| Objectives:Preparation of rat by intraluminal Middle cerebral artery obstruction model, preliminary study of noninvasive limb lschemic Postconditioning on acute cerebral ischemiainjury in rats have a protective effect. And research on ischemic postconditioning on focalcerebral ischemia-reperfusion injury in rats after short-term and long-term neurological effects.And study on the mechanism of TLR4-MyD88signal transduction pathway in the protection ofremot ischemic postconditioning in rats cerebral ischemia. To provide new ideas for the specifictreatment of cerebral infarction.Methods:The experiment is divided into three parts. Part I:Establishment of stable rat model of MCAO suture, Proven non-invasive remote Ischemic Postconditioning on the protective role of ischemic brain tissue.60healthy male Sprague Dawley (SD) rats (250g-300g), Were divided into5groups, each group of12:(1) Sham-operated Group (Sham);(2)2h ischemia reperfusion Group (ischemic reperfusion,I/R Group), for the control group;(3) Ischemia Postconditioning in situ Group (ischemic postconditioning,IP Group);(4) Ischemic Postconditioning Group (remote Ischemic Postconditioning at beginning of ischemic,I-RPC Group);(5) Immediate adaptation group after reperfusion (remote Ischemic Postconditioning at beginning of reperfusion,R-RPC Group). Immediate grant of MCAO in rat2h-reperfusion noninvasive RPC. The rats of neural function defect score in24h after reperfusion respectively. Measuring apoptosis by TUNEL, wet and dry weighing method to measure the water content, TTC staining photograph, Image ProPlus software analysis, calculate the cerebral infarction volume, HE dyed light microscopic observation on morphology. Part II:Many times repeat remote postconditioning on focal cerebral ischemia effect of recovery of neural function in15days.90healthy adult male SD rats were randomly divided into3groups, each group of30, according to the different time in rats of each group (Id,3d,6d,10d,15d) is divided into5subgroups, each subgroup6:(1) Sham-operated Group (Sham);(2) ischemia reperfusion group2h(I/R Group);(3) the Group of remote Postconditioning (RPC Group). And the non-invasive neurological functional scoring is completed corresponding number of days time (Id,3d,6d,10d,15d) in the RPC after6hours and detect the hippocampal cellular apoptosis using TUNEL method after the death of rats. Part Ⅲ:In that remote postconditioning based brain protection effect, and investigate the possible mechanism of its. All the72male Sprague-Dawley (SD) rats, weighing250g-300g, were divided into Control group (C group,n=24),experimental group (which was treated with remote ischemic postconditioning after cerebral reperfusion (P group,n=24)) and Sham group (S group, n=24) stochastically. Each group was randomly divided into4time points:12h,24h,48h,72h (for each time point group, n=6). The focal cerebral ischemia-reperfusion model was established with modified Longa’s method which formats middle cerebral artery occlusion(MCAO). Rats in P group were given reperfusion after2hours of MCAO and treated with remote ischemic postconditioning immediately. Pathological changes were observed in the hippocampus pathological HE staining. To detect the expression of hippocampal TLR4protein using immunohistochemical staining and Western blotting. Expression of TLR4mRNA in the hippocampus were measured in situ hybridization.. Immunohistochemical staining was used to detect the expression of NF-κB, hippocampus MyD88, hs-CRP, IL-6.Results:(1) Compared with the control group, IP Group, I-IPC group, R-RPC Group, levels of apoptotic cells in rats, the degree of cerebral edema and cerebral infarction volume, the degree of pathological changes were significantly reduced, with statistically significant differences (p<0.05); But two two comparison between the three groups in the IP Group, I-IPC group, R-RPC Group has no significant difference (p>0.05).(2) The reperfusion24h nervous system scores indicated in IP Group, I-IPC group, R-RPC group compared with control groups decline, but no statistically significant differences (p>0.05); And two two comparison between the three groups in the IP Group, I-IPC group, R-RPC Group has no significant difference (p>0.05).(3) Compared with the Sham group, I/R and RPC Group NSS score increases, but less increase in RPC Group (p<0.05); After operation3D, the neuronal apoptosis in I/R and RPC groups increased significantly, I/R Group of more serious (p<0.05);(4) To observe by HE dyeing:Sham group:The structure of hippocampus was clear. In hippocampus region, the cell form was normal and in good arrangement and well-bedded. I/R group:The structure of hippocampus was loose. In hippocampus region, neuronal loss was obvious, cells and cell nuclei contracted and were stained deep. Some cells swelled and cavitation existed in endochylema. We also found small necrosis. RPC group:The neuronal loss and swelling was reduced distinctly. The cell form was improved significantly.(5) The TLR4, TLR4mRNA,MyD88and NF-κB expression level in each subgroup of Sham group was low in rats hippocampus region. Compared to Sham group, the expression of TLR4, TLR4mRNA,MyD88in I/R group and P group increased significantly (P<0.05). Compared to I/R group, the expression of TLR4in RPC group reduced at each time point before72h (P<0.05), but the level of NF-κB expression was marginally higher at72h which was not significant (P>0.05).(6) The expression of hs-CRP and IL-6was Only a small amount of expression in each subgroup of Sham group in rats hippocampus region. Compared to S group, the hs-CRP expression level in I/R group and RPC group increased significantly (P<0.05). The hs-CRP expression was lower in each subgroup of RPC group than that in I/R group (P<0.05)Conclusion:Remote ischemia Postconditioning is same as ischemia Postconditioning,can reduce acute ischemia cerebral infarction volume of rats to reduce brain oedema, less number of apoptotic cells, can improve the cerebral ischemic rats15days the degree of neural function defect, has a neuroprotective effect on acute cerebral ischemia in rats. The mechanism might be downregulation of TLR4-MyD88signal transduction pathway and the inhibition of inflammatory cytokine. |
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