38±20 months, P=0.005) compared to patients without these factors. Conclusions: Cirrhotic patients with sarcopenia and myosteatosis have a higher risk of hyperammonemia and overt HE. Further, skeletal muscle abnormalities present concomitantly with HE and hyperammonemia increase the risk of mortality in these patients.
Marc-André Clément, Cristina R. Bosoi, Mariana Oliveira, Mélanie Tremblay, Gabriella A. Ten Have, Nicolaas E. Deutz, Christopher F. Rose.
Background and Aims: Chronic liver disease (CLD) induces numerous complications including muscle mass loss and hepatic encephalopathy (HE) which negatively impact clinical outcomes. Hyperammonemia is considered the central component in the pathogenesis of HE, however recent studies have suggested ammonia to be toxic to other organs/tissues aside the brain, such as the muscle. The aim of this study was to investigate the effect of lowering ammonia on muscle mass in cirrhotic rats treated with an oral formulation of ornithine phenylacetate (OP; OCR-002). Methods: Six-week bile-duct ligated (BDL) and sham rats were used. OP was administered orally by gavage (1g/kg) daily for 5 weeks starting 1 week after surgery. Locomotor activity (day/night) was assessed in infrared beam cages for 24 h. Body weight, fat and lean mass (EchoMRI) were measured. Stable isotope tracers were injected (ip) in order to assess fractional protein synthesis rate (FSR). Blood ammonia and cerebral edema was also evaluaeted. Results: BDL rats demonstrated a 4-fold increase in blood ammonia vs sham-operated controls. This increase was reduced by 40% in OP-treated BDL rats. BDL rats gained less body weight compared to sham-operated controls (body weight of 360.2g ± 13.6 vs 476.8g ± 10.38 p<0.001) which was accompanied with a lower gain of lean mass and a lower muscle FSR. OP-treated BDL rats showed a significant increase in body weight (p<0.001 vs BDL) with a significant higher lean mass (303.1g ± 10.7 in BDL+OP vs 264.4g ± 10.5 in BDL p<0.01). Fat mass remained unchanged between the treated and untreated BDL groups. OP treatment normalized brain water content in BDL rats. In contrast, OP-treatment reduced muscle FSR in SHAM animals, but not in BDL rats. Locomotor activity in BDL rats was reduced compared with sham-operated controls but no significant change was found between BDL+OP and SHAM+OP. Conclusions: This is the first study demonstrating the efficient ammonia-lowering effect of an oral formulation of OP. Long-term treatment with OP is a safe, effective, non-antibiotic alternative demonstrating a significant ammonia-lowering effect, as well as a protective effect on the development of brain edema and muscle mass loss in rats with CLD. Whether the beneficial effect of OP on muscle mass loss is a result of lowering blood ammonia or direct result of OP on muscle metabolism remains to be established.
Constantine Jason Karvellas, Jaime Lynn Speiser, Mélanie Tremblay, William M. Lee, Christopher F. Rose.
Background: Acetaminophen (APAP) - induced Acute Liver Failure (ALF) is associated with significant morbidity and mortality related to cerebral edema (CE) and intracranial hypertension (ICH). Brain type fatty acid binding protein (FABP7) is a small (15 kDa) cytoplasmic protein abundantly expressed in astrocytes where there is active fatty acid metabolism. FABP7 levels have not been previously reported in ALF patients at high risk of CE/ICH. Aims: One: To determine whether serum FABP7 early (day 1) or late (day 3-5) levels are associated with 21-day mortality in APAP-ALF patients in the absence of liver transplant. Two: To determine whether serum FABP7 levels at serial time points are associated with the presence of ICH/CE Methods: Nested case control study 198 APAP-ALF patients (99 survivors, 99 non-survivors) from the US Acute Liver Failure Study Group (ALFSG) registry. Patient samples were analyzed for FABP1 using solidphase enzyme-linked immunosorbent assay (ELISA) and assessed with clinical data. In a second analysis where data were available, patients were stratified based on the presence of ICH/CE (n=46) or absence (n=104) based on death summaries or clinical data (ICP monitor, computed tomography). Multivariable logistic regression was used to determine independent associations with 21-day mortality (n=198) and development of ICH/CE (n=150) Results: APAP-ALF survivors had significantly lower serum FABP7 levels on admission (147.9 vs. 316.5 ng/ml, P=0.0002) and late (87.3 vs. 286.2 ng/ml, P<0.0001) compared with nonsurvivors (Figure 1). However using multivariable logistic regression, a significant association between 21- day mortality and increased serum FABP7 early (Log FABP7 Odds Ratio (OR) 1.16, P=0.32) and late (Log FABP7 OR 1.34, P=0.21) was not detected after adjusting for significant covariates (MELD, vasopressor use). Area under the receiver operating curve for early and late multivariable models were 0.760 and 0.892 respectively. A significant difference in FABP7 levels between patients with or without ICH/CE at early (259.7 vs. 228.2 ng/ml, P=0.61) and late (223.8 vs. 192.0 ng/ml, P=0.19) time points was not identified. FABP7 was not significantly associated with the development of ICH/CE after adjusting for significant covariates (Early OR 1.0, p=0.65; Late OR 1.0, p=0.57 see Table 1). Summary: Brain type FABP (FABP7) levels were elevated in APAP-ALF patients with serum significantly higher serum levels at early and late time points in APAP-ALF non-survivors. However, significant differences in FABP7 levels by 21-day mortality were not ascertained after adjusting for significant covariates reflecting severity of illness (MELD, vasopressor dependence). FABP7 did not discriminate between the APAP-ALF patients with and without ICH/CE. Conclusions: This suggests that while all APAP-ALF patients have some evidence of astrocyte inflammation and increased permeability in the blood brain barrier, FABP7 appears to not discriminate between patients who did and did not have significant intracranial complications of APAP-ALF. Disclosure: The study was sponsored by NIH grant U-01 58369 (from NIDDK), TL1 001451 (from NCATS), and a grant from the University of Alberta Hospital Foundation (UHF).
Constantine Jason Karvellas, Jaime Lynn Speiser, Mélanie Tremblay, William M. Lee, Christopher F. Rose.
Background: Acetaminophen (APAP) - induced acute liver failure (ALF) is associated with significant mortality. To date, traditional prognostic scores lack discrimination in identifying patients with APAP-ALF who will die without liver transplant (LT), and those who will survive with medical management alone. Failure to identify those in need of LT results in a potentially preventable death while wrongly classifying prospective survivors as in need of LT subjects the patient to unnecessary LT. Liver-type fatty acid binding protein (FABP1) is a small (15 kDa) cytoplasmic protein abundantly expressed in hepatocytes. The prognostic value of serum FABP1 levels in APAP-ALF patients has not been reported. Aim: To determine whether serial samples (early; day 1 or late; day 3-5) of serum FABP1 levels are associated with 21-day mortality in the absence of LT. Methods: Nested case control study 198 APAP-ALF patients (99 survivors, 99 non-survivors) from the US Acute Liver Failure Study Group (ALFSG) Registry. Patient samples were analyzed for FABP1 using solid-phase enzyme-linked immunosorbent assay (ELISA) and assessed with clinical data. In addition, 51 healthy controls (University of Alberta) were included. Results: APAP-ALF survivors had significantly lower serum FABP1 levels on admission (238.6 vs. 690.8 ng/ml, P<0.0001) and late (148.4 vs. 612.3 ng/ml, P<0.0001) compared with non-survivors (Figure 1). Using multivariable logistic regression, increased serum FABP1 early (Log FABP1 Odds Ratio (OR) 1.31, P=0.03) and late (Log L-FABP OR 1.50, P=0.005) were associated with significantly increased 21-day mortality after adjusting for significant covariates (MELD, vasopressor use). Area under the receiver operating curve (AUROC) for early and late multivariable models were 0.778 and 0.907 respectively. The addition of FABP1 (Table 1) significantly improved the AUROC of the King’s College Criteria (KCC) (Early: 0.552 alone, 0.711 with L-FABP; Late: 0.604 alone, 0.797 with FABP1, respectively) as well as the ALFSG prognostic index (Early: 0.686 alone, 0.766 with FABP1; Late: 0.711 alone, 0.815 with FABP1, respectively). Summary: Serum levels of FABP1 in APAP-ALF patients were significantly associated with 21-day mortality measured at early and late time points after adjusting for significant covariates. FABP1 improved performance (AUROC) when combined with existing prognostic scores at serial time points. Conclusions: In patients with APAP-ALF, FABP1 showed good potential to discriminate survivors from non- survivors at multiple time-points and significantly improved models currently used in clinical practice. Validation of FABP1 as a clinical prediction tool in APAP-ALF merits further investigation. The study was sponsored by NIH grant U-01 58369 (from NIDDK) and a grant from the University of Alberta Hospital Foundation (UHF).
Nutritional status assessment in patients with chronic liver disease: a pilot study.
Annie Lamoussenerie, Cassandra Picinbono-Larose, Mélanie Tremblay, Catherine Demers, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is one of the most common complications in the increasing number of patients suffering from chronic liver disease (CLD) and is a leading cause of morbidity and mortality. Traditional tools used to evaluate nutritional status are not reliable in CLD due to limitations related to weight, which may be artificially increased by the presence of ascites, underestimating malnutrition. New strategies to assess nutritional status focusing on early malnutrition detection are an unmet clinical need. The aim of this pilot study is to describe the performance of different measures of nutrition including body mass index (BMI), handgrip strength (HGS), mid-arm muscle circumference (MAMC) in relation to subjective global assessment (SGA). Methods: In this ongoing prospective study, patients with and without CLD are recruited at Centre Hospitalier de l’Université de Montréal (St-Luc Hospital) in Canada. We assess nutritional status via: BMI, HGS as measured with a calibrated dynamometer, MAMC calculated with the mid-arm circumference and the tricipital skinfold and a modified version of the SGA for CLD patients, Spearman correlation coefficient is used to assess correlation between different tools. Results : We recruited 36 and 18 patients with and without CLD and 18 patients, respectively. Conclusions: Our preliminary results indicate that BMI and HGS, but not MAMC tend to decrease according to the nutritional status (no malnutrition, mild or moderate malnutrition) determined by SGA. Further patients are required to statistically identify a reliable tool and be able to evaluate nutritional status in cirrhosis as well as detect malnutrition in its early stage.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Marcos Ocana-Sanchez, Christopher F. Rose.
BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: The 6-week bile-duct ligation (BDL) rats, as well as sham-operated controls, were used. Obesity: To induce obesity, high-fat diet (HFD) was given for 3 weeks before BDL. Experimental groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and normal diet (ND) for 6 weeks post-BDL; 2. Lean-BDL rats received ND pre- and post-BDL; 3. Lean-Sham rats received ND pre- and post-sham surgery. Behaviour: Motor coordination, muscular strength, and recognition memory were assessed before surgery, as well as 3 and 6 weeks post-BDL or sham surgery using the RotaRod, grip-strength and object recognition tests, respectively. Body-composition (echoMRI): Fat vs lean mass, as well as free water (ascites, fluid retention), were also monitored. RESULTS: Before the surgery, body weight and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on ND (Lean-BDL and Lean-Sham). Three weeks after surgery, body-weight, fat mass, lean mass and free water were increased in Obese-BDL rats vs Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs Lean-Sham rats. Six weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost body weight, fat and lean mass, and increased free water vs Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats when compared to Lean-Sham rats. SUMMARY: HFD induces obesity features in healthy non-cirrhotic rats, and such effects are maintained in cirrhotic-BDL rats. Obesity also exacerbates and accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats, such as long-term memory, motor coordination and forelimb strength deficits. This new animal model of CLD and obesity suggests a synergic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. CONCLUSION: Obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting that these patients might be more susceptible to neuronal dysfunction and poor neurological performance. Therefore, this model of CLD and obesity will provide important clues to the underlying mechanisms of HE associated with obesity-induced cirrhosis and provide new insights into novel therapeutic strategies.
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.
Cassandra Picinbono-Larose, Annie Lamoussenerie, Mélanie Tremblay, Catherine Demers, Catherine Vincent, Geneviève Huard, Christopher F. Rose, Chantal Bémeur.
Background: Malnutrition is an important prognostic factor potentially influencing clinical outcome of patients suffering from chronic liver disease (cirrhosis; CLD). Malnutrition may increase the risk of developing other complications including hepatic encephalopathy (HE). Malnutrition in cirrhosis may also affect patient’s functional status and wellbeing or health-related quality of life (HRQOL). Management strategies focussing on nutritional status in relation to complications of cirrhosis are an unmet clinical need. We hypothesize sub-optimal nutritional status in cirrhotic patients increases the risk of developing HE and decreases HRQOL. Methods: 33 patients (outpatients and hospitalized; CHUM’s Liver Unit in Montreal, Canada) with liver cirrhosis of different etiologies and 13 healthy controls were assessed for 1) Nutritional status (Subjective Global Assessment (SGA)); 2) HE (Clinical HE Staging Scale (CHESS)); 3) HRQOL (Short-Form-36 (SF-36) questionnaire). Results: This on-going prospective study included 33 cirrhotic patients (58% men) of various etiologies (30% alcohol, 33 % virus, 27% NASH and 33% others), Child-Pugh A/B/C (13A, 9B, 5C and 6 N/A), mean age 55,7±12,9 as well as 13 healthy controls (46% men, mean age 49,4±14,9). SGA analysis revealed that 27% of cirrhotic patients were malnourished in which 22% of them malnourished showed signs of HE as assessed by CHESS. Cirrhotic patients, when compared to controls, displayed a lower score in physical functioning (p=0.03) and general health (p=0.03), both part of the physical health domain of SF-36. Furthermore, cirrhotic malnourished patients show decreased physical aspects of HRQOL compared to well-nourished cirrhotic patients (p<0.01). Conclusion: Our preliminary results suggest that nutritional status does influence particular domains of HRQOL in cirrhotic patients irrelevant of their etiology. Further patients are required to statistically confirm the impact of nutritional status on HE and HRQOL in cirrhotic patients. Identifying factors associated with nutritional status, HRQOL and HE in cirrhotic patients may help improve patient care and guide future research.
