Studies of the pathogenesis of hepatic encephalopathy are hampered by the lack of a satisfactory animal model. We examined the neurological features of rats after bile duct ligation fed a hyperammonemic diet (BDL؉HD). Six groups were studied: sham, sham pair-fed, hyperammonemic, bile duct ligation (BDL), BDL pair fed, and BDL؉HD. The BDL؉HD rats were made hyperammonemic via an ammonia-containing diet that began 2 weeks after operation. One week later, the animals were sacrificed. BDL؉HD rats displayed an increased level of cerebral ammonia and neuroanatomical characteristics of hepatic encephalopathy (HE), including the presence of type II Alzheimer astrocytes. Both BDL and BDL؉HD rats showed activation of the inflammatory system. BDL؉HD rats showed an increased amount of brain glutamine, a decreased amount of brain myo-inositol, and a significant increase in the level of brain water. In coordination tests, BDL؉HD rats showed severe impairment of motor activity and performance as opposed to BDL rats, whose results seemed only mildly affected. In conclusion, the BDL؉HD rats displayed similar neuroanatomical and neurochemical characteristics to human HE in liver cirrhosis. Brain edema and inflammatory activation can be detected under these circumstances. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). H epatic encephalopathy (HE) is a severe neuropsychiatric complication in both acute and chronic liver failure. Although the pathophysiology of this disorder is incompletely understood, there is agreement on the important role of neurotoxins in its development, especially ammonia. 1 Neuropathologically, HE in chronic liver disease is characterized by astrocytic rather than neuronal changes. 2 Histopathology studies of brain sections from patients with cirrhosis who died in hepatic coma show the presence of changes known as Alzheimer type II astrocytosis. These astrocytes display a specific characteristic of swollen cytoplasm containing a large pale nucleus, with prominent nucleolus and patches of heterochromatin associated with the nuclear envelope. 2 Recently, a new pathophysiological hypothesis has been developed, emphasizing the role of low-grade brain edema in the pathogenesis of HE in chronic liver disease. 3 Following this theoretical model, the presence of a lowgrade astrocyte swelling could have important functional consequences despite the absence of clinically overt increases of intracranial pressure. 3

The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (∼30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (∼20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (∼50–60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.