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.
