The investigator proposes, in this revised competitive renewal, to study the pathogenic mechanisms of hepatic encephalopathy. The mechanism which lead to hepatic encephalopathy are not well understood but in recent years there has been substantial evidence which implicates ammonia, astrocytes, and excessive GABAergic activity mediated by elevated levels of endogenous benzodiazepines. During the prior funding period, the investigator studied the role of astrocytes, ammonia and benzodiazepines in hepatic encephalopathy and in their prior studies they found that 1) ammonia upregulates the peripheral benzodiazepine receptor in cultured astrocytes an in animal models of hepatic encephalopathy and 2) peripheral benzodiazepine receptor (PBR) blockers ameliorate ammonia toxicity in vivo. They also found that neurosteroids (which are products of PBR activation) are elevated in animal models of hepatic encephalopathy and in ammonia treated astrocyte cultures, and that some of these neurosteroids exert behavioral and neuropathologic changes similar to hepatic encephalopathy. Their hypothesis is that neurosteroids that positively modulate the GABAA receptor contribute to the pathogenesis of HE. They further propose that the elevation of neurosteroids result from an upregulation of astrocytic PBR by the hyperammonemia associated with liver failure. To test the hypothesis, they plan to investigate the profile of neurosteroids in animal models of HE and ammonia treated astrocyte cultures. They plan to determine whether neurosteroids are elevated in HE and are able to reproduce the clinical, pathological, and neurochemical features of HE. They will also examine whether inhibiting the synthesis or action of neurosteroids that have a positive modulatory effect on the GABAA receptor will improve the outcome of the experimental HE. They plan to study the role of astrocytic PBRs in the generation of neurosteroids from brains of animal models of HE by investigating PBR binding parameters. Measuring PBR mRNA steady-state levels, and localizing changes in the PBR by in situ hybridization. The functional integrity of the PBR will be examined by measuring the rate of pregnenolone synthesis. To establish the role of ammonia in the PBR-dependent production of neurosteroids by astrocytes, similar studies will be carried out in cultured astrocytes. Their proposed mechanism, whereby ammonia upregulates the astrocytic PBR resulting in elevated levels of neurosteroids that act on the neuronal GABAA receptor synthesizes current pathogenic views and more importantly, provides the potential for novel therapeutic approaches through intervention of neurosteroid effects.
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