The brain in obesity-induced liver disease.
Post-doctoral, Neurosciences, Université de Montréal
Direction:
- Dr Christopher Rose
2015 - 2021
Related Publications
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major complication of chronic liver disease (CLD). Defined as a metabolic disorder, HE should resolve following liver transplantation (LT). However, persisting neurological complications have been reported in up to 47% of LT recipients. Retrospective studies have demonstrated an association between a history of HE episodes and neu-rological deficits post-LT. Our aim was to evaluate the impact of multiple episodes (induced by ammonia) on neurological status, integrity and injury in rats with CLD. Methods: 5-week bile-duct ligation (BDL) rats were in-jected (ip) with ammonium acetate (BDL-Ammonia), pre-cipitating an overt episode of HE (loss of righting reflex). Episodes of HE were induced every 4 days from week 3 post-BDL surgery (total; 4 episodes). Sham rats were also injected with a similar dose of ammonia (Sham-Ammonia) and respective controls were injected with sa-line. Three days after the last episode, long-term mem-ory (LTM) was assessed. Upon sacrifice, plasma and brains were collected for oxidative stress measurements and western blot analysis for NeuN and SMI311 (neuro-nal markers), caspase-3 and Bax/Bcl2 ratio (markers of apoptosis), GFAP (astrocyte marker) and 4-HNE (marker of oxidative stress) in frontal cortex, cerebellum and hip-pocampus. Results: LTM was found to be impaired in both BDL-Saline and BDL-Ammonia groups vs respec-tive controls. However, LTM impairment was further ag-gravated in BDL-Ammonia rats. In BDL-Ammonia, higher protein levels of GFAP and apoptotic markers were found in the hippocampus, whereas NeuN and SMI311 levels were reduced compared to all other experimental groups. BDL-Ammonia rats showed increased levels of plasma oxidative stress compared to respective Sham and BDL rats. Decreased levels of total antioxidant capacity as well as increased 4-HNE were found solely in the hip-pocampus (not frontal cortex or cerebellum) of the BDL-Ammonia group compared to controls. Conclusion:Multiple episodes of overt HE leads to a worsening of neurological impairment in BDL rats. LTM impairment in the BDL-Ammonia rats was associated with increased oxidative stress, apoptotic markers and decreased neu-ronal markers in the hippocampus, supporting neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuate gliosis, possibly a result of neuronal loss. These results suggest that repeated HE episodes may cause permanent cell damage which will less likely be revers-ible following LT.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background/Aims: Hepatic encephalopathy (HE) is a neuro¬psychiatric syndrome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypothesize that episodes of HE will accelerate neurological deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neurological status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days by injection of ammonium acetate starting week 3 post-BDL. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis of NeuN, SMI311, caspase-3, Bax/Bcl2 and GFAP. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP, cleaved-caspase-3 and Bax/Bcl2 protein expression were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and Bax/Bcl2 and a decrease in neuronal markers of NeuN and SMI311 in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis because of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background/Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypothesize that episodes of HE will accelerate neurological deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neurological status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days by injection of ammonium acetate starting week 3 post-BDL. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis of NeuN, SMI311, caspase-3, Bax/Bcl2 and GFAP. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP, cleaved-caspase-3 and Bax/Bcl2 protein expression were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and Bax/Bcl2 and a decrease in neuronal markers of NeuN and SMI311 in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis because of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a major complication of chronic liver disease (CLD). HE is defined as a metabolic syndrome and therefore should resolve following liver transplantation (LT). However, persisting neurological complications and poor quality of life have been reported in up to 47% of LT recipients. Several retrospective studies have demonstrated an association between a history of HE episodes and neurological deficits following LT. However, the impact of HE episodes on the brain remains unknown. Purpose: Our aim was to evaluate the impact of multiple episodes (induced by ammonia) on neurological status, integrity and brain injury in cirrhotic rats. Method: 5-week bile-duct ligation (BDL) rats and Sham-operated controls (Sham) were used. BDL rats were injected (ip) with ammonium acetate (BDL-Ammonia), precipitating an overt episode of HE (pre-coma; loss of righting reflex) every 4 days from week 3 post-BDL surgery (total; 4 episodes). Sham rats were also injected with ammonia (Sham-Ammonia) and BDL and Sham rats were injected with saline as controls. Three days after the last episode, both short- and long-term memory (LTM) were assessed. Upon sacrifice, plasma and brains were collected for oxidative stress measurements and western blot analysis for Neuronal nuclei (NeuN and SMI311), caspase-3 (apoptotic marker), Bax/Bcl2 ratio (apoptotic marker), GFAP (astrocyte marker) and 4-HNE (oxidative stress marker) in frontal cortex, cerebellum and hippocampus. Result(s): LTM was found to be impaired in both BDL-Saline and BDL-Ammonia groups vs respective Sham controls. However, LTM impairment was further aggravated in BDL-Ammonia rats. In BDL-Ammonia, higher protein levels of GFAP and apoptotic markers were found in the hippocampus, whereas NeuN and SMI311 levels were reduced compared to all other experimental groups. BDL-Ammonia rats showed increased levels of plasma oxidative stress compared to respective Sham and BDL rats. Decreased levels of total antioxidant capacity and increased 4-HNE (oxidative stress marker) were found in the hippocampus (not in frontal cortex or cerebellum) of the BDL-Ammonia group compared to respective sham and BDL rats. Conclusion(s): Multiple episodes of overt HE lead to a worsening of the neurological impairments in BDL rats. LTM impairment in the BDL-Ammonia rats was associated with increased oxidative stress, apoptotic markers (caspase-3, Bax/Bcl2) and decreased neuronal markers (NeuN, SMI311) in the hippocampus, suggesting neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuate gliosis, possibly a result of neuronal loss. These results suggest that cumulative HE episodes may cause permanent cell damage and, therefore, will less likely reversible following LT. The impact of HE on neurological outcomes following LT merits further investigation.
Ammonia-induced episodic HE leads to neuronal cell injury in bile-duct ligated rats
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background/Aims: Hepatic encephalo¬pathy (HE) is a neuro¬psychiatric syn¬drome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypo¬thesize that episodes of HE will accelerate neuro¬logical deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neuro¬logical status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days by injection of ammonium acetate starting week 3 post-BDL. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis of NeuN, SMI311, caspase-3, Bax/Bcl2 and GFAP. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP, cleaved-caspase-3 and Bax/Bcl2 protein expression were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and Bax/Bcl2 and a decrease in neuronal markers of NeuN and SMI311 in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis because of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Episodic hepatic encephalopathy induces neuronal cell injury in rats with chronic liver disease
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalo¬pathy (HE) is a neuro¬psychiatric syn¬drome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications which is believed to be associated with a history of HE pre-LT. We hypo¬thesize that episodes of HE will accelerate neuro¬logical deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes (ammonia induced) on neuro¬logical status and brain injury in rats with chronic liver disease (CLD). Methods: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were grouped into episodic and non-episodic groups. Episodes of HE were induced every 4 days starting the week 3 post-BDL following a reversible injection of ammonium acetate in which BDL rats fell into pre-coma (loss of righting reflex) for 20-30min. Three days following the last injection, neurobehavior was assessed. Upon sacrifice, plasma ammonia was measured, and brains were collected for western blot and immunofluorescence analysis; neuronal markers including NeuN and SMI311, astrocytic marker; GFAP and apoptotic markers such as cleaved caspase-3 and Bax and Bcl2 were measured to evaluate neurological integrity. Results: BDL rats, and not Sham rats, injected with ammonia developed overt HE. Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls. However, LTM impairment was further aggravated in episodic BDL rats. Protein expression levels of GFAP, cleaved caspase-3 and Bax/Bcl2 ratio were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL rats vs non-episodic BDL rats. Presence of cleaved caspase-3 in neuronal layer of CA1 area of hippocampus was confirmed with immunofluorescence imaging. Blood ammonia levels were found to be higher in episodic vs non-episodic BDL rats. Conclusion: Episodes of overt HE exasperate neurological impairment in BDL rats. LTM impairment was associated with an increase in apoptotic markers, decrease in neuronal markers and presence of cleaved caspase-3 in neuronal layer of CA1 area of hippocampus, suggesting neuronal injury/loss. Elevated levels of GFAP (astrocytic marker) in the hippocampus insinuates gliosis, possibly as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to therefore irreversible and persisting neurological complications post-LT.
Hepatic Encephalopathy: From Metabolic to Neurodegenerative.
Rafael Ochoa-Sanchez, Farzaneh Tamnanloo, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome of both acute and chronic liver disease. As a metabolic disorder, HE is considered to be reversible and therefore is expected to resolve following the replacement of the diseased liver with a healthy liver. However, persisting neurological complications are observed in up to 47% of transplanted patients. Several retrospective studies have shown that patients with a history of HE, particularly overt-HE, had persistent neurological complications even after liver transplantation (LT). These enduring neurological conditions significantly affect patient's quality of life and continue to add to the economic burden of chronic liver disease on health care systems. This review discusses the journey of the brain through the progression of liver disease, entering the invasive surgical procedure of LT and the conditions associated with the post-transplant period. In particular, it will discuss the vulnerability of the HE brain to peri-operative factors and post-LT conditions which may explain non-resolved neurological impairment following LT. In addition, the review will provide evidence; (i) supporting overt-HE impacts on neurological complications post-LT; (ii) that overt-HE leads to permanent neuronal injury and (iii) the pathophysiological role of ammonia toxicity on astrocyte and neuronal injury/damage. Together, these findings will provide new insights on the underlying mechanisms leading to neurological complications post-LT.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome observed in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids and brain lipid alterations to neurological impairment, we hypothesize that obese-induced brain lipid dysregulation increases the progression of HE in bile-duct ligated (BDL) rats. Aim: Study the impact of obesity on brain sphingolipids and the development of neurological impairment in a rat model of CLD and obesity. Method: CLD and HE model: 5-week BDL rats and Sham-controls, were used. Groups: Obese-BDL (O-BDL) and Obese-Sham (O-Sham) received high-fat diet (HFD) for 25-days pre-BDL surgery and then proceeded with high-carbohydrate diet (HCD) for 5 weeks post-BDL; Lean-BDL (L-BDL) and Lean-Sham (L-Sham) received regular-diet (RD) pre-BDL surgery and then HCD post-BDL. Body weight (BW), fat-mass, behavior, plasma liver markers and brain hippocampal sphingolipids were measured at 0 (pre-BDL), 3 and 5 weeks post-BDL. Results: Pre-BDL, HFD lead to increased BW and fat mass vs RD which was sustained with HCD in O-BDL vs L-BDL at 3 and 5 weeks. Ammonia, albumin, AST, ALT, ALP and bilirubin were impaired in O-BDL and L-BDL vs Shams at 3 and 5 weeks. High-density and low-density (LDL) lipoprotein levels were detected in pre-BDL rats which persisted in O-BDL and L-BDL vs controls at 3 and 5 weeks. LDL and total-cholesterol were higher in O-BDL vs L-BDL at 5 weeks. Short- and long-term (LTM) memory were impaired in both BDL groups at 5 weeks, whereas at 3 weeks, LTM impairment was found solely in O-BDL. Furthermore at 3 weeks, motor coordination was reduced in O-BDL, but not in L-BDL. At 5 weeks, motor coordination decreased in both BDL groups, with worse performance in O-BDL vs L-BDL. At 3 and 5 weeks, skill learning improved in L-Shams and L-BDL, but not in O-Shams and O-BDL. Muscle-strength was reduced in L-BDL but not in O-BDL vs Shams. Hippocampal sphingomyelin lipids were increased in O-BDL vs L-BDL rats at 0, 3 and 5 weeks, while ceramides were only reduced at 5 weeks. Conclusion: CLD-obesity rat model is characterized by increased fat mass and hyperlipidemia pre- and post-BDL. Neurological decline in O-BDL rats developed earlier and was more severe vs L-BDL rats. In addition, different neurological impairments developed in O-BDL vs L-BDL. Brain sphingolipid alteration in obese-cirrhotic rats may contribute to accelerate/worsen HE.
Ammonia-induced episodic HE leads to neuronal cell injury in bile-duct ligated rats.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalo¬pathy (HE) is a neuro¬psychiatric syn¬drome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypo¬thesize that episodes of HE will accelerate neuro¬logical deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes on neuro¬logical status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats, and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days starting the week 3 post-BDL following injection of ammonium acetate. 3 days following the last injection, neurological status was assessed. Upon sacrifice brains were collected for western blot analysis; Neuronal nuclei (NeuN), caspase-3 and GFAP were measured. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP and caspase-3 protein expression were significantly increased, whereas NeuN was significantly decreased in hippocampus of episodic BDL rats vs non-episodic BDL rats. Conclusion: HE episodes exacerbates neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and a decrease in NeuN in the hippocampus which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Ammonia-induced episodic HE leads to neuronal cell injury in bile-duct ligated rats.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications associated with a history of overt HE episodes. We hypothesize that episodes of HE will accelerate neurological deterioration. Our goal was to evaluate the impact of cumulative HE episodes on neurological status and brain injury in cirrhotic rats. Method: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were divided into episodic and non-episodic groups. Episodes of HE were induced every 4 days starting week 3 post-BDL following injection of ammonium acetate. 3 days following the last injection, neurological status was assessed. Upon sacrifice, brains were collected for western blot analysis; Neuronal nuclei (NeuN), caspase-3, and GFAP were measured. Results: Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls and was further aggravated in episodic BDL rats. Both GFAP and caspase-3 protein expression were significantly increased, whereas NeuN was significantly decreased in the hippocampus of episodic BDL rats vs non-episodic BDL rats. Conclusion: HE episodes exacerbate neurological impairments in BDL rats. LTM impairment was associated with an increase in caspase-3 and a decrease in NeuN in the hippocampus, which suggests neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to persisting neurological complications post-LT.
Dysregulation of sphingolipids in obese-cirrhotic rats aggravates hepatic encephalopathy.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome observed in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids and brain lipid alterations to neurological impairment, we hypothesize that obese-induced brain lipid dysregulation increases the progression of HE in bile-duct ligated (BDL) rats. Aim: Study the impact of obesity on brain sphingolipids and the development of neurological impairment in a rat model of CLD and obesity. Method: CLD and HE model: 5-week BDL rats and Sham-controls, were used. Groups: Obese-BDL (O-BDL) and Obese-Sham (O-Sham) received high-fat diet (HFD) for 25-days pre-BDL surgery and then proceeded with high-carbohydrate diet (HCD) for 5 weeks post-BDL; Lean-BDL (L-BDL) and Lean-Sham (L-Sham) received regular-diet (RD) pre-BDL surgery and then HCD post-BDL. Body weight (BW), fat-mass, behavior, plasma liver markers and brain hippocampal sphingolipids were measured at 0 (pre-BDL), 3 and 5 weeks post-BDL. Results: Pre-BDL, HFD lead to increased BW and fat mass vs RD which was sustained with HCD in O-BDL vs L-BDL at 3 and 5 weeks. Ammonia, albumin, AST, ALT, ALP and bilirubin were impaired in O-BDL and L-BDL vs Shams at 3 and 5 weeks. High-density (HDL) and low-density (LDL) lipoprotein levels were detected in pre-BDL rats which persisted in O-BDL and L-BDL vs controls at 3 and 5 weeks. LDL and total-cholesterol were higher in O-BDL vs L-BDL at 5 weeks. Short- and long-term (LTM) memory were impaired in both BDL groups at 5 weeks, whereas at 3 weeks, LTM impairment was found solely in O-BDL. Furthermore at 3 weeks, motor coordination was reduced in O-BDL, but not in L-BDL. At 5 weeks, motor coordination decreased in both BDL groups, with worse performance in O-BDL vs L-BDL. At 3 and 5 weeks, skill learning improved in L-Shams and L-BDL, but not in O-Shams and O-BDL. Muscle-strength was reduced in L-BDL but not in O-BDL vs Shams. Hippocampal sphingomyelin lipids were increased in O-BDL vs L-BDL rats at 0, 3 and 5 weeks, while ceramides were only reduced at 5 weeks. Conclusion: CLD-obesity rat model is characterized by increased fat mass and hyperlipidemia pre- and post-BDL. Neurological decline in O-BDL rats developed earlier and was more severe vs L-BDL rats. In addition, different neurological impairments developed in O-BDL vs L-BDL. Brain sphingolipid alteration in obese-cirrhotic rats may contribute to accelerate/worsen HE.
Repeated hyperammonemic insults leads to irreversible brain injury in bile-duct ligated rats.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Background: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD). Defined as a metabolic disorder, HE is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications. Retrospective studies have revealed that patients with a history of overt HE are associated with a poor neurological outcome post-LT. However, the impact of HE episodes on neuro¬logical in¬tegrity is unknown. We hypothesize that episodes of HE will accelerate and/or exasperate neurological deterioration. Purpose: Our goal was to characterize an animal model of episodic HE and to evaluate the impact of cumulative episodes induced by hyperammonemia on neurological status and brain injury in cirrhotic rats. Method: For animal model of CLD and HE, 5-week bile-duct ligation (BDL) rats, and Sham-operated controls were used. Rats, both Sham and BDL, were divided in two groups, episodic and non-episodic. Injection (ip) of ammonium acetate was used to induce episodes of overt HE (pre-coma; loss of righting reflex) every 4 days starting at 3-weeks post-BDL surgery (total 4 episodes). Saline was injected as vehicle for non-episodic group. Three days following the last episode of HE, neurological status was assessed. Upon sacrifice, plasma ammonia was measured, and brains were collected for western blot analysis; Neuronal nuclei (NeuN), caspase-3 (apoptotic marker) and GFAP (astrocyte marker) were measured to evaluate neurological integrity. Results: BDL rats (vs Sham) following ammonia insult developed overt HE (loss of righting reflex). Long-term memory (LTM) was impaired in both non-episodic and episodic BDL groups vs respective controls. However, LTM impairment was further aggravated in episodic BDL rats. Both GFAP and cleaved caspase-3 protein expression were significantly increased, whereas NeuN was significantly decreased in hippocampus of episodic BDL rats vs non-episodic BDL rats. Blood ammonia levels were found to be higher in episodic vs non-episodic BDL rats. Conclusion: HE episodes exasperate neurological impairments in BDL rats. LTM impairment was associated with an increase in the apoptotic marker (caspase-3) and a decrease in neuronal marker (NeuN) in the hippocampus, suggesting neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuates gliosis as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage and therefore will less likely be reversible following LT justifying persisting neurological complications post-LT.
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.
An Investigation of PS‐b‐PEO Polymersomes for the Oral Treatment and Diagnosis of Hyperammonemia
Simon Matoori, Yinyin Bao, Aaron Schmidt, Eric J. Fischer, Rafael Ochoa-Sanchez, Mélanie Tremblay, Mariana M. Oliveira, Christopher F. Rose, Jean‐Christophe Leroux.
Ammonia-scavenging transmembrane pH-gradient poly(styrene)-b-poly(ethylene oxide) polymersomes are investigated for the oral treatment and diagnosis of hyperammonemia, a condition associated with serious neurologic complications in patients with liver disease as well as in infants with urea cycle disorders. While these polymersomes are highly stable in simulated intestinal fluids at extreme bile salt and osmolality conditions, they unexpectedly do not reduce plasmatic ammonia levels in cirrhotic rats after oral dosing. Incubation in dietary fiber hydrogels mimicking the colonic environment suggests that the vesicles are probably destabilized during the dehydration of the intestinal chyme. The findings question the relevance of commonly used simulated intestinal fluids for studying vesicular stability. With the encapsulation of a pH-sensitive dye in the polymersome core, the local pH increase upon ammonia influx could be exploited to assess the ammonia concentration in the plasma of healthy and cirrhotic rats as well as in other fluids. Due to its high sensitivity and selectivity, this polymersome-based assay could prove useful in the monitoring of hyperammonemic patients and in other applications such as drug screening tests.
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.
Developing a New Animal Model of Episodic Hepatic Encephalopathy.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). The primary cause of hospital admissions for cirrhotic patients is an overt episode of HE. Precipitating factors of HE frequently lead to an increase in blood ammonia. Patients who have experienced multiple episodes of HE are associated with persisting neurological complications post-liver transplantation. Currently, the impact of HE episodes on neurological integrity is unknown. We hypothesize that multiple episodes of HE will accelerate and/or intensify neurological deterioration. To date, an animal model of episodic HE is lacking. Therefore, our goal was to characterize an animal model of episodic HE (precipitated with ammonia) and to evaluate the impact of cumulative episodes on neurological status in cirrhotic rats. Material and Methods: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, and Sham-operated controls were used. BDL and Sham rats were divided in two groups, episodic and non-episodic. Injection (i.p) of ammonium acetate was used to induce episodes of overt HE (pre-coma; loss of righting reflex) every 4 days starting 3-weeks post-BDL surgery (total 5 episodes). Saline was injected as vehicle for non-episodic groups. Two days following the last HE episode, we assessed motor-coordination (RotaRod), anxiety (elevated plus maze, EPMT), as well as short-term and long-term memory (novel object recognition) in all groups. Upon sacrifice, plasma ammonia was measured. Results: The concentration of ammonia required to induce an episode of overt HE in BDL rats lessened with each subsequent episode, ranging from 7 to 4.5 mmol/kg. Short-term memory (p<0.05) and motor-coordination (p<0.05) were impaired in both non-episodic and episodic BDL groups compared to respective Sham-operated controls. Long-term memory impairment (p=0.06) and increased anxiety (+60.0%, p<0.05) were exclusively found in episodic BDL rats compared to non-episodic BDL rats. Moreover, there was an increase in blood ammonia (+30.4%, p=0.06) in episodic compared to non-episodic BDL rats, suggesting that although episodic-BDL rats recover from each HE episode, baseline (pre-episode) ammonia remain higher than non-episodic BDL rats. Conclusion: Cumulative HE episodes escalate neurological impairments in cirrhotic-BDL rats. Thus, this new episodic HE model represents a good approach to explore the pathological mechanism arising from multiple episodes, as well as further investigate whether higher hyperammonemia and/or increased brain sensitivity to ammonia is responsible for more complex neurological manifestations in episodic-BDL rats. Moreover, this model is an excellent platform to investigate novel therapies to prevent/treat episodic HE.
Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mylene Perreault, Bill Querbes, Caroline B. Kurtz, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome observed in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Investigate the synergistic effect of obesity and CLD on the development of neurological impairment in a novel rat model of cirrhosis and obesity. M&M: Animal model of CLD and HE: 5-week bile-duct ligation (BDL) rats and Sham-operated controls, were used. Groups: Obese-BDL and Obese-Sham received high-fat diet (HFD) for 25-days pre-BDL and high-carbohydrate diet (HCD) for 5-weeks post-BDL; Lean-BDL and Lean-Sham received regular-diet (RD) pre-BDL and HCD post-BDL. Body-weight and fat-mass (EchoMRI) were monitored pre-BDL as well as 3- and 5-weeks post-BDL. Behavior: Motor-coordination, motor skill-learning, and muscular-strength were assessed at 3- and 5-weeks post-BDL. Locomotion and anxiety were measured at 5-weeks. Plasma ammonia, liver enzymes, and lipids were measured at 3- and 5-weeks. RESULTS: Before BDL surgery, body-weight and fat-mass of rats on HFD increased compared to rats on RD. 3-week post-BDL, body-weight and fat-mass decreased in Lean-BDL and Obese-BDL vs respective Shams, while at 5-weeks this was only found in Lean-BDL. These parameters were higher in Obese-BDL vs Lean-BDL at 3- and 5-weeks. Plasma ammonia, bilirubin, albumin, ALT, AST, and ALP were impaired in Obese- and Lean-BDL vs respective Shams at 3- and 5-weeks. AST and ALP increased in Obese-BDL vs Lean-BDL at 5-weeks. Elevated HDL-cholesterol and decreased LDL-cholesterol were detected in Obese-BDL and Lean-BDL vs respective Shams at 3- and 5-weeks, while LDL-cholesterol was higher in Obese-BDL vs Lean-BDL at 5-weeks. Total-cholesterol increased in Obese-BDL vs all groups at 5-weeks. At 3 weeks; motor-coordination was reduced in Obese-BDL, but not in Lean-BDL vs respective Shams, while at 5-weeks, motor-coordination decreased in both Lean-BDL and Obese-BDL vs respective Shams, with worse performance in Obese-BDL vs Lean-BDL. At 3-weeks, skill-learning improved in all Shams and Lean-BDL, but not in Obese-BDL; at 5-weeks contrary to Sham-groups, both BDL groups did not improve performance. Muscle-strength decreased in Lean-BDL and Obese-BDL vs respective Shams at 3- and 5-weeks. Hypolocomotion and anxiogenic effects were detected in Obese-BDL, but not in Lean-BDL vs Shams at 5-weeks. CONCLUSION: HFD induces obesity pre-BDL which is maintained post-BDL with a HCD-diet which was accompanied with increase fat-mass and hyperlipidemia. Neurological decline in obese-cirrhotic rats developed earlier and was more severe versus Lean-BDL rats. Besides, some neurological impairments developed in Obese-BDL but not in Lean-BDL. These results suggest a synergistic effect, which accelerates/worsens the disease-associated abnormalities in CLD and HE.
Rafael Ochoa-Sanchez, Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mélanie Tremblay, Mylene Perreault, Bill Querbes, Caroline B. Kurtz, Christopher F. Rose.
Background: Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) production that contributes to systemic hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering circulating NH3. As a therapeutic strategy, Escherichia coli Nissle 1917 bacterium (EcN), a well characterized probiotic, was genetically modified to consume and convert NH3 to arginine (SYNARG), and its administration to thioacetamide-treated mice resulted in a significant reduction of NH3 levels1. SYNARG was further modified to synthesize butyrate (SYNARG+BUT), a short-chain fatty acid with anti-inflammatory/anti-oxidant properties, and both strains were tested in an experimental model of cirrhosis and HE, the bile duct ligation (BDL). Methods: One week post surgery, BDL rats were gavaged with SYNARG, SYNARG+BUT (3x1011 CFU/day, BID) or vehicle until they were sacrificed at 3- or 5-weeks along with respective sham controls. Plasma NH3 and liver markers were measured at 3 and 5 weeks. Recognition-memory, motor-coordination, muscle-strength, locomotion and anxiety were assessed in the 5-week BDL groups. Results: BDL significantly increased NH3 over time, with levels of 109.1±9.2µM (Shams 56.7±3.5µM, p<0.001) and 150.2±25.6µM (Shams 58.3±3.0µM, p<0.001) at 3- and 5-weeks, respectively. In addition, plasma liver markers alanine-transaminase, aspartate-transaminase, bilirubin, and gamma-glutamyl transferase were significantly increased in BDL rats at both timepoints while albumin was significantly lowered. As compared to BDL-Veh rats, hyperammonemia was attenuated by SYNARG (103.9±12.3µM) and SYNARG+BUT (110.8±8.5µM) at 5, but not 3 weeks post-surgery, while liver fibrosis (hydroxyproline content) was attenuated at 3, but not 5 weeks post-surgery. None of the circulating liver markers were changed by the treatments at any timepoint. Motor-coordination, muscle-strength, locomotion and anxiety were affected in all BDL groups without protective effect of treatments. Short-term memory (STM) was impaired in BDL-Veh (p<0.001) and BDL-SYNARG (p<0.05) versus Shams, while STM was resolved in BDL-SYNARG+BUT (p<0.05 vs BDL-Veh). Long-term memory (LTM) was impaired in BDL-Veh vs Shams (p<0.05), but BDL-SYNARG and BDL-SYNARG+BUT were protected. Conclusion: EcN, engineered to consume NH3 in the gut and synthesize butyrate, is an effective approach to lower plasma NH3 in a model of cirrhosis and HE. Moreover, the attenuation of hyperammonemia in cirrhotic rats is associated with a protective effect on memory in this model. The therapeutic potential of these engineered EcN strains should be further evaluated in patients with CLD and HE.
Characterization of a novel animal model of episodic hepatic encephalopathy.
Farzaneh Tamnanloo, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). The primary cause of hospital admissions for cirrhotic patients is an overt episode of HE. Precipitating factors of HE are frequently due to increased blood ammonia. Patients with history of multiple episodes of HE experienced persisting neurological complications post-liver transplantation. Nevertheless, the impact of HE episodes on neurological integrity is unknown. We hypothesize that multiple episodes of HE will accelerate and/or intensify neurological deterioration. To date, an animal model of episodic HE is lacking. Therefore, our goal was to characterize an animal model of episodic HE (precipitated with ammonia) and to evaluate the impact of cumulative episodes on neurological status in cirrhotic rats. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, and Sham-operated controls were used. Ammonium acetate was used to induce HE episodes, every 4 days starting at 3-weeks post-surgery (total 5 episodes). After the last episode, we assessed motor-coordination (RotaRod), anxiety (elevated plus maze), as well as, short-term and long-term memory (novel object recognition). Rats were then sacrificed, and plasma ammonia measured. Results: Short-term memory (p<0.05) and motor-coordination (p<0.05) were reduced in both non-episodic and episodic BDL groups when compared Sham-operated controls. Long-term memory impairment (p=0.06) and increased anxiety (+60.0%, p<0.05) were found only in episodic vs non-episodic BDL rats. Moreover, there was an increase in blood ammonia (+30.4%, p=0.06) in episodic BDL vs non-episodic BDL rats. Conclusion: The induction of HE episodes escalates neurological impairments in cirrhotic rats. Thus, this new episodic HE model represents a good approach to explore the pathological mechanism arising from multiple episodes, as well as further investigate brain sensitivity to ammonia. Moreover, this model is an excellent platform to investigate novel therapies to prevent or treat episodic HE.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. Aim: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Model of CLD and HE: 6-week bile-duct ligation (BDL) rats and Sham-controls were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Obese-BDL received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; Lean-BDL received RD pre-/post-BDL; Lean-Sham received RD pre-/post-Sham. Recognition memory, motor-coordination, muscular-strength and body-composition (fat vs lean mass) were assessed before, 3 and 6 weeks post-surgery. Results: Before surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased vs rats on RD (Lean-BDL, Lean-Sham). 3 weeks post-surgery, BW, fat and lean mass were increased in Obese-BDL vs. Lean-BDL. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham. 6 weeks post-surgery, similar to Lean-BDL, Obese-BDL lost BW, fat and Lean mass vs. Lean-Sham. Motor-coordination, forelimb strength and long-term memory were impaired in Obese-BDL vs Lean-BDL or Lean-Sham, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL. Conclusion: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats, while others are exacerbated. A synergistic effect of obesity and CLD accelerates/worsens the disease-associated abnormalities observed in HE, suggesting more susceptibility to poor neurological performance in obese-induced cirrhotic patients.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, as well as Sham-operated controls, were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; 2. Lean-BDL rats received RD pre- and post-BDL; 3. Lean-Sham rats received RD pre- and post-Sham surgery. Behaviour: Recognition memory, motor coordination and muscular strength were assessed before surgery, as well as 3 and 6 weeks post-surgery using the novel object recognition, rotarod and grip-strength tests, respectively. Body-composition (echoMRI): Fat vs. lean mass and free water (ascites) were also monitored. RESULTS: Before the surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on RD (Lean-BDL and Lean-Sham). 3 weeks after surgery, BW, fat mass, lean mass and free water were increased in Obese-BDL rats vs. Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham rats. 6 weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost BW, fat and Lean mass, while free water increased vs. Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats, compared to Lean-Sham rats. CONCLUSION: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Obesity also accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats (long-term memory), while others are exacerbated (motor coordination, forelimb strength). This new animal model of CLD and obesity suggests a synergistic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. Thus, obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting more susceptibility to poor neurological performance.
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.
Rafael Ochoa-Sanchez, Mariana Oliveira, Grégory Petrazzo, Yossi Dagon, Kip West, Lauren Renaud, Caroline Kurtz, Christopher F. Rose.
Background: Hyperammonemia associated with chronic liver disease (cirrhosis) plays a major role in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) that contributes to systemic hyperammonemia in HE. Probiotic bacteria have shown benefits in the treatment of HE although the underlying mechanism(s) are not completely understood. We engineered Escherichia coli Nissle 1917 bacterium (EcN) to consume NH3 and convert it to arginine in the gut. To enhance the beneficial effect of NH3 consumption, we further engineered the EcN to synthesize the short chain fatty acid butyrate in the gut. The resulting strains, SYNARG (arginine producing) and SYNARG+BUT (arginine and butyrate producing) were tested in two experimental models of cirrhosis and HE: thioacetamide (TAA) or bile duct ligation (BDL). Methods: Cirrhosis was induced in BALB/c mice by intraperitoneal treatment with TAA for 4 weeks, and in Sprague-Dawley rats by BDL for 5 weeks. TAA-treated mice were gavaged with a daily dose of 1 x 1010 colony forming units (CFU) of SYNARG. BDL rats were gavaged with 1 x 1012 CFU with SYNARG or SYNARG+BUT for 4 weeks. Plasma NH3 was measured in both models at baseline (BL) and after treatment, with an additional measurement at 2 weeks post treatment (3 weeks post-BDL). Results: TAA mice developed hyperammonemia (BL: 22.4 ± 3.3 umol/L to 4 weeks: 82.4 ± 8.7 umol/L, p<0.05) which was attenuated after SYNARG treatment (45.5 ± 4.7 umol/L, p<0.05). Longitudinal analysis in Vehicle-BDL rats developed hyperammonemia at 3 weeks (BL: 68.8 ± 5.7 to 121.6 ± 9.8 umol/L, p<0.01) which was further increased after 5 weeks (158.8 ± 22.0 umol/L, p<0.001 vs BL and 3 weeks). At 3 weeks, SYNARG+BUT prevented a significant increase in blood NH3 in BDL rats (99.6 ± 8.7 umol/L, p<0.05 vs BL). Both SYNARG+BUT (115.9 ± 17.2 umol/L) and SYNARG (127.9 ± 15.5 umol/L) were protective in preventing further increase in blood NH3 from 3 to 5 weeks, as observed in Vehicle-BDL rats. Moreover, the analysis between groups at 5 weeks showed that SYNARG+BUT reduces NH3 compared to Vehicle-BDL rats (p<0.05). Conclusion: Our data suggest that EcN, engineered to consume NH3 in the gut, is an effective approach to lower plasma NH3 in models of cirrhosis and hyperammonemia. Based on these results, the therapeutic potential of these engineered EcN strains should be further evaluated in patients with liver disease and HE.
Progressive resistance training prevents loss of muscle mass and strength in bile duct ligated rats.
Mariana M. Oliveira, Christopher F. Rose, Luise Aamann, Rafael Ochoa-Sanchez, Mariana Oliveira, Mélanie Tremblay, Chantal Bémeur, Gitte Dam, Hendrik Vilstrup, Niels Kristian Aagaard, Christopher Rose.
Loss of muscle mass and strength is common in cirrhosis and increases the risk of hyperammonemia and hepatic encephalopathy. Resistance training optimizes muscle mass and strength in several chronic diseases. However, the beneficial effects of resistance training in cirrhosis remains to be investigated. Bile duct ligated (BDL)-rats develop chronic liver disease, hyperammonemia, reduced muscle mass and strength. Our aim was to test the effects of resistance training on muscle mass, function and ammonia metabolism in BDL-rats. A group of BDL-rats underwent a progressive resistance training program and a group of non-exercise BDL-rats served as controls. Resistance training comprised of ladder climbing with a progressive increase in carrying weights attached to the tail. Training was performed five days a week during four weeks. Muscle strength and body composition were assessed using grip strength and EchoMRI. Weight and circumference of the gastrocnemius muscle (normalized to bodyweight), plasma ammonia and glutamine synthetase protein expression and activity were assessed. BDL+exercise rats had significantly larger gastrocnemius circumference compared to non-exercise BDL-rats: ratio 0.082 vs. 0.075 (p<0.05). Gastrocnemius muscle weight was higher in exercisers than controls: 0.006 vs. 0.005 (p<0.05). A tendency towards a lower plasma ammonia in the exercise group compared to controls was observed (p=0.10). There were no differences in lean body mass, GS protein expression and activity between the groups. Resistance training in rats with chronic liver disease beneficially effects muscle mass and strength. The effects were followed by non-significant reduction in blood ammonia, however a tendency was observed. This article is protected by copyright. All rights reserved.
Pathogenesis of Hepatic Encephalopathy in Chronic Liver Disease.
Rafael Ochoa-Sanchez, Christopher F. Rose.
Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that occurs during chronic liver disease (CLD). While ammonia and other precipitating factors in liver disease including inflammation, bile acids, oxidative stress, and lactate play a role in the pathogenesis of HE, the exact mechanism that leads to HE is not fully understood. Notably, accumulating evidence points toward a synergic effect rather than independent actions among precipitating factors that contributes to the development and severity of HE in CLD. Hence, this review is aimed to briefly discuss the single and synergic interplay of pathological factors in the progression and severity of HE.
Grégory Petrazzo, Mariana M. Oliveira, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a common and severe complication of liver failure. The pathogenesis of HE is linked with gut-derived ammonia (NH3). The standard care for patient experiencing episodes of HE is lactulose but observance is poor due to uncomfortable side-effects. Rifaximin is a potent candidate but there is no clinical study that assess its efficiency solely. This study aim to assess, in a bile-duct ligation (BDL) model of HE, the efficiency of rifaximin to reduce plasma NH3 and ameliorate HE status. 3 wks after BDL surgery, all animals were sorted in 5 grps: SHAM-Veh, BDL-Veh, BDL-Lac, BDL-Rif, BDL-Lac+Rif. Ttm was given by gavage for 3 wks with vehicle, lactulose, rifaximin and the combination of both ttm. Survival, body-weight, food consumption and body composition was assess every wk. During the ttm, behavioral analysis was done to assess the HE status, including OFT, EPM, Rotarod, NOR and night activity. At the end of the study, brain water was measured and plasma sample was taken. No difference was seen in the survival, growth, food consumption or body composition between BDL-grps. In the last wk the free water was increased in all BDL rats. No difference was seen in any grps regarding behavioral tests nor was any evidence of brain edema. NH3 level was increased in BDL treated with either lactulose or rifaximin compare to SHAM but surprisingly not with vehicle. The biochemistry parameters confirm the onset of cirrhosis in BDL grps. This study don’t present strong, reliable and sufficient data to conclude on the efficacy of rifaximin. Future direction will aim to increase the dose of drug; reduce the duration of the model; trigger an episode of HE before ttm.
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.
Christopher F. *Rose, Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, as well as Sham-operated controls, were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; 2. Lean-BDL rats received RD pre- and post-BDL; 3. Lean-Sham rats received RD pre- and post-Sham surgery. Behaviour: Recognition memory, motor coordination and muscular strength were assessed before surgery, as well as 3 and 6 weeks post-surgery using the novel object recognition, rotarod and grip-strength tests, respectively. Body-composition (echoMRI): Fat vs. lean mass and free water (ascites) were also monitored. RESULTS: Before the surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on RD (Lean-BDL and Lean-Sham). 3 weeks after surgery, BW, fat mass, lean mass and free water were increased in Obese-BDL rats vs. Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham rats. 6 weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost BW, fat and Lean mass, while free water increased vs. Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats, compared to Lean-Sham rats. CONCLUSION: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Obesity also accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats (long-term memory), while others are exacerbated (motor coordination, forelimb strength). This new animal model of CLD and obesity suggests a synergistic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. Thus, obesity-induced cirrhosis in patients may result in more complex neurological
Nouveau modèle d’encéphalopathie hépatique épisodique due à la cirrhose.
Kim Phat Pham, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction :L'encéphalopathie hépatique (EH) est une complication neuropsychiatrique majeure de la maladie du foie chronique, telle que la cirrhose du foie. L’une de ses formes est l’EH épisodique dont les symptômes surgissent épisodiquement. Notre hypothèse était que les épisodes d’EH aggraveraient les dommages neurologiques de l’EH et accentueraient ainsi les troubles neurologiques. Toutefois, il n’existe aucun modèle animal d’EH épisodique à ce jour. Nous avions pour but de caractériser un modèle d’EH épisodique due à la cirrhose et d’évaluer l’impact de multiples épisodes sur le dysfonctionnement neurologique. Matériel et méthodes :Une cirrhose hépatique a été provoquée par ligatures du conduit biliaire (BDL) et a été combinée à des injections d’acétate d’ammonium afin d’induire l’EH épisodique dans le rat. La coordination motrice et l’apprentissage moteur ont été évalués avec le test RotaRod et la locomotion avec l’analyse de l’activité nocturne. Résultats et discussion :les déficits de coordination et de locomotion détectés chez les rats BDL ne se sont pas aggravés après des épisodes d’EH. Cependant, les rats cirrhotiques ayant eu des épisodes d’EH présentaient des déficits de coordination motrice après un épisode, alors que les rats cirrhotiques sans épisode ne présentaient pas de déficit de coordination. Un effet synergique de la cirrhose et des épisodes d’EH pourrait causer ces déficits de coordination motrice déjà après un seul épisode. Conclusion :Ce nouveau modèle de l’EH épisodique permettra d’explorer son mécanisme pathologique et d’y tester des traitements.Organisme subventionnaire : PREMIER-Département de médecine
Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Development of an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: 6-week bile-duct ligation (BDL) rats, as well as Sham-operated controls, were used. Inducing obesity: High-fat diet (HFD) was given for 3 weeks before BDL or Sham surgery. Groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and regular diet (RD) for 6 weeks post-BDL; 2. Lean-BDL rats received RD pre- and post-BDL; 3. Lean-Sham rats received RD pre- and post-Sham surgery. Behaviour: Recognition memory, motor coordination and muscular strength were assessed before surgery, as well as 3 and 6 weeks post-surgery using the novel object recognition, rotarod and grip strength tests, respectively. Body-composition (echoMRI): Fat vs. lean mass and free water (ascites) were also monitored. RESULTS: Before the surgery, body weight (BW) and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on RD (Lean-BDL and Lean-Sham). 3 weeks after surgery, BW, fat mass, lean mass and free water were increased in Obese-BDL rats vs. Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs. Lean-Sham rats. 6 weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost BW, fat and Lean mass, while free water increased vs. Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats, compared to Lean-Sham rats. CONCLUSION: HFD induces obesity features in healthy non-cirrhotic rats. Such effects are maintained in cirrhotic-BDL rats. Obesity also accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats (long-term memory), while others are exacerbated (motor coordination, forelimb strength). This new animal model of CLD and obesity suggests a synergistic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. Thus, obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting more susceptibility to poor neurological performance.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Christopher F. Rose.
BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and HE: The 6-week bile-duct ligation (BDL) rats and sham-operated controls, were used. Obesity: To induce obesity, high-fat diet (HFD) was given for 3 weeks before BDL. Experimental groups: 1. Obese-BDL rats received HFD for 3 weeks pre-BDL and normal diet (ND) for 6 weeks post-BDL; 2. Lean-BDL rats received ND pre- and post-BDL; 3. Lean-Sham rats received ND pre- and post-sham surgery. Behaviour: Motor coordination, muscular strength, and recognition memory were assessed before surgery, as well as 3 and 6 weeks post-BDL or sham surgery using the RotaRod, grip-strength and object recognition tests, respectively. Body-composition (echoMRI): Fat vs lean mass, as well as free water were also monitored. RESULTS: Before the surgery, body weight and fat mass of rats on HFD (Obese-BDL) were increased in comparison to rats on ND (Lean-BDL and Lean-Sham). Three weeks after surgery, body-weight, fat mass, lean mass and free water were increased in Obese-BDL rats vs Lean-BDL rats. Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs Lean-Sham rats. Six weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost body weight, fat and lean mass, and increased free water vs Lean-Sham rats. Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL or Lean-Sham rats, whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats vs Lean-Sham rats. SUMMARY: HFD induces obesity features in healthy non-cirrhotic rats, and such effects are maintained in cirrhotic-BDL rats. Obesity also worsens and accelerates the accumulation of free water in cirrhotic-BDL rats. Interestingly, some neurological impairments are detected in Obese-BDL but not in Lean-BDL rats, such as long-term memory, motor coordination and forelimb strength deficits. This new animal model of CLD and obesity suggests a synergic effect, which accelerates and worsens the disease-associated abnormalities observed in CLD and HE. CONCLUSION: Obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting that these patients might be more susceptible to neuronal dysfunction and poor neurological performance. Therefore, this model of CLD and obesity will provide important clues to the underlying mechanisms of HE associated with obesity-induced cirrhosis and provide new insights into novel therapeutic strategies.
Nouveau modèle d’encéphalopathie hépartique épisodique due à la cirrhose.
Kim Phat Pham, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Introduction : L'encéphalopathie hépatique (EH) est une complication neuropsychiatrique majeure de la maladie du foie chronique, telle que la cirrhose du foie. L’une de ses formes est l’EH épisodique dont les symptômes surgissent épisodiquement. Notre hypothèse était que les épisodes d’EH aggraveraient les dommages neurologiques de l’EH etaccentueraient ainsi les troubles neurologiques. Toutefois, il n’existe aucun modèle animal d’EH épisodique à ce jour. Nous avions pour but de caractériser un modèle d’EH épisodique due à la cirrhose et d’évaluer l’impact de multiples épisodes sur le disfonctionnement neurologique.Matériel et méthodes : Une cirrhose hépatique a été provoquée par la chirurgie du bile duct ligation (BDL) et a été combinée à des injections d’acétate d’ammonium afin d’induire l’EH épisodique dans le rat. Les niveaux d’ammoniaque ont été mesurés. Les mémoires à court et long terme ont été évaluées avec le Novel Object Recognition test ; l’anxiétéavec le Elevated Plus Maze test ; et la coordination motrice avec le RotaRod test. Résultats : Les déficits de mémoire à court terme et de coordination motrice chez les rats BDL avec épisodes d’EH n’étaient pas altérés comparés aux rats BDL sans épisodes. Toutefois, leur mémoire à long terme a été réduite, et leurs niveaux d’anxiété et d’ammoniaque plasmique ont été augmentés. Discussion et conclusion : Un effet synergique de la cirrhose, des épisodes d’EH et des niveaux élevés d’ammoniaquepourraient causer ces altérations. Ce nouveau modèle de l’EH épisodique permettra d’explorer son mécanisme pathologique et d’y tester des traitements. Projet subventionné par la COPSE et la CLF.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Marc-André Clément, Marcos Ocana-Sanchez, Christopher F. Rose.
BACKGROUND: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the impact of obesity on the development of neurological impairment in cirrhotic rats. M&M: Obesity: To induce obesity, high-fat diet (HFD) was given for 3 weeks before surgical intervention (bile-duct ligation (BDL) or Sham). Experimental groups: 1. Obese-BDL: rats received HFD for 3 weeks pre-BDL and normal diet (ND) for 6 weeks post-BDL; 2. Lean-BDL: rats received ND pre- and post-BDL; 3. Lean-Sham: rats received ND pre- and post-sham surgery. HE parameters: Motor coordination, muscular strength, and recognition memory were assessed pre-pre-, 3 and 6 weeks post-surgical intervention using RotaRod, grip- strength and object recognition test respectively. Body-composition (echoMRI): Fat, lean mass and free water were monitored. RESULTS: Three weeks HFD lead to an increase in fat mass in comparison to rats on ND (p<0.01). Three weeks after surgery, fat, and lean mass and free water were increased in Obese-BDL vs Lean-BDL rats (p<0.05). Long-term memory was reduced in Obese-BDL, but not in Lean-BDL, vs Lean-Sham rats (p<0.05). Six weeks after surgery, similar to Lean-BDL rats, Obese-BDL rats lost fat and lean mass, and increased free water vs Lean-Sham rats (p<0.5). Motor coordination, forelimb strength and long-term memory were impaired in Obese-BDL rats in comparison to Lean-BDL and Lean-Sham rats (p<0.01), whereas hind-limb strength and short-term memory were impaired in both Obese- and Lean-BDL rats when compared to Lean-Sham rats (p<0.01). CONCLUSION: Obesity exacerbates and accelerates the accumulation of free water and motor coordination deficits in cirrhotic-BDL rats. Interestingly, long-term memory and forelimb strength deficits were impaired in Obese-BDL but not in Lean-BDL rats. This novel animal model of CLD and obesity suggests obesity accelerates CLD-induced neurological impairment. Therefore, this model of CLD and obesity will provide important clues to the underlying mechanisms of HE associated with obesity-induced cirrhosis and provide new insights into novel therapeutic strategies.
The bile duct ligated rat: A relevant model to study muscle mass loss in cirrhosis.
Cristina R. Bosoi, Mariana M. Oliveira, Rafael Ochoa-Sanchez, Mélanie Tremblay, Gabriella A. Ten Have, Nicolaas E. Deutz, Christopher F. Rose, Chantal Bémeur.
Muscle mass loss and hepatic encephalopathy (complex neuropsychiatric disorder) are serious complications of chronic liver disease (cirrhosis) which impact negatively on clinical outcome and quality of life and increase mortality. Liver disease leads to hyperammonemia and ammonia toxicity is believed to play a major role in the pathogenesis of hepatic encephalopathy. However, the effects of ammonia are not brain-specific and therefore may also affect other organs and tissues including muscle. The precise pathophysiological mechanisms underlying muscle wasting in chronic liver disease remains to be elucidated. In the present study, we characterized body composition as well as muscle protein synthesis in cirrhotic rats with hepatic encephalopathy using the 6-week bile duct ligation (BDL) model which recapitulates the main features of cirrhosis. Compared to sham-operated control animals, BDL rats display significant decreased gain in body weight, altered body composition, decreased gastrocnemius muscle mass and circumference as well as altered muscle morphology. Muscle protein synthesis was also significantly reduced in BDL rats compared to control animals. These findings demonstrate that the 6-week BDL experimental rat is a relevant model to study liver disease-induced muscle mass loss.
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.
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.
Rafael Ochoa-Sanchez, Mélanie Tremblay, Mariana Oliveira, Cristina Bosoi, Marc-André Clément, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, a major complication of chronic liver disease (CLD, cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence on obesity affecting neurological function, we hypothesize that obesity increases the risk, severity and accelerates the progression of HE in non-alcoholic fatty liver disease (NAFLD)-related cirrhosis. AIM: To develop and characterize an animal model of cirrhosis and obesity to investigate the synergistic effect of obesity and CLD on the development of neurological impairment and HE. M&M: Animal model of CLD and obesity: The 6-week bile-duct ligation (BDL) rat is a surgical model in which obstruction of the bile duct leads to cirrhosis and HE. We induced obesity with a high-fat diet (HFD). Previously, HFD was given after BDL, but it was not well accepted, and the body weight did not increased. Now we are pre-feeding the rats with HFD to see if that is a better model. Thus, rats will be fed HFD for 3 weeks (pre-BDL) and 6 weeks post-BDL. Food consumption, weight gain, as well as lean vs fat mass will be monitored. Preliminary results: 3-week HFD increases body weight (12.1%) and fat (30.5 vs 44.1g) mass compared to rats fed normal diet. Food consumption was decreased (HFD 12g/day vs normal diet 27g/day), while the calorie intake was not affected. Discussion: obesity-induced cirrhosis in patients may result in more complex neurological manifestations, suggesting these patients might be more susceptible to neuronal loss and poor neurological performance. Thus, this animal model of CLD and obesity will give important clues about psychiatric diseases including, HE and how they might be treated. Funded: CIHR
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