Hepatic encephalopathy sensibilize brain to neuronal death induced by hypotension/reperfusion in cirrhotics rats.
M.Sc., Biomedical sciences, Université de Montréal
Direction:
- Dr Christopher Rose
2017 - 2019
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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.
Evaluate the efficiency of Rifaximin treatment in a bile-duct ligated model of cirrhosis.
Grégory Petrazzo, Mariana M. Oliveira, Mélanie Tremblay, Christopher F. Rose.
Hepatic encephalopathy (HE) is a common and potentially severe complication of liver failure. HE is related to impaired quality of life. The pathogenesis of HE is linked with gut-derived ammonia (NH3) therefore treatment aim to reduce the blood level of NH3. The standard care of HE is Lactulose, a non-absorbable disaccharide. Nonetheless, patient observance is poor due to some uncomfortable side-effect. Among new therapies that have arisen this past decade, Rifaximin is a potent candidate. The aim of this study is to assess the efficiency of Rifaximin in reducing plasma NH3 and ameliorate the HE in a murine bile duct model. Three weeks after BDL surgery, BDL and SHAM-operated rats were sorted into five groups according to their treatments : SHAM-vehicle, BDL-vehicle, BDL-Lactulose, BDL-Rifaximin, BDL-Lactulose+Rifaximin. Treatment was given by gavage for 3 weeks. Survival, body-weight, food consumption and body composition were assess every week during the six weeks of the study. Behavioral analysis was conduct to assess the HE status. At sacrifice, brain water was measured to assess potential brain edema and plasma sample was taken to measure NH3 and hepatic biochemistry parameters. No difference was seen in the survival, growth pattern, food consumption or body composition between BDL-groups. No difference was seen in the performance during EPM, OFT, Rotarod, NOR or night activity. No difference was seen in brain water between SHAM or BDL groups. Surprisingly, NH3 level was increase in all but BDL-vehicle compare to SHAM. The biochemistry parameters confirm the establishment of cirrhosis in BDL groups compare to SHAM. Overall, this study didn’t present strong, reliable and enough data to conclude on the efficacy of Rifaximin as sole treatment in the context of HE. Some results fail to reach significance probably because of the low number of animals. Future direction on this project will aim to : 1) increase the antibiotic dose; 2) reduce the duration of the model to five weeks; 3) trigger an episode of HE by injecting a dose of NH3 before treating the rats.
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.
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.
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.
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