| Background:Stroke has become a serious disease which threatens the health of our people. It is the largest cause of disability and the second largest cause of death in China, a heavy blow and burden to individuals, families and communities, among which the incidence of ischemic stroke, which accounts for 60% to 80%, has long been the lack of effective treatment. How to effectively mitigate damage and promote the rehabilitation of stroke patients is an important issue in neuroscience. Cerebral ischemia can start regeneration reaction for the ischemia surrounding tissue, including nerve regeneration and angiogenesis. Angiogenesis is a dynamic complex process involved by a variety of cytokines and cellular components. The central part of the initial is proliferation, migration, differentiation, and tube formation for the vascular endothelial cell. Under the microenvironment of the endothelial cell, the pro-angiogenic and anti-angiogenic factors are in dynamic equilibrium.. angiogenesis plays an important role in the repair process after brain injury, but the post compensatory angiogenesis of ischemic stroke is often inadequate. It needs further discussion about the cerebral ischemic angiogenesis mechanism. canonical Wnt pathway is a key link in the regulation of cell proliferation and differentiation. Developmental biology studies show that, Wnt play an important regulatory role in vascular endothelial cell proliferation, migration, vascular development and other aspects. A recent study on the development of cerebrovascular also shows, Wnt/ β-catenin signaling pathway is a key regulatory pathway in the regulation of brain angiogenesis, the formation of the blood brain barrier and the maintenance of its biological characteristics. Moreover, the formation of Wnt/β-catenin signaling pathway has an important effect on the formation and maintenance of the blood-brain barrier, the specific structure of cerebral vascular. Although Wnt/β-catenin signaling pathway has a crucial role in the development of cerebral blood vessels, whether the signaling pathway is involved in the adult animal brain angiogenesis after ischemia has not been reported.Objective:Using a rat model of MCAO, research the role of the canonical Wnt signaling pathway in angiogenesis after the rat ischemic strokeMethods:60 experimental Wistar rats were randomly divided into four groups: sham operation group (Sham group), cerebral ischemia control groups (MCAO group), cerebral ischemia plus lithium chloride intervention group (LiCl group), cerebral ischemia plus DKK1 intervention group (DKK1 group). Adopt suture method to occlude rat middle cerebral artery, prepare focal cerebral ischemia model, with reperfusion after 90min. Post operation at the point-in-time of 2h, Id,7d,14d give the neurological function score of the model. At postoperative 1d,7d,14d, test the change of the whole cells of the surrounding ischemic brain tissue and the change of nucleus β-catenin content by using immunoblotting. After exogenously adding lithium chloride and DKK1, test the change of the whole cell and nuclear β-catenin content and the change of the p-GSK-3β/GSK-3β ratio by using the Western blot after 7 days of the pose operation. After exogenously adding lithium chloride and DKK1 and 14 days of the post operation, measure the ischemic peripheral vascular density and the blood flow volume for the Doppler blood flow measurement instrument by FITC-dextran, a confocal microscopy viewer to reflect the angiogenesis circumstance.Results:suture occlusion method can establish a stable model for the focal cerebral ischemia reperfusion, and there’s a small difference between groups; 2h and 1d after cerebral ischemia reperfusion the adult male Wistar rats, the neurological score is higher than the normal group (P<0.01), and 7d,14d of reperfusion, the neurological score is still higher than the normal group (P<0.05); 1day after cerebral ischemia, the nucleus protein and (3-catenin expression level of total protein in ischemic brain tissue surrounding are significantly decreased (P<0.01), and recovery in 7days (P<0.05), and close to normal in 14 days (P> 0.05). After the intervention of the canonical Wnt pathway activator LiCl, β-catenin expression level and p-GSK-3β/GSK-3β ratio have a significant increase (P<0.01), and ischemic peripheral vascular density and blood flow volume are significantly increased (P<0.05); while after the intervention of the canonical Wnt pathway inhibitor DKK1, changes contrast, β-catenin expression level and p-GSK-3β/GSK-3β ratio are significantly lower (P<0.01), vascular density and blood flow volume are significantly decreased (P<0.05).Conclusion:The use of suture method of middle cerebral artery occlusion 90min can establish a simple, stable and ideal model of cerebral ischemia-reperfusion injury, which is repeatable and suitable for a wide range of application on the basis of experimental studies; this test uses immunoblotting for the first time to the detection of cerebral ischemia Wnt signaling pathway important signaling molecule β-catenin and p-GSK-3β/GSK-3β ratio changes, and uses FITC-dextran to measure the density of the ischemic vascular surrounding area, and use doppler blood flow instrument to measure the blood flow volume in ischemic region etc. Canonical Wnt signaling pathway participates in the adjustment of peripheral ischemic angiogenesis of ischemic strok. Further study on the canonical Wnt signaling pathway may open a new target for the treatment of ischemic stroke angiogenesis. |
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