【作者】 苏颖；

【导师】 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2007， 博士

【摘要】 第一部分实验性脑缺血再灌注损伤的磁共振成像研究目的:应用不同的磁共振成像序列,活体观察实验大鼠脑缺血再灌注损伤不同时期的病理生理过程,为脑保护治疗提供精确的神经影像学信息。方法:14只健康雄性Wistar大鼠随机分为手术组和假手术组,每组各分为再灌注24h和再灌注5d两个亚组。线栓法建立大鼠大脑中动脉闭塞1.5h再灌注模型。所有大鼠分别于线栓插入及拔出后立即行PWI;再灌注0h、3.5h及24h行DWI;再灌注3.5h、24h及5d行常规T₂WI和Gd-DTPA增强T₁WI;再灌注24h及5d行T₂*WI。测定不同时间点MRI异常信号体积和增强扫描信号强化区域体积及平均信号强度值。结果以体积比（异常信号体积/同侧半球体积×100 %）,强度比（异常信号平均信号强度值/对侧镜像区平均信号强度值×100 %）表示。结果:成功大鼠模型PWI显示造影剂首次通过时,双侧大脑半球信号衰减于线栓置入时呈不对称性改变,线栓拔出后立即恢复。手术组大鼠:缺血1.5h后DWI即显示异常高信号,高信号体积随再灌注时间延长而增加（P<0.05）;脑缺血再灌注3.5h后T₂WI和T₁WI增强扫描均显示异常高信号,高信号体积随再灌注时间延长而增加,再灌注5d后有所下降（P<0.05）,强化区域的平均信号强度值随再灌注时间延长递增（P<0.05）;再灌注24h及5d T₂*WI上均无异常信号出现。假手术组大鼠所有MRI序列上均无异常改变。结论:合理应用不同的磁共振成像序列可以为超早期脑梗死的诊断,BBB损伤的范围及程度、血管源性水肿、脑梗死灶体积的评价提供实时、详尽、个体化的影像学信息,有利于我们动态观察缺血脑组织的病理变化,实时监测药物干预的治疗效果。第二部分脑缺血再灌注后明胶酶活性与血脑屏障损伤的相关性分析目的:探讨脑缺血再灌注不同时期体内明胶蛋白酶活性与BBB损伤之间的相关性,明确其在脑缺血再灌注微血管损伤中的作用及地位。方法: 34只健康雄性Wistar大鼠随机分为手术组和假手术组,每组各分为再灌注24h和再灌注5d两个亚组。线栓法建立大鼠大脑中动脉闭塞1.5h再灌注模型。所有大鼠分别于再灌注3.5h、24h及5d行Gd-DTPA增强T₁WI扫描。明胶酶谱分析法检测缺血再灌注24h及5d后脑组织、血清MMP-2、MMP-9活性,并与MRI作相关回归分析。免疫组织化学染色检测缺血脑组织MMP-9的表达与分布。结果:脑缺血再灌注24h,手术组大鼠脑组织MMP-2、MMP-9活性显著升高（P<0.05）;再灌注5d,MMP-2活性继续升高（P<0.05）,MMP-9则下降至检测水平以下。假手术组大鼠脑组织中仅检测到少量MMP-2,未见MMP-9水解条带,两者活性随时间变化不明显（P>0.05）。脑缺血再灌注24h,手术组大鼠血清MMP-2、MMP-9活性显著高于假手术组（P<0.05）;再灌注5d,两者活性继续升高（P<0.05）。假手术组大鼠血清MMP-2、MMP-9活性随时间变化不明显（P>0.05）。脑缺血再灌注24h,脑组织MMP-9活性与T₁WI上信号强化范围呈明显正相关,（r=0.96,p<0.001）; MMP-9免疫组化阳性染色分布与T₁WI上信号强化区一致。结论:脑组织明胶蛋白酶活性增高是导致缺血再灌注早期血脑屏障通透性升高、脑水肿加重的重要因素,其中MMP-9与脑缺血再灌注导致的神经血管损伤关系尤为密切。第三部分脑缺血再灌注后MMPs及其组织抑制因子TIMPs的表达分析目的:检测脑缺血再灌注不同时期脑组织MMP-2、MMP-9及其组织抑制因子TIMP-1、TIMP-2转录及翻译的变化,探讨其表达特点。方法:56只健康雄性Wistar大鼠随机分为手术组和假手术组,每组各分为再灌注24h和再灌注5d两个亚组。线栓法建立大鼠大脑中动脉闭塞1.5h再灌注模型。应用RT-PCR检测缺血再灌注24h及5d后脑组织MMP-2、MMP-9 mRNA表达的变化;Western blot检测缺血再灌注24h和5d脑组织MMP-2、MMP-9及其组织抑制因子TIMP-2、TIMP-1蛋白含量的变化。结果:手术组大鼠脑缺血再灌注24h MMP-2、MMP-9 mRNA表达显著升高（P<0.05）;再灌注5d后MMP-9 mRNA表达迅速下降（P<0.05）,MMP-2 mRNA表达则继续升高（P<0.05）。假手术组大鼠脑组织内仅检测到少量MMP-2 mRNA的表达,且随时间变化不明显（P>0.05）。手术组大鼠脑缺血再灌注24h TIMP-1、TIMP-2蛋白表达较假手术组明显升高（P<0.05）,再灌注5d TIMP-1表达迅速下降（P<0.05）,TIMP-2表达仍继续增加（P<0.05）。假手术组大鼠脑组织TIMP-1、TIMP-2呈低水平表达,随时间变化不明显。结论:缺血再灌注导致脑组织MMP-2、MMP-9 mRNA及蛋白表达明显升高,再灌注早期以MMP-9升高为主,晚期则以MMP-2升高为主。TIMP-1和TIMP-2的表达模式与MMP-2、MMP-9表达一致,这可能与机体维护内环境稳定的自我调节有关。第四部分抗-MMPs治疗对脑缺血再灌注损伤的影响目的:观察抗-MMPs治疗对脑缺血再灌注损伤的影响,为缺血性脑卒中的神经保护治疗寻找新的切入点。方法:28只健康雄性Wistar大鼠随机分为BB-94治疗组和缺血对照组,每组各分为再灌注24h和再灌注5d两个亚组。线栓法建立大鼠大脑中动脉闭塞1.5h再灌注模型。BB-94组大鼠于不同时间点腹腔注射广谱MMPs抑制剂BB-94（50mg/kg）;缺血对照组大鼠相同时间点腹腔注射等体积生理盐水。两组大鼠分别于再灌注0h、3.5h及24h行DWI扫描;再灌注3.5h、24h及5d行T₂WI和Gd-DTPA增强T₁WI扫描。脑缺血再灌注24h和5d后对所有大鼠进行神经功能缺陷评分。结果:BB-94组大鼠脑缺血再灌注3.5h及24h DWI上异常高信号体积明显小于缺血对照组（P<0.05）;再灌注3.5h、24h及5d BB-94组大鼠T₂WI上异常高信号体积及T₁WI信号强化的范围和强度亦明显小于缺血对照组（P<0.05）。BB-94显著降低了缺血再灌注24h和5d大鼠神经功能缺陷评分（P<0.05）。结论:抗-MMPs治疗能明显减轻缺血再灌注后血脑屏障损伤的范围和程度,缩小脑梗死灶体积,促进神经功能恢复,对脑缺血再灌注损伤具有显著的神经保护作用。第五部分米诺环素对脑缺血再灌注损伤保护作用及其机制的研究目的:观察米诺环素对脑缺血再灌注后BBB通透性、梗死灶体积以及神经功能恢复的影响进行,探讨其作用机制。方法:56只健康雄性Wistar大鼠随机分为米诺环素组和缺血对照组,每组各分为再灌注24h和再灌注5d两个亚组。线栓法建立大鼠大脑中动脉闭塞1.5h再灌注模型。两组大鼠分别于再灌注0h、3.5h及24h行DWI扫描;再灌注3.5h、24h及5d行T₂WI和Gd-DTPA增强T₁WI扫描。脑缺血再灌注24h和5d后对所有大鼠进行神经功能缺陷评分和血清、脑组织明胶酶活性测定。应用RT-PCR和Western blot分别考察米诺环素对缺血再灌注后脑组织MMP-2、MMP-9转录和翻译水平的影响,以及对TIMP-1、TIMP-2蛋白表达的调控。结果:米诺环素组大鼠缺血再灌注3.5h及24h DWI上异常高信号体积明显低于缺血对照组（P<0.05）;再灌注3.5h、24h及5d米诺环素组大鼠T₂WI上异常高信号体积及T₁WI信号强化的范围和强度亦明显小于缺血对照组（P<0.05）。米诺环素显著降低了缺血再灌注24h和5d大鼠神经功能缺陷评分（P<0.05）和脑组织明胶酶活性（P<0.05）,同时对缺血再灌注24h血清MMP-9及再灌注5d血清MMP-2的水解活性亦具有抑制作用（P<0.05）。脑缺血再灌注24h,米诺环素组大鼠脑组织MMP-2、MMP-9 mRNA及蛋白表达较缺血对照组显著减少（P<0.05）,TIMP-2表达高于对照组（P<0.05）,TIMP-1表达无组间差异（P>0.05）;脑缺血再灌注5d,米诺环素组大鼠脑组织MMP-2 mRNA及蛋白表达仍低于对照组（P<0.05）,TIMP-1、TIMP-2蛋白含量无明显组间差异（P>0.05）,两组大鼠脑组织中均未再检测到MMP-9 mRNA及蛋白的表达。结论:米诺环素能显著减轻缺血再灌注早、晚期血脑屏障损伤的范围和程度,缩小梗死体积,促进神经功能恢复,其保护机制可能与米诺环素抑制血清明胶蛋白酶活性,减少脑组织明胶蛋白酶的转录、翻译和活化,上调TIMP-2的表达有关。更多还原

【Abstract】 PartⅠMRI study in an experimental model of focal cerebral ischemia/reperfusion injuryObjective: To explore time characteristics and dynamic changes of pathology in a rat model of focal cerebral ischemia-reperfusion at different time points with MRI techniques.Methods: Fourteen male Wistar Rats were randomized into ischemic group and sham-operated group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion （MCAO） was induced by an intraluminal suture method. Perfusion-weighted imaging was performed to confirm complete occlusion and reperfusion. Diffusion-weighted imaging was carried out at 0h, 3.5h, 24h after reperfusion. T₂WI and contrast enhanced T₁WI were carried out at 3.5h, 24h and 5d after reperfusion respectively. T₂*-weighted imaging was used at 24h and 5d after reperfusion. MRI lesion volume and signal intensity on postcontrast T₁WI were evaluated at different time point, and the results were expressed in forms of rV-MRI （lesion volume / volume of the ipsilateral hemisphere×100 %） and rSI-MRI （mean grey value of ROIs in the ipsilateral hemisphere/ that of the contralateral hemisphere×100 %）.Results: signal attenuation during the first passage of a bolus-contrast indicated a complete occlusion of the artery. On the contrary, a failed occlusion was lack of such a signal change. In the ischemic group hyper-intensity on DWI was first detectable at 1.5h after reperfusion, and the lesion volume on DWI increased with the time of reperfusion （P<0.05）. Hyper-intensity on T₂WI and postcontrast T₁WI was detected at 3.5h after reperfusion in the ischemic group.The lesion volume increased at 24h after reperfusion and declined at 5d after reperfusion （P<0.05）, but the signal intensity increased all the time （P<0.05）. No abnormal signal was found on T₂*WI at any time point. The rats from sham-operated group showed no changes on MRI during the experiment.Conclusions: The apply of MRI in focal cerebral ischemia-reperfusion at different time points can afford dynamic and individual neuroimaging informations of BBB disruption, cerebral edema and infarct size following cerebral ischemia. MRI techniques have shown latent potency in elucidating time characteristics in reperfusion injury and provided a foundation of diagnosis and treatment for cerebral ischemic injury.PartⅡCorrelation analysis of BBB permeability and activity of gelatinases following transient focal cerebral ischemiaObjective: To explore the relationship between gelatinases activity and disruption of BBB in different phases following cerebral ischemia and reperfusion, and confirm the effects of gelatinases on microvascular damage after transient focal cerebral ischemia in rats.Methods: Thirty-four male Wistar Rats were randomized into ischemic group and sham-operated group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion （MCAO） was induced by an intraluminal suture method. Gd-DTPA enhanced T₁WI was carried out at 3.5h, 24h and 5d after reperfusion. Brain and serum gelatinases activities were analyzed by gelatin zymography at 24h and 5d after reperfusion, respectively. Correlation between MRI findings and gelatinases activities was analyzed. Immunehistochemistry was used to confirm the distribution of MMP-9 expression in the ischemic cerebral tissues.Results: In ischemic group activities of brain MMP-2 and MMP-9 were increased at 24h after reperfusion （P<0.05）. MMP-9 was no longer visible on gelatin zymograms after 5d of reperfusion, while MMP-2 activity remained increased at 5d after reperfusion （P<0.05）. In sham-operated group, activity of brain MMP-2 remained low all the time, and no MMP-9 was detectable at any time point. Activities of serum MMP-2 and MMP-9 were increased correspondingly at 24h after reperfusion （P<0.05）, and kept increased at 5d after reperfusion （P<0.05）. In sham-operated group, activities of serum MMP-2 and MMP-9 changed little during the experiment （P>0.05）. A significant correlation was observed between activity of brain MMP-9 and rV-T₁WI at 24h of reperfusion （r=0.96, p<0.001）. Furthermore, immunehistochemistry results showed distribution of MMP-9 expression corresponded to the area of enhancement on postcontrast T₁WI.Conclusions: Enhanced activities of brain gelatinases play an important role in the disruption of BBB and formation of edema after cerebral ischemia and reperfusion. Activity of brain MMP-9 shows remarkable correlation with reperfusion induced neurovascular damage in cerebral ischemia.PartⅢStudy on expression of MMPs and TIMPs in rat brain following transient focal cerebral ischemiaObjective: To study the mRNA and protein expression of MMPs and TIMPs in different phases of cerebral ischemia and reperfusion.Methods: Fifty-six male Wistar Rats were randomized into ischemic group and sham-operated group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion （MCAO） was induced by an intraluminal suture method. After 24h and 5d of reperfusion mRNA expression of MMP-2 and MMP-9 was observed by RT-PCR, and the protein expression of MMP-2/TIMP-2 and MMP-9/TIMP-1 was also defined by western blot.Results: mRNA expression of MMP-2 and MMP-9 at 24h and 5d after reperfusion was elevated in the ischemic group（P<0.05）, and mRNA level of MMP-9 declined dramatically after 5d of reperfusion （P<0.05）, while expression of MMP-2 mRNA kept increased （P<0.05）. Only low level of MMP-2 mRNA was detected in the sham-operated group at any time point without obvious fluctuation （P>0.05）. Expression of TIMP-1 and TIMP-2 was significant higher in the ischemic group at 24h after reperfusion （P<0.05）. After 5d of reperfusion expression of TIMP-1 declined dramatically （P<0.05） while protein level of TIMP-2 still increased （P<0.05）. Low expression of TIMP-1 and TIMP-2 was confirmed in the sham-operated group.Conclusions: Cerebral ischemia/reperfusion induces expression of MMP-2 and MMP-9 in rat brain. In the early stage of reperfusion, expression of gelatinases is charactered with high level of MMP-9, whereas in the late phase of reperfusion, expression of MMP-2 seems to be predominant. Expression of TIMP-1 and TIMP-2 after cerebral ischemia changes correspondingly with expression of gelatinases, which indicates an auto-regulation in vivo.PartⅣEffects of anti-MMPs therapy on cerebral ischemia/reperfusion induced injury in rat brainObjective: To observe the effects of anti-MMPs therapy on reperfusion induced injury in a rat model of transient focal cerebral ischemia.Methods: Twenty-eight male Wistar Rats were randomized into BB-94 group and the control group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion （MCAO） was induced by an intraluminal suture method. BB-94, an inhibitor of MMPs, was injected intraperitoneally （50mg/kg） at different time point in the BB-94 group, and equal volume of saline was injected intraperitoneally at the same time in the control group. Diffusion-weighted imaging was carried out at 0h, 3.5h, 24h after reperfusion. T₂WI and contrast enhanced T₁WI were carried out at 3.5h, 24h and 5d after reperfusion respectively. All rats received neurological evaluation before sacrifice.Results: After 3.5h and 24h of reperfusion rV-DWI in the BB-94 group was significant lower than that of control （P<0.05）. BB-94 reduced rV-T₂WI, rV-T₁WI and rSI-T₁WI at 3.5h, 24h and 5d of reperfusion, respectively （P<0.05）. Neurological outcome after 24h and 5d of reperfusion was also improved by BB-94 as compared with the control group （P<0.05）.Conclusions: Anti-MMPs therapy shows significant neuroprotective effects by attenuating BBB disruption, reducing infarct volume and improving neurological outcome after transient focal cerebral ischemia in rats. PartⅤStudy on neuroprotective effects of minocycline in ischemia- reperfusion induced injury and its mechanismObjective: To observe the effects of minocycline on reperfusion induced BBB disruption, infarct size and neurological outcome following transient focal cerebral ischemia in rats, and explore the underlying mechanism.Methods: Fifty-six male Wistar Rats were randomized into minocycline group and the control group, which were further divided into two subgroups with 24 hours and 5days of reperfusion, respectively. 1.5 hours of middle cerebral artery occlusion （MCAO） was induced by an intraluminal suture method. Diffusion-weighted imaging was carried out at 0h, 3.5h, 24h after reperfusion. T₂WI and contrast enhanced T₁WI were carried out at 3.5h, 24h and 5d after reperfusion respectively. All rats received neurological evaluation before sacrifice. At 24h and 5d of reperfusion, activities of brain and serum gelatinases were analysed by zymography. Effects of minocycline on brain mRNA level of MMP-2, MMP-9 and protein expression of MMP-2/TIMP-2, MMP-9/TIMP-1 were defined by RT-PCR and western blot, respectively. Results: After 3.5h and 24h of reperfusion rV-DWI in the minicycline group was significantly lower than that of control （P<0.05）. Minocycline reduced rV-T₂WI, rV-T₁WI and rSI-T₁WI at 3.5h, 24h and 5d of reperfusion, respectively （P<0.05）. Neurological outcome after 24h and 5d of reperfusion was also improved by minocycline as compared with the control group （P<0.05）. Brain gelatinases activities were inhibited by minocycline at 24h and 5d after reperfusion （P<0.05）. Serum MMP-9 activity at 24h and serum MMP-2 activity at 5d after reperfusion were also inhibited by minocycline （P<0.05）. After 24h of reperfusion brain mRNA and protein level of MMP-2 and MMP-9 were significantly reduced by minocycline as compared with the control group （P<0.05）. Expression of brain TIMP-2 was increased in the minocycline group （P<0.05）, while expression of TIMP-1 showed no difference between the two groups （P>0.05）. After 5d of reperfusion brain mRNA and protein level of MMP-2 remained lower than that of control （P<0.05）. There was no difference in the expression of TIMP-1 and TIMP-2 between the two groups any more （P>0.05）. Expression of MMP-9 mRNA and protein was invisible in both minocycline and control group at 5d after reperfusion.Conclusions: Minocycline attenuates BBB disruption in both the early and late phases of cerebral ischemia and reperfusion. Treatment with minocycline reduces infarct volume and improves neurological outcome in a rat model of transient focal cerebral ischemia. Inhibiting the activities of brain and serum gelatinases, reducing their expression in the brain, and elevating the level of brain TIMP-2, which is capable of inhibiting the activity of gelatinases, may be responsible for the neurovascular protection of minocycline.更多还原