A Study On Mechanism Of Microvascular Impairment In Rat Model Of Cerebral Ischemic-reperfusion

Ischemic stroke is a common disease in aged people, it is a major source of increased morbidity, mortality and disability rat, which is a threat to people's health and results in heavy pressure of spirits and economy on the society and families. In the field of ischemic stroke treatment, thrombolysis is the only effective treatment that was generally adopted to acute cerebral infarction. But the time window of thrombolysis is short. There is a potential risk of secondary cerebral hemorrhage. Therefore, the clinical application of thrombolysis limited. It was the brain protection that become a hot topic appeared in clinical. The combination of thrombolysis and brain protection is the direction of ischemic stroke treatment in the future.At present, the cerebral protection treatment of ischemic stroke is mostly focused on dilating blood vessel, anti-free radical, antagonism of excitatory amino acid(EAA), etc. The microvascular impairment can aggravate brain edema, cause hemorrhagic transformation after cerebral infarction,leukocyte infiltration and phagocytosi and further release inflammation factor, and form inflammation magnification and positive feedback. So the blood-brain barrier(BBB) composed of blood capillary play an extremely important role in the neural impairment of cerebral ischemic stroke and have been regarded as a new key target.Rho-kinase(ROCK) is a kind of serine/threonine kinase, which regulates cell movement including cytodieresis, contraction, adherence, migration, secretion, etc through downstream effector molecule. In ischemic cerebrovasculardisease, ROCK mediate inflammatory after ischemia and lead to nerve blood vessel impairment of cerebral ischemic stroke by many ways. At present, the study on mechanism of microvascular Impairment is rarely reported.In the study, we used an established middle cerebral artery occlusion(MCAO) model described initially by Zea-Longa to explore the relationship between Rho-kinase,matrix metalloproteinase-9(MMP-9),S100B and microvascular impairment, and furthermore to inquire that Rho-kinase mediate microvascular impairment in rat model of cerebral ischemic-reperfusion by regulating the expressing of MMP and S100B. The study provide the experimental foundation for Rho-kinase being regarded as a new key target of microvascular impairment in ischemic stroke.Part I The relationship between Rho-kinase and microvascular impairmentObjective:To observe the relationship between Rho-kinase and microvascular impairment by middle cerebral artery occlusion(MCAO) model.Methods:All male SD rats bought from the laboratory animals centre in our university were randomly divided into following five groups:sham group,MCAO 6hours,24 hours,48 hours and 72 hours group.There are fifteen SD rats in every groups. The right middle cerebral artery ischemia-repefusion models described initially by Zea-Longa were established. After 90 minutes of temporary MCAO, The rats were respectively subjected reperfusion of 6h,24h,48h and 72h. In this part of experient the expression levels of Rho-kinase mRNA were detected by RT-PCR. And we killed the rats and draw the ischemic penumbra brain tissue of infarct side to observe the change in permeability of BBB by detecting the contentof Evan's blue(EB), to measure the expression levels of laminin by immumofluores-cence method and to observe the relationship between them.Results:There are obvious statistics difference (P<0.01) between the ischemia-repefusion 6 hours,24 hours,48 hours,72 hours groups and sham group in the neurological functional decect; Among the four MCAO groups the neurological functional decect have no statistics difference (P>0.05). The expression levels of Rho-kinase mRNA,the content of EB and the expression level of laminin have obvious statistics difference between the five groups(P<0.05); There are obvious statistics difference (P< 0.01) between the four MCAO groups and sham group. After cerebral ischemia-repefusion, the expression levels of Rho-kinase mRNA and the content of EB begin to increase at the time of 6h and will get its peak at the time of 48h and decrease gradually 48h later. The expression level of laminin begin to decrease at the time of 6h and will get its minimum at the time of 48h and increase gradually 48h later. The expression levels of Rho-kinase mRNA presents positive correlation with the content of EB, r= 0.925, P= 0.025<0.05; The expression levels of Rho-kinase mRNA presents negative correlation with the expression level of laminin, r=-0.955, P= 0.011<0.05; The content of EB presents negative correlation with the expression level of laminin, r=-0.982, P= 0.003<0.01.Conclusion:The expression levels of Rho-kinase mRNA presents linear correlation with microvascular impairment. Part II The Mechanism of microvascular impairment after the cerebral ischemia-reperfusion in ratsObjective:In this part of experiment, we explore the relationship between Rho-kinase,matrix metalloproteinase-9(MMP-9),S100B to inquire that Rho-kinase mediate microvascular impairment in rat model of cerebral ischemic-reperfusion by regulating the expressing of MMP and S100B and observe the protective effects of Rho kinase inhibitor to microvascular impairment of cerebral ischemic-reperfusion.Method:The thirty male SD rats bought from the laboratory animals centre in our university were randomly divided into following three groups:sham group,ischemia-repefusion 24 hours group and Rho kinase Inhibitor group. Focal cerebral ischemia-reperfusion model in rats was made by transient occlusion of middle cerebral artery for 1.5h followed by 24h reperfusion. In this part of experiment, we observe the mRNA expression variation and relationship of MMP-9,S100B in sham group,ischemia-repefusion 24 hours group and Rho kinase Inhibitor group. RT-PCR was employed to detect the expression of mRNA levels of MMP-9,S100B. At the same time, we observe the neurological functional decect,the modification of the content of Evan's blue(EB),the expression levels of laminin,the water content of brain measured with dry-wet weight in Rho kinase Inhibitor group and compare with ischemia-repefusion 24 hours group.Result:The neurological functional decect,the content of EB,the expression levels of laminin and the water content of brain have obvious statistics difference in the ischemia-repefusion 24 h group compared with sham group (P<0.05); There are obvious statistics difference (P<0.05) between Rho kinase Inhibitor group and ischemia-repefusion 24 h group. The expression levels of MMP-9 mRNA and S100B mRNA increases significantly in ischemia-repefusion 24 hours group compared with sham group (P<0.05). Rho kinase inhibitor inhibited the expressions of MMP-9 mRNA and S100B mRNA significantly (P<0.05 vs ischemia-repefusion 24 hours group).Conclusion:1. Rho kinase inhibitor can alleviate microvascular impairment and brain tissue edema, decrease the permeability of BBB, present the protective effects for microvascular impairment.2. Rho-kinase mediate microvascular impairment in rat model of cerebral ischemic-reperfusion by regulating the expressing of MMP-9 and S100B.