The Study Of Central Muscarinic Cholinergic Receptor Involved In Activing Astrocytes And Producing Cytokines In Rats With Endotoxin

Objective To investigate the probable mechanism and interaction between central muscarine cholinergic receptor and the inflammation immune response in brain and periphery through by the effects of centrally administered muscarine receptor agonist/antagonist on the activation and multiplication of astrocytes and the levels of cytokines in brain and periphery during endotoxin in rats.Methods The study have two parts1. The effect of muscarine cholinergic receptor agonist/antagonist administered intracerebroventricularly on the activation and multiplication of astrocytes in rats with endotoxin.Twenty-four male SD rats were divided into four groups by randomly(n=6 each): saline group(N):inject saline intravenously and intracerebroventricularly;control group(C): inject LPS intravenously,saline intracerebroventricularly; antagonist group(A):inject LPS intravenously ,muscarine receptor antagonist atropine intracerebroventricularly; agonist group(M):inject LPS intravenously, muscarine receptor agonist muscarine intracerebroventricularly. Administer compound or saline intracerebroventricularly one hour before making rat models which were induced by LPS. Take the rat brains to measure the expression changes of GFAP in the hippocampus CA1 area by immunohistochemistry four hours after making the rat models.2. The effect of muscarine cholinergic receptor agonist/antagonist administered intracerebroventricularly on the the levels of cytokines in brain and periphery during endotoxin in rats.The grouping is the same to part one. The rats in each group were administered compound or saline intracerebroventricularly one hour before making rat models which were induced by LPS. Take the blood about 5ml through the abdominal aorta four hours after making the rat models. Blood was centrifuged and the supernatants were collected for cytokines determination. Sera were used for TNF and IL-6 protein analysis by ELISA. At the same time, got the two sides of hippocampus from the brains, tissue homogenated and collected the supernatants. Protein was quantitied by BCA at first, then analysis TNF,IL-6 protein by ELISA.Results1. The results of immunohistochemistryThe expression of GFAP in hippocampus was significantly enhanced in group C than that of group N. Compared with the group N, the GFAP-positive cells in group C stained deeply , appeared hypertrophia. The branches of these cells were growed in number and coarsened. Group M showed that the muscarine receptor agonist i.c.v can significantly suppress the activation of astrocytes. Conversely, group A didn't have the same effect. Image analysis showed that compared with group N, the number of GFAP-positive cells and average optical value were notablely increased in other groups. The results had significantly difference between group N and other groups.(P<0.05). Compared with group C, the number of GFAP-positive cells and average optical value were notablely decreased in group M. The results had significantly difference between group C and group M in statistics.(P<0.05). But group A and group C had no significantly difference in statistics.2. The results of ELISASerum and supernatants TNF,IL-6 productions in group C were significantly higher than those of group N. The results had significantly difference between group C and group N in statistics.(P<0.05). Serum and supernatants TNF,IL-6 productions in group M were significantly lower than those of group C. The results had significantly difference between group M and group C in statistics.(P<0.05). There was no significantly difference between group A and group C in serum and supernatants TNF,IL-6 productions.Conclusion1. Our study showed that LPS can active astrocytes, increase the expression of GFAP. Muscarine receptor agonist muscarine which was administered intracerebroventricularly can significantly suppress the activation of astrocytes induced by LPS. It showed that the number of GFAP-possitive cells and average optical value were notablely decreased. There was no same effect, when we administered muscarine receptor antagonist atropine intracerebroventricularly.2. LPS can increase the secretion of serum and supernatants TNF-α,IL-6. Muscarine receptor agonist muscarine which was administered intracerebroventricularly can significantly suppress the level of serum and supernatants TNF-α,IL-6. There was no same effect, when we administered muscarine receptor antagonist atropine intracerebroventricularly..