The blood-brain barrier in cirrhotic rats: a male and female comparison.
Licence, Université de Poitiers, France
Direction:
- Dr Christopher Rose
Winter 2019
Related Publications
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.
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