Background: Hepatic encephalopathy (HE) is a major complication of chronic liver disease (CLD). Defined as a metabolic disorder, HE should resolve following liver transplantation (LT). However, persisting neurological complications have been reported in up to 47% of LT recipients. Retrospective studies have demonstrated an association between a history of HE episodes and neu-rological deficits post-LT. Our aim was to evaluate the impact of multiple episodes (induced by ammonia) on neurological status, integrity and injury in rats with CLD. Methods: 5-week bile-duct ligation (BDL) rats were in-jected (ip) with ammonium acetate (BDL-Ammonia), pre-cipitating an overt episode of HE (loss of righting reflex). Episodes of HE were induced every 4 days from week 3 post-BDL surgery (total; 4 episodes). Sham rats were also injected with a similar dose of ammonia (Sham-Ammonia) and respective controls were injected with sa-line. Three days after the last episode, long-term mem-ory (LTM) was assessed. Upon sacrifice, plasma and brains were collected for oxidative stress measurements and western blot analysis for NeuN and SMI311 (neuro-nal markers), caspase-3 and Bax/Bcl2 ratio (markers of apoptosis), GFAP (astrocyte marker) and 4-HNE (marker of oxidative stress) in frontal cortex, cerebellum and hip-pocampus. Results: LTM was found to be impaired in both BDL-Saline and BDL-Ammonia groups vs respec-tive controls. However, LTM impairment was further ag-gravated 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 as well as increased 4-HNE were found solely in the hip-pocampus (not frontal cortex or cerebellum) of the BDL-Ammonia group compared to controls. Conclusion:Multiple episodes of overt HE leads to a worsening of neurological impairment in BDL rats. LTM impairment in the BDL-Ammonia rats was associated with increased oxidative stress, apoptotic markers and decreased neu-ronal markers in the hippocampus, supporting neuronal injury/loss. Elevated levels of GFAP in the hippocampus insinuate gliosis, possibly a result of neuronal loss. These results suggest that repeated HE episodes may cause permanent cell damage which will less likely be revers-ible following LT.
