Background: Liver failure/disease leads to hyperammonemia which is central in the pathogenesis of hepatic encephalopathy. However, it is believed systemic oxidative/nitrosative stress may play an important role in exacerbating the neuropsychological effects of hyperammonemia observed in patients with liver disease. The present study aims to investigate the role of oxidative/nitrosative stress in 2 rat models of liver disease/hepatic encephalopathy; 1) portacaval anastomosis (PCA), and 2) bile-duct ligation (BDL). Methods: Rats were sacrificed 4 weeks following PCA and 6 weeks following BDL. Arterial plasma and cerebrospinal fluid (CSF) were collected to measure ammonia, reactive oxygen species (ROS), hydrogen peroxide (H2O2) (only measured in plasma) and nitric oxide (NO) using a commercially available kit, the DCFDA and Amplex Red fluorescent techniques and the Griess reaction respectively. S-nitrosylation of cystein thiols was measured in the frontal cortex and plasma proteins by immunoblotting. Brain water content was measured in the frontal cortex using a specific gravimetric technique. Results: Hyperammonemia developed in both PCA (PCA: 181.0 ± 8.5uM vs sham: 60.5 ± 7.0uM; p<0.0001) and BDL (BDL: 101.1 ± 11.0uM vs sham: 41.7 ± 9.2uM; p<0.05) rats with PCA rats being significantly higher compared to BDL rats (p<0.001). Consequently, ammonia increased in CSF in both PCA (PCA: 159.5 ± 15.8uM vs sham: 32.5 ± 3.7uM; p<0.0001) and BDL (BDL: 123.5 ± 21.7uM vs sham: 23.3 ± 6.1uM; p<0.05), but no significant change was observed between PCA and BDL. Only rats with BDL demonstrated an increase in plasma levels of ROS (5.8-fold increase vs sham; p<0.001), H2O2 (3.2-fold increase vs sham; p<0.05) and NO (2.9-fold increase vs sham; p<0.05). This was accompanied by an increase in S-nitrosylation of plasma proteins. No significant changes in CSF ROS and NO, nor in S-nitrosylation of frontal cortex proteins were found in BDL or PCA compared to their respective control groups. In addition, an increase in brain water content was observed in rats with BDL (BDL: 81.91 ± 0.19% vs sham: 81.23 ± 0.20, p<0.05). No significant change in brain water content was found in PCA rats vs sham-operated controls. Conclusion: Systemic oxidative stress together with hyperammonemia results in an increase in brain water in rats with BDL which is not demonstrated in PCA rats where only hyperammonemia is observed. Our findings suggest systemic oxidative stress is implicated in the pathogenesis of brain edema and its synergistic effect with ammonia may lead to progression of hepatic encephalopathy.
