| Objective:To study intracellular calcium signal transduction mechanisms during focal cerebralischemia reperfusion and the mechanisms of XingNaoKaiQiao (XNKQ) acupuncture method in modulating [Ca2+]i in neurons.Methods:1. SD rats are randomly divided into 6 groups: control group, sham operated group,model group, non-acupoint group, traditional acupuncture group and XNKQ acupuncture group. The last 4 groups are divided into ischemia 1h, reperfusion lh, 3h, 6h, 12h, 24h after lh ischemia, totally 6 time points. The model is established on rats by left middle cerebral artery occlusion (MCAO) induced by thread ligature. The 3 acupuncture group rats are stimulated with electro-acupuncture needles for 10 minutes. The rats are decapitated at corresponding time points. Then prepare living hippocampal slices and observe the dynamic changes of neuron [Ca2+]i in hippocampal CA1 area at different time points with laser scanning confocal microscope technique.2. Make brain paraffin section, using HE staining method, dynamicly observe the hippocampus histological structure and the neurons pathological changes.3. rats is respectively injected neomycin, heparin, dantrolene, thapsigargin(TG), carbonyl cyanide 3-chlorophenylhydrazone(CCCP) through left cerebral ventricle, after 20 minutes establish MCAO model, acupuncture intervention is the same with the above. Rats are decapitated at 6h ischemia reperfusion time point, then prepare living hippocampal slices to observe [Ca2+]i changes.Results:1. Compared with the control group, intracellular Ca2+ concentration of the neurons ([Ca2+]i, expressed by intracellular Ca2+relative fluorescent intensity) of hippocampal CA1 area in model group is elevated after 1h ischemia, and continue to increase at 1h, 3h reperfusion time point after 1h ischemia; the [Ca2+]i is increased at the maximal level at 6h time point, at 12h time point [Ca2+]i is decreased; the [Ca2+]i is increased again at 24htime point, but is still lower than that of 6h time point ( PO.05 ). Compare with control group, sham operated group has no difference (P>0.05). There is no difference between non-acupoint group and model group at the same time point (P>0.05).The [Ca2+]i in both traditional acupuncture group and XNKQ acupuncture group are lower than that of model group at corresponding time point ( P<0,05, PO.01 ), compare with traditionalacupuncture group., XNKQ acupuncture method has more significance ( PO.05 ).2. Observe the hippocampal neuron changes with HE staining method: thehistological structure is regular in control and sham operated group, the number of neurons are normal, structure of the neurons are clearly and distributed in order without injury. The neurons of hippocampal area has no significant changes at lh ischemia and lh, 3h reperfusion time point, at 6h reperfusion time point, hippacampal area begin to emerge the focal ischemic changes: the nuclear of some neurons are dyed deeply with pyknosis, the injury become aggravated with the reperfusion time extended; necrosis cells are significantly increased at 24h time point. The changes in non-acupoint group are similar to model group. The neuron damage area is diminished at both traditional acupuncture and XNKQ acupuncture group, just a small quantity of the nuclear of the cells are dyed deeply with pyknosis, necrosis cells are significantly decreased compared with model group.3. After inhibiting PLC activeity, IP3R or RyR channels respectively, [Ca2+]i in hippocamal neurons in both sham operated and XNKQ groups have no significantly changes before and after medication (P>0.05), [Ca2+]i in model group is significantly lowed after medication ( P<0.05) and XNKQ acupuncture group is lower than that of model group ( PO.05 ).4. After inhibiting Ca~' pump on plasma and ER or mitochondrial function respectively, the [Ca2+]i in sham operated group-has no significantly changes before and after medication ( P>0.05 ) while both model group and XNKQ group increase significantly ( PO.05 ). XNKQ acupuncture group is lower than that of model groupafter medication (PO.05). Conclusions:...
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