| Matrix metalloproteinases (MMPs) are a group of zinc-containing protease that can proteolytically degrade a variety of molecules that constitute the extracellular matrix components such as collagens, laminin, gelatin, elastin, fibronectin, and proteoglycans. Normally MMPs exist as zymogen in the inactive form and their modulations are controlled at multi-levels. Their activations are mediated by serine protease or fibrinase. In addition, they can also be activated by autocatalysis and modulated by their specific tissue inhibitor of MMPs (TIMPs). Recently, MMPs have been implicated in the pathogenesis of brain damage resulting from cerebral ischemia. It has been reported that MMP-2 and MMP-9 are activated after onset of stroke in clinical practice as well as in experimental brain ischemia and inhibiting the activityof MMP-9 can reduce brain infarct. These data indicate that MMPs are involved in the process of brain damage after ischemia and MMPs may bee a potential target in the treatment of stroke. To determine the role of MMPs in the brain ischemia, we conducted our experiments focusing on the following aspects: 1. Establish the animal models of focal cerebral ischemia and reperfusion in mice by intraluminal occlusion using monofilament, with the help of microsurgical technology and multi-physiological monitoring. 2. Examine the expression of MMPs in the permanent focal cerebral ischemic model and test the neuroprotective effect of MMP inhibitor KB-R7785 on this model. 3. By using technology of RT-PCR , Northern-blot and zymography, we investigate the expression of MMPs and its tissue inhibitor (TIMP) after transient focal cerebral ischemia/ reperfusion and detect the effect of U-0126, a newly developed inhibitor of mitogen-activated protein kinase, on the expression of MMPs and its tissue inhibitor (TIMP) after transient focal cerebral ischemia/ reperfusion. 4. Examine the effects of KB-R7785 and U-0126 on the formation of brain infarct and brain edema in animal model of transient focal cerebral ischemia/ reperfusion by the methods of Evans blue extravasation and TTC staining. These experiments will elucidate the further mechanisms of brain damage after brain ischemia and reperfusion, and will establish a reliably theoretical foundation for the development of neuroprotective drug that targets the MMPs.1. Establishing the animal model of focal cerebral ischemia and reperfusion in mice by intraluminal occlusion using monofilament. Under microscope, the left carotid artery and external/internal carotid artery were exposed and dissociated; the proximal ends of left carotid artery and external carotid arterywere ligated by 5-0 thread; an 8-0 segment(llmm long) of monofilament was inserted into the proximal position of left middle cerebral artery through carotid artery; the regional cerebral blood flow (rCBF) in the area of left middle cerebral artery was monitored by laser Doppler Flowmetry; when rCBF dropped down to the degree of less than 25% of that in normal condition, the model was thought success for permanent occlusion of middle cerebral artery (pMCAO). For transient ischemia/reperfusion model, we inserted the segment through external carotid artery; after 2h of occlusion the monofilament was withdrew and blood supply was restored; only those mice whose reperfusion rates were higher than 95% were thought success. 24h after surgery, brain was made into thick slices and stained with TTC, infarct volume was calculated by computer-assisted image analysis system. By repeating our practice and experience, we got the following ideas, which would be the key points to determine the reproducibility, reliability and stability of the models: 1. The preparation and selection of monofilament. First, to prepare the filament, it requires quite uniform coating of silicon gel resin and the head of segment should be round and blunt, avoiding the formation of curve and hook, which would perforate the blood vessel; in addition, because the disparity of blood vessels in diameter was positively correlated with their...
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