Ischemia- reperfusion (IR) injury occurs in a wide spectrum of clinically important disorders such as myocardial infarction, stroke, hemorrhagic shock, and organ transplantation. IR leads to an acute inflammatory tissue injury in which the inflammatory mediator cytokines play an important role. The Pl's lab has previously reported that inhibition of cytokine release from the macrophages significantly attenuated IR liver injury in mice (Mosher et al 2001). Recent studies have indicated that acetylcholine, the principal vagus nerve neurotransmitter, inhibited cytokine release by macrophages, through a novel physiological mechanism, termed the cholinergic antiinflammatory pathway (Tracey KJ, 2002).Thus, the objective of this proposal is to evaluate the effect of pharmacological stimulation of cholinergic anti-inflammatory pathway in liver injury following hepatic IR. The hypothesis is that the pharmacological stimulation of cholinergic pathway via acetylchofine receptors (AChRs) inhibits cytokine release, thereby inhibiting the development of inflammatory liver injury following IR. To test this hypothesis, the following specific aims are proposed: I. Determine the inhibitory effect of AChRs stimulation on liver injury in response to hepatic IR in mice. The liver injury will be evaluated by liver histopathology and changes of the plasma alanine transaminase (ALT) levels. II. Determine the effect of AChRs stimulation on production of inflammatory cytokines in mice subjected to hepatic IR; tissue mRNA expression and plasma levels of the cytokines will be measured. III. Determine the effect of AChRs stimulation on neutrophil infiltration into the ischemic liver of mice subjected to IR. IV. Determine if liver macrophages (i.e., Kupffer cells) are the major target of cytokine inhibition via AchR stimulation in hepatic IR. An ex vivo perfused liver model will be applied. An in vivo partial hepatic IR model in mice, with 90 min of ischemia followed by specific reperfusion periods, will be applied. Selective AChR agonists and antagonists will be used as the pharmacological stimulants to evaluate the role of anti-inflammatory cholinergic pathway in hepatic IR injury. The liver injury will be evaluated by monitoring changes in plasma ALT levels, neutrophil infiltration, and liver histopathology. The local (tissue) and systemic (plasma) cytokine levels will be measured using the Western and ELISA techniques, respectively. The ex vivo liver perfusion model will determine the role of Kupffer cells in cytokine production and inhibition by AChR agonists. The results of these studies will provide new insight into the role of the cholinergic anti-inflammatory pathway in hepatic IR injury. The generated knowledge, which may be applied to a wide range of other IR related diseases, will assist in the development of novel therapeutic approaches for inflammatory disease.
