Protective Effects Of Cerebral Arteries-released Endothelium Derived Hyperpolarizing Factor On Rats Insulted By Cerebral Ischemia-reperfusion And Relevant Mechanisms

Aim To study the neuroprotective effects, with the possible mechanisms, of acetylcholine(ACh) mediated - presumptive endothelium derived hyperpolarizing factor (EDHF), released from cerebral arteries, on both rat brain suffered from cerebral ischemia-reperfusion and cultured neurons subjected to hypoxia- reoxygenation injury.Methods1. In vivo: rat middle cerebral artery occlusion (MCAO) injury model was used through out the whole experiments, EDHF releasing from cerebral arteries was mediated by injection of 1μmol·L-1 ACh into rat common carotid artery(CCA) in the presence of NG - nitro - L - argininemethyl ester(L - NAME, a NOS inhibitor) and indomethcacin (Indo, a COX inhibitor), meanwhile, KC(l25 mmol L-1)was served as the inhibitor of EDHF. Influence of cerebral arteries-released EDHF on rat cerebral infarction size, water content, Lactate dehydrogenase(LDH) activity and content of malonaldehyde (MDA) in the blood serum were detected.2. In vitro: injury model of PC12 cells and Primarily cultured hippocampal neurons insulted by hypoxia-reoxygenation were utilized in these experiments respectively. EDHF releasing from rat middle cerebral arteries (MCAs) segments was mediated by 1μmol·L-1 ACh in the presence of NG-nitro-L-argininemethyl ester(L-NAME) and indomethcacin (Indo), in addition, KCl ( 25 mmol L-1 ),BAPTA-AM (a membrane-permeable Ca²⁺ chelator) and Chlorpromazine (a calmodulin antagonist) were used as the EDHF inhibitors. The impact of MCA segments-released EDHF on MTT absorbance and LDH activity in the supernate culture fluid of both PC12 cells and primarily cultured hippocampal neurons were observed; Moreover, Hoechst dying and flow Cytometry technique were utilized to exam whether EDHF delivered by rats MCA segments can influence apoptosis rate of primarily cultured hippocampal neurons; Also, Confocal laser scanning microscope (CLSM) and Western blot technique were employed respectively to investigate the concentration of free calcium and erk1/2 expression in primarily cultured hippocampal neurons.ResultsIn vivo Compared with normal group, the activity of LDH and the content of MDA in rats blood serum, also the cerebral infraction size and cerebral water content of rats in the group of MCAO model increased significantly;Contrasted with the group of MCAO model, the activity of LDH and the content of MDA in the blood serum, the cerebral infraction size and cerebral water content in the group of"ACh"and"ACh+L-NAME+Indo"decreased distinctively, while these indexes were not changed a lot in the"ACh+L-NAME+Indo+KCl"group. These results mentioned above indicate that injection of ACh into rats cerebral arteries can produce a protection of reducing the damages of MCAO rats and this kind of protection contains a non - NO and non - PGI2 mechanism which have deep relationship with EDHF secreted by cerebral arteries endothelium.In vitro1) PC12 cell experiments: Compared with normal cultured cells, MTT absorbance decreased significantly, LDH activity of the supernate culture fluid increased significantly in model group insulted by hypoxia-reoxygenation; Contrasted with model group, MTT absorbance increased significantly, LDH activity of the supernate culture fluid decreased significantly in the group of"ACh+MCA/endo"and"ACh+MCA/endo+L-NAME+Indo", while these indexes have no significant change in the"ACh"group, the"MCA/endo"group and the"ACh+MCA/-endothelium"group; In compare with"ACh+MCA/endo+L-NAME+Indo"group, MTT absorbance deceased significantly, LDH activity of the supernate culture fluid increased significantly in the group of"ACh+MCA/endo+L-NAME+Indo+KCl","ACh+MCA/endo(BAPTA-AM pre-bathed) +L-NAME+Indo"and"ACh+MCA/endo(CPZ pre-bathed)+L-NAME+Indo". These outcomes illustrate that contribution of ACh to MCA segments can lead to a neuroprotective effect against PC12 cell injuries insulted by hypoxia-reoxygenation, this kind of effect is vessel endothelium dependent and can neither be produced by ACh alone nor MCA segments alone, moreover, it contains a non - NO and non - PGI2 mechanism which have much concerns with EDHF released from MCA segments endothelium.2) primarily cultured rat hippocampal neurons experiments:â‘ MTT and LDH detecting: Compared with normal group, MTT absorbance decreased distinctively and LDH activity in the supernate culture fluid increased significantly in hypoxia-reoxygenation model group; In compare with model group, MTT absorbance increased significantly, LDH activity of the supernate culture fluid decreased significantly in the group of"ACh+MCA/endo"and"ACh+MCA/endo+L-NAME+Indo", while these two indexes did not change a lot in the groups of"ACh"alone,"MCA/endo"alone and the group of"ACh+MCA/-endothelium"; Contrasted with"ACh+MCA/endo+L-NAME+Indo"group, MTT absorbance deceased significantly, LDH activity of the supernate culture fluid increased significantly in the group of"ACh+MCA/endo+L-NAME+Indo+KCl","ACh+MCA/endo(BAPTA-AM pre-bathed)+L-NAME+Indo"and"ACh+ MCA/endo(CPZ pre-bathed)+L-NAME+Indo".â‘¡Cell apoptosis detecting: the apoptosis rate of primarily cultured hippocampal neurons was very low in control group normal cultured, apoptosis rate soared a lot in model group subjected to hypoxia - reoxygenation; Compared with model group, the neurons apoptosis rate changed little in the group of"ACh"alone,"MCA/endo"alone and the group of "ACh+MCA/-endothelium", while it decreased significantly in the group of"ACh+MCA/endo"and"ACh+MCA/endo+L-NAME+Indo".â‘¢Ca²⁺ concentration detecting: Contrasted with normal group, calcium fluorescence intensity enhanced distinctively in hypoxia-reoxygenation model group; In compare with model group, Ca²⁺ fluorescence intensity weakened significantly in"ACh+MCA/endo"group and"ACh+MCA/endo+L-NAME+Indo"group, while it have no conspicuous change in"ACh+MCA/endo+L-NAME+Indo+KCl"group.â‘£erk1/2 and p-erk1/2 expression detecting: comparatively, normal cultured hippocampal neurons had a relative big expression of p-erk1/2, while the p-erk1/2 expression in hypoxia - reoxygenation injury neurons had a small decrease; In compare with model group, the expression of p-erk1/2 had no change in the group of"ACh"alone,"MCA/endo"alone and the group of"ACh+MCA/-endothelium", but it increased a little in the group of"ACh+MCA/endo"and"ACh+MCA/endo+L-NAME+Indo". The expression of non - phosphorylated erk1/2 basically had no variation in every group. These results above manifest that contribution of ACh to MCA segments can generate protective effects on primarily cultured rat hippocampal neurons against damages insulted by hypoxia-reoxygenation, this protection is endothelium dependent and can only be produced by cooperation of ACh and MCA/endo, can neither results from ACh alone nor MCA/endo alone. Additionally, this protection has a non - NO and non - PGI2 mechanism which have many connections with EDHF released from MCA segments, and this EDHF - related protective mechanism concerns much with decreasing Ca²⁺ concentration and up-regulation of p - erk in neurons.Conclusion EDHF released from cerebral arteries has neuroprotective effects on both rat brain suffered from cerebral ischemia-reperfusion and cultured neurons subjected to hypoxia-reoxygenation injury, the mechanisms may be related to decreasing intracellular free Ca²⁺ concentration, attenuating oxidative damage and upregulating of p - erk1/2 expression.