Eye For Focal Cerebral Ischemia Reperfusion Injury Protective Effect And Mechanism Of Apoptosis

Objective and background:The key to the treatment of Ischemic Cerebrovascular Disease is to rescue the dying neurons in ischemic penumbra and promote the recovery of injured nerve function. Cerebral ischemia-reperfusion injury is the pathological phenomenon that after a short time of ischemia, the brain tissue restores the blood supply while to make the ischemic injury more serious. The mechanism of cerebral ischemic injury is much complicated. The death of the neurons in ischemic penumbra of cerebral ischemia-reperfusion injury takes the apoptosis as the main form for dying. Eye-acupuncture therapy is effective to cure stroke. During that, a lot of experiences are accumulated. But the function mechanism is still in study. The subject is to probe the protective mechanism of eye-acupuncture in the treatment of cerebral ischemia-reperfusion injury by restricting neuron apoptosis and regulating the death receptor signal path of neuron apoptosis. At the same time, find the function mechanism of eye-acupuncture treatment to Ischemic Stroke, in order to provide scientific evidence for the clinical experience.Method:Group division: Divided the healthy male SD rats into 3 groups, that is, the sham-operated group, model group and eye-acupuncture group. Each group was divided into groups with different test period like 3h, 24h, or 72h etc. for the cerebral ischemia-reperfusion.Model Making: Make the model rat of focal cerebral ischemia-reperfusion according to the improved method of Longa.Experimental method: The points searching of eye-acupuncture group: Shangjiao area, Xiajiao area, liver area, kidney area . Method of acupuncturing: Acupuncture the point area levelly with 2mm distance from the internal orbital margin with the 0.25mmx40mm tiny needles, from starting to the ending in the point area. Until 0.3cm into the area, change to take the twisting practices for 30 minutes. During that time, the needle should be practiced for 1 minute every 15 minutes. Acupuncturing timing: Give the assessment to neurological function deficits at the starting moment of reperfusion. Give the assessment to neurological function deficits at the starting moment of reperfusion. The rats are divided into every group at random. The test hours for acupuncture should be chosen at the moment of reperfusion and before death. Give the acupuncture every 12 hours in the period of living. The rats of sham-operated and model group are fed as usual without any treatment.Method of testing and indicators:Make the scores of neurological function deficits according to Longa 5-score standards; adopt the TTC staining to measure the infarct volume; observe the pathological changes and the ultra-structure of the brain tissue in ischemic penumbra by micro-spectroscope and electron microscope; detect neuronal apoptosis of the brain tissue in ischemic penumbra by the TUNEL method; test the expression of Fax/Faxl of the brain tissue in ischemic penumbra by immunohistochemistry; test the protein expression of caspase-3mRNA and caspase-8mRNA by RT-PCR of the brain tissue in ischemic penumbra by RT-PCR methods; test the content of TNF-αin ischemic penumbra of the brain tissue and in serum by using the Elisa method.Method of data statistic: All the data, in the expression form of±SD, is analyzed by spss10.0 software. And the group comparison adopts the single factor variance analysis. Results:1.The effect of eye-acupuncture on neurological function deficitsNo neurological function deficits appear after the operation in sham-operated group. At the point of 3h is chemia-reperfusion in model group and eye-acupuncture group,The symptom of neurological function deficits is the most serious one in the 24h and 72h ischemia-reperfusion in model group, so the difference is remarkable between eye-acupuncture group and model group.2. The effect of eye-acupuncture on Infarct volumesNo infarct lesion is found in sham-operated group after 24h following ischemia-reperfusion. The infarct lesion in model and eye-acupuncture group lies in the right part of forehead, temporal, parietal cortex, caudate nucleus and Putamen region. The Infarct volume in eye-acupuncture group is obviously less than that in model group.3.The effect of eye-acupuncture on the pathological changes and the ultra-structure of the neurons in ischemic penumbraThe model group shows the typical infarct changes. The necrotic foci can be seen clearly in the parietal part of the ischemic side, the lateral striatum and the prefrontal. Fewer cells exist in the necrotic foci.Observation by micro-spectroscope:There are rich organelles in neuron cytoplasm in penumbra of the sham-operated group. The nucleus is big and elliptic, with the clear and smooth structure of two membranes. The chromatin in the nuclear has uniform distribution.4.The effect of eye-acupuncture on neuron apoptosis in ischemia penumbraApoptotic cells of model group and eye-acupuncture group just begin to increase after 3h following ischemia-reperfusion. It shows no statistically significant difference at 3h in eye-acupuncture group, compared with the model group. The number of apoptotic cells is obviously less at 24h and 72h in eye-acupuncture group, compared with the model group. There is significant difference by statistics .5. The effect of eye-acupuncture on the expression of Fax/Faxl of the neurons in ischemicpenumbraThe model group, eye needle group masculine goal expression After Nao ischemia pours into again 3h to start to increase, various time model group, the eye needle group and the sham-operation group compare the masculine cell expression to have the significance difference; The eye needle group and the model group compare, a 3h time masculine goal expresses the non-significance difference; 24h, the 72h time masculine goal expression remarkable reduction, has the significance difference6.The effect of eye-acupuncture on the expression of caspase-3mRNA caspase-8mRNA in penumbraVarious time model group, the eye needle group and the sham-operation group compare Caspase-3, Caspase-8 the mRNA expression to have the remarkable difference; The eye needle group and the model group compare, 3 hour non-significance difference; 24 hour, 72 hours have the unusual significance difference.7. The effect of eye-acupuncture on TNF-αof blood and brain tissue in penumbraVarious time model group, the eye needle group and the sham-operation group compare Caspase-3, Caspase-8 the mRNA expression to have the remarkable difference; The eye needle group and the model group compare, 3 hour non-significance difference; 24 hour, 72 hours have the unusual significance difference. Conclusion:1. When making the model rat of cerebral ischemia-reperfusion with the improved line-tying method, it appears the symptom of neurological deficit and the cerebral infarction lesion. It proves that the model making is successful.2. Stroke disease lies in the brain and has close relationship with the liver and kidney. Eye- acupuncture can improve the scores of neurological function deficit of the cerebral ischemia-reperfusion rats and many molecular biology dictators. It proves that eye-acupuncture can regulate the actual situation of viscera and clear the block of the meridian. Thus, we have the belief that"The eye connects with the brain with the meridian and regulates the viscera."3. Eye- acupuncture can reduce the scores of neurological function deficit of the cerebral ischemia-reperfusion rats, decrease the infarct volume, improve the injury of impaired neuron in penumbra and at the same time protect the impaired brain caused by ischemia-reperfusion.4. Apoptosis cells have serious condition in penumbra of the cerebral ischemia-reperfusion rats in model group. It shows that can exist in the cerebral ischemia-reperfusion injury, while eye- acupuncture can restrict the apoptosis in the penumbra to protect the impaired brain caused by ischemia-reperfusion.5. The expression of Fax and Faxl in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases obviously, which proves that the high expression of the promoted apoptosis signals exist in the cerebral ischemia-reperfusion injury. Eye- acupuncture may decrease the expression of Fax and Faxl in the ischemic penumbra to restrict the conduct of the apoptosis signals, so that it can protect the impaired brain caused by ischemia-reperfusion.6. The expression of Caspase-3 mRNA and Caspase-8mRNA in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases remarkably, which proves that the high expression of the promoted apoptosis signals exist in the cerebral ischemia-reperfusion injury. Eye- acupuncture may decrease the expression of Caspase-3 mRNA and Caspase-8mRNA in the ischemic penumbra to restrict the conduct of the apoptosis signals, so that it can protect the impaired brain caused by ischemia-reperfusion.7. The content of TNF-αof blood and brain tissue in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases remarkably, which proves that the content of TNF-αexisting in the cerebral ischemia-reperfusion injury increase obviously. Eye-acupuncture can decrease the expression. Eye- acupuncture may decrease the content of TNF-αof blood and brain tissue in the ischemic penumbra of the cerebral ischemia-reperfusion to restrict the apoptosis and the inflammatory response, so that it can protect the impaired brain caused by ischemia-reperfusion.8. The research reveals the partial mechanism of eye-acupuncture in treating ischemia brain vascular disease. Also it offers the molecular biology evidence for the clinical treatment of the brain vascular disease by eye-acupuncture.