Background: Hepatic encephalo¬pathy (HE) is a neuro¬psychiatric syn¬drome and it is understood to be reversible following liver transplantation (LT). However, up to 47% of LT patients have been documented to have persisting neurological complications which is believed to be associated with a history of HE pre-LT. We hypo¬thesize that episodes of HE will accelerate neuro¬logical deterioration. Our goal was to evalu¬ate the impact of cumulative HE episodes (ammonia induced) on neuro¬logical status and brain injury in rats with chronic liver disease (CLD).
Methods: Five-week bile-duct ligation (BDL) rats and Sham-operated controls were grouped into episodic and non-episodic groups. Episodes of HE were induced every 4 days starting the week 3 post-BDL following a reversible injection of ammonium acetate in which BDL rats fell into pre-coma (loss of righting reflex) for 20-30min. Three days following the last injection, neurobehavior was assessed. Upon sacrifice, plasma ammonia was measured, and brains were collected for western blot and immunofluorescence analysis; neuronal markers including NeuN and SMI311, astrocytic marker; GFAP and apoptotic markers such as cleaved caspase-3 and Bax and Bcl2 were measured to evaluate neurological integrity.
Results: BDL rats, and not Sham rats, injected with ammonia developed overt HE. Long-term memory (LTM) was impaired in both non- and episodic BDL groups vs respective controls. However, LTM impairment was further aggravated in episodic BDL rats. Protein expression levels of GFAP, cleaved caspase-3 and Bax/Bcl2 ratio were significantly increased, whereas NeuN and SMI311 were significantly decreased in hippocampus of episodic BDL rats vs non-episodic BDL rats. Presence of cleaved caspase-3 in neuronal layer of CA1 area of hippocampus was confirmed with immunofluorescence imaging. Blood ammonia levels were found to be higher in episodic vs non-episodic BDL rats.
Conclusion: Episodes of overt HE exasperate neurological impairment in BDL rats. LTM impairment was associated with an increase in apoptotic markers, decrease in neuronal markers and presence of cleaved caspase-3 in neuronal layer of CA1 area of hippocampus, suggesting neuronal injury/loss. Elevated levels of GFAP (astrocytic marker) in the hippocampus insinuates gliosis, possibly as a consequence of neuronal loss. These results suggest that multiple episodes of HE may cause permanent cell damage, leading to therefore irreversible and persisting neurological complications post-LT.