Effects Of MMP-9 On Focal Cerebal Ischemical Reperfusion Injuery In Rats

Objective To explore the regular expression of Matrix Metalloproteinase-9 (MMP-9), Tissue Inhibitors of Matrix Metalloproteinase-1 (TIMP-1) laminin(LN) and fibronectin (FN) as well as the disruption of blood brain barrier after focal cerebral ischemical reperfusion jury in rat.Methods: Totally 42 wistar rats were randomly divided into 2 groups: Sham-operated group and ischemical reperfusion group (I/R group). The middle cerebral artery occlusion model was constructed in 42 wistar rats with Longa's method. The expression of MMP-9, TIMP-1 LN and FN were investigated by immunohistochemistry . The water content was calculated by dry and wet weight method , The disruption of BBB were detected by the EB content at 6, 24, 48, 72h and 7d of reperfusion.Results: The expressions of MMP-9 and TIMP-1 did not appear in sham-operated group, however the expression of LN and FN were normal in sham-operated group. The MMP-9 began to express on MMP-9 positive cells at 6h reperfusion, then it increased at 24h and reached peak level at 48h, after this, the level of MMP-9 began to decrease up to 7d. The TIMP-1 began to express on TIMP-1 positive cells at 6h reperfusion and reached peak level at 24hrs, compared with 24h the level of TIMP-1 began to decrease at 48hr and the decrease continued up to 7d group. The positive situs of MMP-9 and TIMP-1 were located in vascular endothelial cells, neuron and gitter cells. Otherwise the positive situs of TIMP-1 were located in non-ischemical area. The expression of LN and FN began to decrease at 24h of reperfusion and reached peak level at 48h, up to 7d of reperfusion it decreased back to normal level. The water content increased at 6h,then it were obviously increased at 24 48h, there was significance difference between sham group and I/R group(P<0.01). The permeation of EB were detected at 6h and become severity at 24-72h of reperfusion, there was significance difference between sham group and I/R group at different stages of reperfusion (P<0.01).Conclusion: The expressions of MMP-9 and TIMP-1 increased in the early stage of reperfusion, and it decreased at following stage; in contrast the expression of LN and FN were nearly normal in the early stage of reperfusion, at following stage it suffered change of decrease and increase and up to normal level. Their patterns of expressions were depending on different stage of reperfusion. This study suggestinged that MMP-9 may play an important role in early cerebral ischemical reperfusion injury, and this phenomenon may has been mainly caused by degradation of LN and FN which induce BBB lesion after cerebral ischemical reperfusion injury.Part II The Effect of Resveratrol, Deoxycycline and Allied Application of iton Cerebral Ischemia-Reperfusion Injury in RatsObjective: The purpose of this study was to evaluate the neuroprotective effect of resveratrol deoxycycline and the allied application of both resveratrol and deoxycycline.Methods: 174 male adult wistar rats were randomly divided into 5 groups, the sham-operated group the Cerebral ischemia-reperfusion (control group) the resveratrol-treated group the deoxycycline- treated group and the allied application group. Rats were intraperitoneal injected with 50mg/kg of 2% of resveratrol in resveratrol-treated group; rats were intragastric injected with administration of 4ml/kg of 0.75% deoxycycline in deoxycycline-treated group ; rats were both intraperitoneal injected with 50mg/kg of 2% of resveratrol and intragastric injected with administration of 4ml/kg of 0.75% deoxycycline in allied application group. And the sham group was pretreated with normal saline for 6days before the model of middle cerebral artery occlusion (MCAO) was established. The rats were decapitated at 6h, 24h, 48h, 72h and 7d after reperfusion. The neurological deficit score was graded by Garcia's methods, the volume of the cerebral infarction was estimated by 2%TTC staining, the water content was calculated by dry and wet weight method, the disruption of BBB was detected by the EB content, and the expression of MMP-9 TIMP-1 LN and FN were investigated by immunohistochemistry, and the fine structure of brain was detected by electron microscope.Results: 1) According to the neurological deficit scores, the significant difference existed on the scores between the control group and the drug groups at 24,48h,and the allied application group at 72h (P<0.05). 2) The drug groupS obviously cut down cerebral infarct sizes (P<0.01). 3) The water contents were significant differenT existed on the drug groups and control group at 24,48and 72h. 4) The EB contents were significant different on the drug groups and control group at 6h, 24h, 48h, 72h and 7d (P<0.01, P<0.05). 5) The situs and regular pattern of expression of MMP-9 and TIMP-1 in the drug groups were similar to the control group, but the positive cells significantly decreased at 6h, 24h, 48h and 72h compared with control group (P<0.01). 6) The expression of LN was significant different on the drug groups and control group at 6h, 24h, and 48h (P<0.01).7) The expression of FN was significant difference on the drug groups and control group at 6h, 24h, 48h and 72h (P<0.01, P<0.05). 8) The level of edema of endothelial cell, the increasing intermembrance space of basal membrane and the apoptosis of neuron were cut down in the drug groups compared the control group at 24h.Conclusion:1. Resveratrol and Deoxycycline can lighten the level of vasogenic brain edema, cut down the volume of cerebral infarct sizes and improve the neurological deficit scores, so it have a neuroprotective effect on impaired neuron in cerebral ischemia-reperfusion injury.2. Resveratrol and Deoxycycline can reduce the patho-expression of TIMP-1 by suppressing the expression of MMP-9 after focal cerebral ischemia-reperfusion injury. 3. One of the mechanisms of neuroprotective effect of Resveratrol and Deoxycycline was that either suppresses the expression of MMP-9 and lessens the degradation of LN and FN so that either can maintain integrity of blood brain barrier.4. Allied application of Resveratrol and Deoxycycline significantly diminish the the volume of cerebral infarct sizes, it indicate that Resveratrol and Deoxycycline may be have a synergism of neuroprotective effect on focal cerebral ischemia-reperfusion injury.