| IntroductionIn the process of focal cerebral ischemia and reperfusion, extracellular pro-teolytic cascades triggered by MMP - 9 disrupt the extracellular matrix, contribute to cell detachment, and lead to a form of apoptotic cell death known as anoikis. MMP - 9 has been admitted as one of the main factors that lead to neuron damage in the process of reperfusion. So it could be a potential therapy to maintain the vitality of neuron by inhibitting the expression of MMP -9. bcl — 2 gene is the first gene that been acknowaged to have the function of suppress eu-karyon cell apoptosis. It can not only develop neuroprotection directly, but also inhibit the activation of MMPs through many cell factor routes, which could relieve the microcirculation disturbance after cerebral ischemia. This experiment is that, in certain time window, plasmid pLXSN - bcl - 2 cDNA was injected through internal carotid artery to focal ischemia /reperfusion injured rats. In order to study the role of MMP - 9 in focal cerebral reperfusion rats, we observe the effects of bcl - 2 gene on infarction volume, MMP - 9 exoression and neuro-nal apoptosis in ischemic region. Therefore, to approach the possible regulation mechanisms of bcl - 2 gene on MMP - 9, and assess the neuroprotective potential in the gene therapy with pLXSN - bcl - 2 cDNA after brain ischemic insult.Materials and Methods99 adult male Wistar rats were assigned randomly into 3 groups;physiologi-cal saline group (n =33) ,pLXSN group (n =33) and pLXSN -bcl -2 group (n = 33) . Then each group was devided into 3 subgroups:24h,48h,72h,according to the time after reperfusion onset. Thereafter, middle cerebral artery occlusion ( MCAO) was performed with the help of intraluminal thread to each rat. Two hours after the ischemia insult, reperfusion was implemented to those rats. Then three hours later, 120 ul physiological saline,pLXSN and pLXSN - bci -2 cD-NA were respectively injected into internal carotid artery. Following determinations were taken to those succeeded models-.1. Determination of cerebral infarction volume: Six rats in each subgroups were euthanized with narcotic overdose at the desired end point. The brains were subjected to TTC staining and paraformaldehyde fixation, and then used to calculate infarction volume though image analysis system.2. Immunohistochemistry determination and TUNEL techniques;The rest rats in each subgroup were anaesthetized again. Immediately after that, transear-diac perfusion was performed to each of them ( heparinized saline followed by paraformaldehyde ) . Then the brains were removed, postfixed, embedded in paraffin, and consecutively sectioned (6jAm thick coronal sections). The sections were subject to staining procedures after deparaffination. Using SABC Immunohistochemistry kit to observe Bcl - 2/Bax and MMP - 9 expression, and TUNEL techniques to check out the distribution of apoptosis neurons. At last,all sections were disposed by routine dehydration, dimethylbenzene transparentized and gum sealed.Determination of the result;Taken two sections of each sample , examined and photographed using computerized video imaging microscopy. Data were acquired from different eight fields in corpus striatum region by analysing the positive area percentage at high power magnification (400 x ).All the results were transformed to datas through image analysis system, which were expressed as x ± s. The values were analyzed , using ANOVA and t test of SPSS software. A value of P <0. 05 was considered significant, obvious significance was set at P <0. 01.ResultsTTC staining showed that the infarction volume of pLXSN - bcl - 2 group was respectively much smaller than that of two control groups at the three time points(P<0.05).Immunohistochemistry staining results were that, in experimental group, Bcl - 2 protein was increased obviously and Bax protein was greatly attenuated compared with that of control groups( P < 0.05 ) .Detection of MMP - 9 activity was performed at three time points after MCAO. The summit of MMP - 9 expression was about 48 hours after ischemic/ reperfusion insult in each group. The elevated levels of MMP - 9 were significantly attenuated in the bcl - 2 group as compared to the two control groups (P < 0.05).The result of TUNEL techniques was that the number of apoptosis neurons in bcl -2 group reduced greatly compared with that of control groups (P <0. 01).While those dates in physiological saline and pLXSN group at each time point ,including infarction volume,Bcl — 2/Bax and MMP —9 expression , number of apoptosis neuron, showed no statistical significance( P >0.05).ConclusionIn few hours after cerebral I/R injury, injection of pLXSN - bcl - 2 cDNA via internal carotid artery could bring about an altered Bcl - 2/Bax rheostat and downregulation the expression of MMP - 9 favoring resistance to neuron apoptosis in ischemic region,which may significantly diminish the size of infarction. These postinsult regulations of bcl - 2 gene to Bcl - 2/Bax and MMP - 9, could provide a potential therapeutic strategies to the treatment of ischemic stroke.
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