La barrière hémato-encéphalique chez les rats cirrhotiques : une comparaison mâle et femelle.
Licence, Université de Poitiers, France
Direction:
- Dr Christopher Rose
Hiver 2019
Publications connexes
Mariana Macedo de Oliveira, Alexis Monnet-Aimard, Cristina R. Bosoi, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a debilitating neurological complication of chronic liver disease (CLD). Hyperammonemia plays an important role in HE's pathogenesis, acting synergistically with systemic oxidative stress. During CLD, muscle plays a compensatory role in detoxifying ammonia, and therefore muscle loss leads to an increase in the risk of developing HE. With most animal studies involving males, sex's impact on the development of CLD and associated complications such as HE and muscle loss remains unknown. Therefore, we aimed to identify the impact of sex on CLD, HE, and muscle mass loss in a rodent model of CLD. Liver injury markers, hyperammonemia, oxidative stress, muscle mass, and ammonia clearance were measured in female and male bile-duct ligated (BDL) rats. In addition, covert HE was assessed in females while ammonia-precipitated severe HE was assessed in female and male BDL rats, and male BDL rats treated with allopurinol (100 mg/kg), an antioxidant (xanthine oxidase inhibitor). Female BDL developed CLD and HE (impaired motor coordination and night activity) compared to respective SHAM. Hyperammonemia and muscle ammonia clearance were similar between female and male BDL. However, only female BDL rats did not develop muscle loss, brain edema, and short-term memory impairment (vs. female SHAM) and systemic oxidative stress and decreased albumin levels (vs. male BDL). Furthermore, both female BDL and allopurinol-treated male BDL rats were protected against ammonia-induced overt HE. In conclusion, female and male BDL rats develop distinct features of CLD and HE, with systemic oxidative stress playing a pivotal role in the susceptibility to ammonia-precipitated overt HE.
Impact of sex on muscle mass loss and hepatic encephalopathy in rats with chronic liver disease.
Mariana M. Oliveira, Alexis Monnet-Aimard, Cristina Bosoi, Mélanie Tremblay, Christopher Rose.
Background: In chronic liver disease (CLD) loss of muscle mass (sarcopenia) is highly prevalent which leads to an increased risk of hepatic encephalopathy (HE). Muscle plays a compensatory role during CLD in clearing ammonia since it expresses glutamine synthetase (GS). Therefore, diminished muscle mass in CLD leads to a further reduced capacity to clear ammonia. Male rats with CLD due to bile-duct ligation (BDL) have been shown to result in a loss of muscle in association with hyperammonemia and HE. However, these complications have not been explored in female CLD rats. Purpose: Our aim was to identify whether female sex impacts muscle mass loss, blood ammonia levels and HE in ratswith CLD. Method: Five weeks after either BDL (n=8) or Sham (n=8) surgery in male and female rats, we assessed markers of liver injury (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)) and function (albumin and bilirubin). Neurophenotyping was achieved using the open field test and the elevated plus maze test for anxiety, the rota-rod test for motor coordination, the novel object recognition for short-term memory and the nighttime activity test. In addition, brain edema was assessed using the gravimetric technique. Body parameters (weight, composition (MRI)) and muscle (gastrocnemius weight and circumference and grip strength) were also evaluated. In addition, muscle GS activity, ammonia clearance as well as glutamine generation (femoral venous-arterial difference)were evaluated in female vs. male BDL rats. Result(s): Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin and albumin (p<0.001)) and both developed HE (impaired motor-coordination and night activity (p<0.05)) when compared to respective Shams. However, female BDL rats did not develop brain edema and did not have loss of short-term memory. Male BDL rats experienced loss of lean mass as well as reduced muscle circumference, weight and strength (p<0.01) compared to Sham rats, while similar differences between female BDL vs. Sham rats were not found. Male and female BDL rats had comparable blood ammonia levels as well as similar muscle ammonia clearance and glutamine production. However, GS activity was lower in female vs. male BDL rats (p<0.01). Conclusion(s): Our results demonstrate that following BDL surgery, female rats develop similar degrees of CLD compared to male rats. As in male BDL rats, female rats also develop HE but female BDL rats acquire unique features (not observed in males) such as lack of brain edema and intact short-term memory. Contrary to males, female BDL rats did not develop sarcopenia compared to respective controls. However, preserved muscle mass in female BDL did not result in increased muscle ammonia clearance and glutamine production. Therefore, the similar degrees of hyperammonemia in males vs. females may be due to an upregulation in GS found in the muscle of male BDL rats. Thus, the female-induced protection against brain edema and short-term memory in BDL rats likely involves additional factors besides ammonia.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD) loss of muscle mass (sarcopenia) is highly prevalent which leads to an increased risk in hepatic encephalopathy (HE). Muscle plays a compensatory role during liver disease in clearing ammonia since it contains glutamine synthetase (GS). Therefore, diminished muscle mass leads to a further reduced capacity to remove ammonia. Loss of muscle mass has been described in male rats with CLD but has not been explored in female CLD rats. Our aim was to identify whether sex has an effect on muscle mass and blood ammonia levels. Methods: Five weeks after either BDL (n=8) or Sham (n=8) surgery in male and female rats, the following were assessed; markers of liver injury and function, HE (open field test for anxiety, rota-rod test for motor coordination and night-time activity), body parameters (weight, composition (MRI) and gastrocnemius muscle weight/circumference and grip strength). In addition, muscle GS activity and muscle ammonia clearance as well as glutamine generation (femoral venous-arterial difference) were evaluated in female vs male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin and albumin (p<0.001)) and both developed HE (motor-coordination and night activity (p<0.05)) when compared to respective Shams. Male BDL rats experienced loss of lean mass, muscle weight and strength (p<0.01) while no differences were found in female BDL vs Sham rats. Male and female BDL rats had similar ammonia clearance and glutamine production by muscle, while GS activity was lower in female vs. male BDL rats (p<0.01). Discussion: Our results demonstrate that following BDL surgery, female rats develop CLD and HE comparable to male rats. However, contrary to males, female BDL rats did not develop sarcopenia compared to respective controls. Preserved muscle mass in female BDL did not result in lower blood ammonia (higher ammonia clearance) whereas muscle mass loss in male BDL rats was accompanied with an upregulation in GS which may explain the similar blood ammonia levels found in both male and female BDL rats.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD), the muscle plays a compensatory role in clearing ammonia and therefore muscle mass loss increases the risk of hepatic encephalopathy (HE). It has been demonstrated that systemic oxidative stress is implicated in the pathogenesis of brain edema, however only in male rats with CLD. Therefore, our aim is to identify the impact of sex on the pathogenesis of HE and brain, including ammonia-induced overt HE. Methods: Male and female bile-duct ligated (BDL) rats (and respective Shams) were evaluated for minimal HE and ammonia-precipitated overt HE (lethargy/loss of righting reflex). In addition, liver markers, hyperammonemia, systemic OS, muscle mass and ammonia clearance were evaluated. Results: Female and male BDL rats had similar liver impairment (ALP, AST, and bilirubin (p<0.001)) and HE (motor-coordination and night activity (p<0.05)) vs. respective Shams. Contrary to males, females BDLs did not develop muscle loss (p<0.05), brain edema or severe HE episode (p<0.005). Furthermore, degree of hyperammonemia as well as muscle ammonia clearance were similar between male and female BDLs but systemic OS was significantly lower in females (p<0.01). Discussion: Our findings demonstrate that female BDL rats develop CLD, HE and similar degrees of hyperammonemia compared to male rats. Differently from males, female BDL did not develop muscle mass loss, brain edema nor severe HE following ammonia challenge. The protection against brain edema in female BDLs is primarily due to low systemic OS but whether brain edema or systemic OS renders male BDLs most susceptible to ammonia-precipitated insults remains to be determined.
Role of sex on the development of sarcopenia and ammonia metabolism in bile-duct ligated rats.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD) loss of muscle mass (sarcopenia) is highly prevalent which leads to an increased risk in hepatic encephalopathy (HE). Muscle plays a compensatory role during liver disease in clearing ammonia since it contains glutamine synthetase (GS). Therefore, diminished muscle mass leads to a further reduced capacity to remove ammonia. Loss of muscle mass has been described in male rats with CLD but has not been explored in female CLD rats. Our aim was to identify whether sex has an effect on muscle mass and blood ammonia levels. Methods: Five weeks after either BDL (n=8) or Sham (n=8) surgery in male and female rats, the following were assessed; markers of liver injury and function, HE (open field test for anxiety, rota-rod test for motor coordination and night-time activity), body parameters (weight, composition (MRI) and gastrocnemius muscle weight/circumference and grip strength). In addition, muscle GS activity and muscle ammonia clearance as well as glutamine generation (femoral venous-arterial difference) were evaluated in female vs male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin and albumin (p<0.001)) and both developed HE (motor-coordination and night activity (p<0.05)) when compared to respective Shams. Male BDL rats experienced loss of lean mass, muscle weight and strength (p<0.01) while no differences were found in female BDL vs Sham rats. Male and female BDL rats had similar ammonia clearance and glutamine production by muscle, while GS activity was lower in female vs. male BDL rats (p<0.01). Discussion: Our results demonstrate that following BDL surgery, female rats develop CLD and HE comparable to male rats. However, contrary to males, female BDL rats did not develop sarcopenia compared to respective controls. Preserved muscle mass in female BDL did not result in lower blood ammonia (higher ammonia clearance) whereas muscle mass loss in male BDL rats was accompanied with an upregulation in GS which may explain the similar blood ammonia levels found in both male and female BDL rats.
Sex impacts muscle loss and ammonia metabolism in rats with chronic liver disease.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD), muscle wasting is highly prevalent and leads to an increased risk of hepatic encephalopathy (HE). Since muscle’s glutamine synthetase (GS) compensates for ammonia detoxification during CLD, less muscle leads to a lower capacity to remove ammonia. Muscle loss has been described in male rats but not in female rats with CLD. We aimed to identify whether sex influences muscle mass and blood ammonia levels. Methods: Five weeks after either bile duct ligation (BDL) (n=8) or Sham surgery (n=8) in male and female rats, the following were assessed; markers of liver injury and function, HE (behavioral tests), body weight, and composition (MRI) and, muscle weight, circumference and strength, as well as muscle GS and glutamine generation were evaluated in female vs. male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin, and albumin (p<0.001)) and HE (motor-coordination and activity (p<0.05)) compared to respective Shams. Male BDL, but not female, experienced loss of lean mass, muscle weight, and strength (p<0.01). Male and female BDL had similar muscle ammonia clearance and glutamine production, with lower GS activity in female vs. male BDL (p<0.01). Discussion: Our results demonstrate that although sex did not influence CLD and HE, female rats had preserved muscle mass, which did not result in lower blood ammonia. Contrarily, muscle loss in male rats upregulated GS, which may explain the similar blood ammonia levels found in both male and female BDL rats.
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: In chronic liver disease (CLD), muscle wasting is highly prevalent and leads to an increased risk of hepatic encephalopathy (HE). Since muscle’s glutamine synthetase (GS) compensates for ammonia detoxification during CLD, less muscle leads to a lower capacity to remove ammonia. Muscle loss has been described in male rats but not in female rats with CLD. We aimed to identify whether sex influences muscle mass and blood ammonia levels. Methods: Five weeks after either bile duct ligation (BDL) (n=8) or Sham surgery (n=8) in male and female rats, the following were assessed; markers of liver injury and function, HE (behavioral tests), body weight, and composition (MRI) and, muscle weight, circumference and strength, as well as muscle GS and glutamine generation were evaluated in female vs. male BDL rats. Results: Female and male BDL rats had similar levels of impaired liver markers (ALP, AST, bilirubin, and albumin (p<0.001)) and HE (motor-coordination and activity (p<0.05)) compared to respective Shams. Male BDL, but not female, experienced loss of lean mass, muscle weight, and strength (p<0.01). Male and female BDL had similar muscle ammonia clearance and glutamine production, with lower GS activity in female vs. male BDL (p<0.01). Discussion: Our results demonstrate that although sex did not influence CLD and HE, female rats had preserved muscle mass, which did not result in lower blood ammonia. Contrarily, muscle loss in male rats upregulated GS, which may explain the similar blood ammonia levels found in both male and female BDL rats.
Sex disparity in the development of brain edema and hepatic encephalopathy severity in BDL rats.
Mariana Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background and Aims: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome with symptoms ranging from impaired reaction time, poor memory to asterixis, gross disorientation, lethargy and coma. HE is a major complication of chronic liver disease (CLD) and develops when the hepatic capacity to detoxify ammonia is decreased. During this condition, muscle plays a compensatory role removing ammonia, but muscle mass loss further reduces the capacity to metabolise ammonia and hyperammonemia prevails. HE is marked by an increase in brain water caused via the synergistic effect of ammonia and oxidative stress (reactive oxygen species (ROS)). Brain edema and ammonia-related cognitive impairments as well as muscle mass loss have been well described in male rats with CLD but have not been explored in female CLD rats. Therefore, our aim was to identify whether sex influences brain edema and ammonia-mediated cognitive impairments. Methods: Five weeks after either bile-duct ligation (BDL) (n=8) or Sham (n=8) surgery in male and female rats, we assessed markers of liver injury and function, body parameters (weight, composition (MRI), gastrocnemius muscle weight/circumference and grip strength), HE (open field test for anxiety, rota-rod test for motor coordination and night-time activity) and brain edema (by gravimetric density method). Finally, we assessed susceptibility to develop severe cognitive impairment (mild-moderate lethargy, ataxia and loss of righting reflex) in male and female BDL rats after an ammonia challenge (6 mmoles/kg of ammonium acetate injected subcutaneously). Both plasma ammonia (Randox kit) and ROS (2',7'-dichlorodihydrofluorescein diacetate test (DCFDA)) were investigated. Results: Female BDL rats, similar to male BDL rats, had CLD, with impaired liver markers (ALP (p<0.001), AST (p<0.001), bilirubin (p<0.0001) and albumin (p<0.001)) compared to respective Sham controls. Male BDL rats experienced loss of lean mass (p<0.001), muscle weight (p<0.01) and strength (p<0.01) while similar differences were not found in female BDL vs respective Sham controls. Both female and male BDL developed HE (impaired motor-coordination (p<0.05) and reduced night activity (p<0.05)), compared to respective Shams. However, contrary to male BDLs, female BDLs did not develop brain edema compared to respective Sham controls. When compared to male BDL, baseline plasma ammonia levels did not differ to female BDL rats. However, plasma ROS levels were lower in females compared to males (p<0.05). Following ammonia challenge, male BDL rats had progressive worsening of mental status, reaching ataxia and loss of righting reflex while female BDL were protected, reaching only moderate lethargy (p<0.05). During the ammonia challenge, female BDL rats had similar ammonia levels but lower levels of ROS compared to male BDL rats (p<0.01). Discussion: We demonstrated BDL surgery in females leads to hepatic and neurological impairment comparable to male BDL rats. Contrary to males, female BDL did not develop loss of muscle mass nor brain edema compared to respective controls. However, sustained muscle mass in females did not lead reduced blood ammonia therefore the protection versus brain edema in females in due to protection against systemic oxidative stress. Female BDLs did not developed severe HE following ammonia challenge. Whether brain edema or systemic oxidative stress renders the females resistant to ammonia insults remains to be determined.
Rafael Ochoa-Sanchez, Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mélanie Tremblay, Mylene Perreault, Bill Querbes, Caroline Kurtz, Christopher F. Rose.
Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) production that contributes to systemic hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering systemic NH3. As a therapeutic strategy, Escherichia coli Nissle 1917 bacterium (EcN), a well characterized probiotic, was genetically modified to consume and convert NH3 to arginine (SYNARG), and its administration to thiaoacetamide-treated mice reduced NH3 levels. SYNARG was further modified to synthesize butyrate (SYNARG+BUT), a short-chain fatty acid with anti-inflammatory properties, and both strains were tested in an animal model of CLD and HE, the bile duct ligation (BDL). Methods: One week (wk) post surgery, BDL rats were gavaged with SYNARG, SYNARG+BUT (3x1011 CFU/day, BID) or vehicle until they were sacrificed at 3- or 5-weeks along with respective sham controls. Plasma NH3 and liver markers were measured at 3 and 5 wk. Recognition memory, motor coordination, muscle strength, locomotion and anxiety were assessed in the 5-week groups. Results: BDL increased NH3 over time, with levels of 109.1±9.2µM (Shams 56.7±3.5µM, p<0.001) and 150.2±25.6µM (Shams 58.3±3.0µM, p<0.001) at 3- and 5-wk, respectively. In addition, plasma liver markers ALT, AST, bilirubin, and GGT as well as liver fibrosis (hydroxyproline) were increased in BDL rats at both timepoints while albumin was lowered. As compared to BDL-Veh rats, NH3 was attenuated by SYNARG (103.9±12.3µM) and SYNARG+BUT (110.8±8.5µM) at 5, but not 3-wk post-BDL, while liver fibrosis was attenuated at 3, but not 5-wk post-BDL. None of the systemic liver markers were changed by the treatments at any timepoint. Motor coordination, muscle strength, locomotion and anxiety were affected in all BDL groups without protective effect of treatments. Short-term memory (STM) was impaired in BDL-Veh (p<0.001) and BDL-SYNARG (p<0.05) vs Shams, while STM was improved in BDL-SYNARG+BUT (p<0.05 vs BDL-Veh). Long-term memory was impaired in BDL-Veh vs Shams (p<0.05), but BDL-SYNARG and BDL-SYNARG+BUT were partially protected. Conclusion: EcN, engineered to consume NH3 in the gut, is an effective approach to lower plasma NH3 in a model of CLD and HE. Moreover, the attenuation of NH3 in BDL rats is related to a protective effect on memory in this model. The therapeutic potential of these strains should be further evaluated in patients with CLD and HE.
Mariana Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background The impact of sex differences on chronic liver disease (CLD) and hepatic encephalopathy (HE) is unknown. The majority of animals used in research are male since the main difficulty with using female animals is the potential impact of the estrous cycle, increasing intragroup variability. The bile duct ligated (BDL) rat is a well-characterized model of CLD and HE in males which has not been investigated in females. Therefore, we aimed to characterize a female BDL model of CLD and HE and compare to male BDL rats. Material and Methods We assessed BDL or Sham female rats for estrous cycle phase, behavior (anxiety, motor incoordination and activity), body parameters (weight and composition, muscle weight/circumference, grip strength), liver parameters (enzymes and ammonia). We than compared to historical laboratory data from male BDL rats. Results Female BDL rats had impaired liver markers (P<0.0001) and ammonia (p<0.001) compared to female Shams. These results were comparable to male BDL rats except ammonia which was lower in females (p< 0.01). Female BDL rats did not differ in body weight, muscle circumference/weight and grip strength and had increased lean mass (p<0.005) compared to female shams. Whereas, male BDL rats have decreased lean mass, muscle circumference/weight and grip strength. Similar to male BDL rats, female BDL rats had increased anxiety (p<0.005), motor incoordination (p<0.05), and decreased activity (p<0.05) independent of the estrous cycle phase. Discussion and Conclusion We demonstrated BDL surgery in females leads to hepatic and neurological impairment comparable to male BDL rats (similar intra-group variability). Interestingly, contrary to male BDL vs Shams, body weight and muscle mass does not differ between female BDL and Shams. Since Mmuscle plays an important compensatory role in clearing ammonia during CLD, maintenance of muscle mass in females which could explain the lower blood ammonia levels in female BDL rats compared to male BDL rats. We conclude that this model provides new insights on the impact of sex on the pathogenesis of CLD and HE.
Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mylene Perreault, Bill Querbes, Caroline B. Kurtz, Rafael Ochoa-Sanchez, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome observed in chronic liver disease (CLD/cirrhosis). With an increasing prevalence of obesity-induced cirrhosis and evidence linking blood-derived lipids to neurological impairment, we hypothesize that obesity increases the risk, severity and progression of HE. AIM: Investigate the synergistic effect of obesity and CLD on the development of neurological impairment in a novel rat model of cirrhosis and obesity. M&M: Animal model of CLD and HE: 5-week bile-duct ligation (BDL) rats and Sham-operated controls, were used. Groups: Obese-BDL and Obese-Sham received high-fat diet (HFD) for 25-days pre-BDL and high-carbohydrate diet (HCD) for 5-weeks post-BDL; Lean-BDL and Lean-Sham received regular-diet (RD) pre-BDL and HCD post-BDL. Body-weight and fat-mass (EchoMRI) were monitored pre-BDL as well as 3- and 5-weeks post-BDL. Behavior: Motor-coordination, motor skill-learning, and muscular-strength were assessed at 3- and 5-weeks post-BDL. Locomotion and anxiety were measured at 5-weeks. Plasma ammonia, liver enzymes, and lipids were measured at 3- and 5-weeks. RESULTS: Before BDL surgery, body-weight and fat-mass of rats on HFD increased compared to rats on RD. 3-week post-BDL, body-weight and fat-mass decreased in Lean-BDL and Obese-BDL vs respective Shams, while at 5-weeks this was only found in Lean-BDL. These parameters were higher in Obese-BDL vs Lean-BDL at 3- and 5-weeks. Plasma ammonia, bilirubin, albumin, ALT, AST, and ALP were impaired in Obese- and Lean-BDL vs respective Shams at 3- and 5-weeks. AST and ALP increased in Obese-BDL vs Lean-BDL at 5-weeks. Elevated HDL-cholesterol and decreased LDL-cholesterol were detected in Obese-BDL and Lean-BDL vs respective Shams at 3- and 5-weeks, while LDL-cholesterol was higher in Obese-BDL vs Lean-BDL at 5-weeks. Total-cholesterol increased in Obese-BDL vs all groups at 5-weeks. At 3 weeks; motor-coordination was reduced in Obese-BDL, but not in Lean-BDL vs respective Shams, while at 5-weeks, motor-coordination decreased in both Lean-BDL and Obese-BDL vs respective Shams, with worse performance in Obese-BDL vs Lean-BDL. At 3-weeks, skill-learning improved in all Shams and Lean-BDL, but not in Obese-BDL; at 5-weeks contrary to Sham-groups, both BDL groups did not improve performance. Muscle-strength decreased in Lean-BDL and Obese-BDL vs respective Shams at 3- and 5-weeks. Hypolocomotion and anxiogenic effects were detected in Obese-BDL, but not in Lean-BDL vs Shams at 5-weeks. CONCLUSION: HFD induces obesity pre-BDL which is maintained post-BDL with a HCD-diet which was accompanied with increase fat-mass and hyperlipidemia. Neurological decline in obese-cirrhotic rats developed earlier and was more severe versus Lean-BDL rats. Besides, some neurological impairments developed in Obese-BDL but not in Lean-BDL. These results suggest a synergistic effect, which accelerates/worsens the disease-associated abnormalities in CLD and HE.
Rafael Ochoa-Sanchez, Alexis Monnet, Farzaneh Tamnanloo, Mariana M. Oliveira, Mélanie Tremblay, Mylene Perreault, Bill Querbes, Caroline B. Kurtz, Christopher F. Rose.
Background: Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia (NH3) production that contributes to systemic hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering circulating NH3. As a therapeutic strategy, Escherichia coli Nissle 1917 bacterium (EcN), a well characterized probiotic, was genetically modified to consume and convert NH3 to arginine (SYNARG), and its administration to thioacetamide-treated mice resulted in a significant reduction of NH3 levels1. SYNARG was further modified to synthesize butyrate (SYNARG+BUT), a short-chain fatty acid with anti-inflammatory/anti-oxidant properties, and both strains were tested in an experimental model of cirrhosis and HE, the bile duct ligation (BDL). Methods: One week post surgery, BDL rats were gavaged with SYNARG, SYNARG+BUT (3x1011 CFU/day, BID) or vehicle until they were sacrificed at 3- or 5-weeks along with respective sham controls. Plasma NH3 and liver markers were measured at 3 and 5 weeks. Recognition-memory, motor-coordination, muscle-strength, locomotion and anxiety were assessed in the 5-week BDL groups. Results: BDL significantly increased NH3 over time, with levels of 109.1±9.2µM (Shams 56.7±3.5µM, p<0.001) and 150.2±25.6µM (Shams 58.3±3.0µM, p<0.001) at 3- and 5-weeks, respectively. In addition, plasma liver markers alanine-transaminase, aspartate-transaminase, bilirubin, and gamma-glutamyl transferase were significantly increased in BDL rats at both timepoints while albumin was significantly lowered. As compared to BDL-Veh rats, hyperammonemia was attenuated by SYNARG (103.9±12.3µM) and SYNARG+BUT (110.8±8.5µM) at 5, but not 3 weeks post-surgery, while liver fibrosis (hydroxyproline content) was attenuated at 3, but not 5 weeks post-surgery. None of the circulating liver markers were changed by the treatments at any timepoint. Motor-coordination, muscle-strength, locomotion and anxiety were affected in all BDL groups without protective effect of treatments. Short-term memory (STM) was impaired in BDL-Veh (p<0.001) and BDL-SYNARG (p<0.05) versus Shams, while STM was resolved in BDL-SYNARG+BUT (p<0.05 vs BDL-Veh). Long-term memory (LTM) was impaired in BDL-Veh vs Shams (p<0.05), but BDL-SYNARG and BDL-SYNARG+BUT were protected. Conclusion: EcN, engineered to consume NH3 in the gut and synthesize butyrate, is an effective approach to lower plasma NH3 in a model of cirrhosis and HE. Moreover, the attenuation of hyperammonemia in cirrhotic rats is associated with a protective effect on memory in this model. The therapeutic potential of these engineered EcN strains should be further evaluated in patients with CLD and HE.
Uncovering sex-based differences in a rat model of chronic liver disease and hepatic encephalopathy
Mariana M. Oliveira, Alexis Monnet-Aimard, Mélanie Tremblay, Christopher F. Rose.
Background The impact of sex differences on chronic liver disease (CLD) and hepatic encephalopathy (HE) is unknown. The majority of animals used in research are male since the main difficulty with using female animals is the potential impact of the estrous cycle, increasing intragroup variability. The bile duct ligated (BDL) rat is a well-characterized model of CLD and HE in males which has not been investigated in females. Therefore, we aimed to characterize a female BDL model of CLD and HE and compare to male BDL rats. Material and methods Female rats underwent either BDL (n=8) or Sham (n=8) surgery. After 5 weeks, we assessed estrous cycle phase (by cellular cytology), anxiety (open field test), motor incoordination (rota-rod test) and night-time activity. We also assessed body weight, body composition (MRI), gastrocnemius muscle weight/circumference, grip strength, and ammonia and liver enzymes in plasma. Results from female BDL rats were compared to historical laboratory data from male BDL rats. Results Female BDL rats had increased liver enzymes (ALP (P=0.001) and AST (P<0.0001) (but not ALT)), bilirubin (P<0.0001) and ammonia (p<0.001), and decreased albumin (P<0.0001) compared to female Shams. These results were comparable to male BDL rats except ALT and ammonia which were lower in females (p< 0.01). Female BDL rats did not differ in body weight, muscle circumference/weight and grip strength but had decreased fat mass (p<0.0001), increased lean mass (p<0.005) compared to female shams. Whereas, male BDL rats have decreased fat mass, muscle circumference/weight and grip strength. BDL in female rats induced a dysregulated estrous cycle compared to Sham (increased metestrus phase (p<0.01)). Similar to male BDL rats, female BDL rats had increased anxiety (p<0.005), motor incoordination (p<0.05), and decreased night activity (p<0.05) independent of the estrous cycle phase. Discussion We demonstrated BDL surgery in females leads to hepatic and neurological impairment comparable to male BDL rats (similar intra-group variability). Interestingly, female BDL rats developed unique features. Contrary to male BDL vs Shams, body weight and muscle mass does not differ between female BDL and Shams. Since muscle mass plays an important compensatory role in regulating ammonia levels, this could explain why the increase in blood ammonia levels in female BDL rats (vs. female Shams) was lower compared to male BDL. We expect that this model will provide new insights on the effect of sex differences on the pathogenesis of CLD and HE and help to personalize HE treatment.
Nouvelles connexes
Présentations au ISHEN 2019
5 years ago
