缺血后适应对树鼩血栓性脑缺血磁共振成像及脑保护的分子机制研究

【作者】 何波；

【导师】 李树清；

【作者基本信息】 昆明医科大学 ， 外科学， 2013， 博士

【摘要】 目的：建立树鼩血栓性脑缺血及缺血后适应动物模型并进行磁共振(magnetic resonance imaging,MRI)成像研究,包括MRI平扫、弥散加权成像(diffusion weighted imaging,DWl)、二乙三胺五醋酸钆(gadolinium-diethylene-triaminepentaacetate, Gd-DTPA)增强等系列,观察缺血区72h不同时间点内演变规律,探讨树鼩血栓性脑缺血的MRI特点及其在脑缺血后适应过程中的监测价值,为临床提供实验依据和理论基础。方法：采用光化学反应诱导建立树鼩血栓性脑缺血模型,在造模成功4h后夹闭右侧颈总动脉5min,再灌注5min,共计交替进行3个循环实施缺血后适应。应用3.0T MRI在缺血后4h、24h、48h及72h对树鼩脑部行冠状扫描,观察树鼩脑缺血及缺血后适应后缺血灶改变特点。在工作站上测量并计算出以下数值：Tl加权图像(T1weighted image, T1WI). T2WI和表面扩散系数(apparent diffusion coefficient,ADC)图上感兴趣区(region of intrest,ROI)的T1WI相对信号强度(relative signal intensity,rSI)、T2WI rSI及相对表面扩散系数(relative apparent diffusion coefficient,rADC)值；异常信号中心区和边缘区rADC值；DWI和T2加权图像(T2weighted image,T2WI)上异常信号体积；增强后信号强度(signal intensity,SI)增加率。分别应用HE染色、2,3,5氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride, TTC)染色技术显示脑缺血组及缺血后适应组缺血灶不同区域细胞改变和皮层梗死面积的变化。结果：假手术组各时间点MRI及TTC未见异常。缺血组4h MRI可见信号改变，T2WI rSI在缺血后可见增高,24h中心区T2WI rSI值最高(P<0.05),缺血后适应组各时间点中心区T2WI rSI值与缺血组比较,无统计学差异(P>0.05)。缺血组4h及24h缺血病灶中心区rADC均下降,48h及72h明显升高(P<0.05)；缺血后适应组各时间点中心区rADC与缺血组比较,无统计学差异(P>0.05)。缺血组病灶边缘区rADC值在4h也出现降低,24h时最低(P<0.05),降至66.16±4.98%,随后逐渐升高；缺血后适应组边缘区rADC随时间逐渐升高,与缺血组各时间点边缘区rADC值比较,在24h及48h两组之间具有统计学差异(P<0.05),4h及72h两组之间无统计学差异(P<0.05)；缺血组缺血病灶中心区rADC在4h低于边缘区(P<0.05),24h、48h、72h无统计学差异(P>0.05)。TTC染色所测梗死区的体积与DWI高信号区体积比较,4h时DWI所测体积大于TTC(P<0.05),24h、48h及72h DWI所测体积与TTC体积之间无统计学差异(P>0.05)。缺血组脑梗塞体积在4h时最小(P<0.05),随后增大；缺血后适应组脑梗塞体积从4h至24h未见明显增大,4h体积为64.9±6.8mm3,24h体积为69.1±5.7mm3,病灶体积随时间的变化无统计学差异(P>0.05)。缺血组及缺血后适应组所有动物不同时间点注入Gd-DTPA后均出现病变异常强化,缺血组24h时增强后SI增加率最高(P<0.05)；增强后所测强化体积小于T2WI所测体积(P<0.05)；缺血后适应组SI增加率与缺血组比较,在24h、48h及72h增强后SI增加率小于缺血组(P<0.05)。光镜示缺血后4h病灶中心区神经元可见皱缩,缺血组织与正常组织之间可见水肿带,缺血半暗带神经元肿胀、固缩,部分血管腔内见血小板积聚,血栓形成；缺血24h缺血中心区组织溶解,呈疏松网结构,神经元脱失,固缩明显；缺血后48h及72h,神经元脱失明显,出现嗜酸神经元,整个细胞均染为红色。结论：MRI能动态反应树鼩血栓性脑缺血动物模型缺血后不同时间点的病理演变过程；MRI可用于脑缺血后适应效果的评价,缺血后适应抑制缺血区的扩大,结合半定量化参数rADC值,可能与缺血后适应改善局部脑血流及挽救半暗带有关；增强后缺血后适应组SI增加率小于缺血组,可能系由于缺血后适应减轻血脑屏障的破坏。目的：通过采用超顺磁性氧化铁颗粒(superparamagnetic iron oxide,SPIO)增强MRI对树鼩血栓性脑缺血及缺血后适应动物模型进行动态扫描,观察病灶的改变特点,并与普鲁士蓝染色及抗白介素-1β(Interleukin-1beta, IL-1β)与单核/巨噬细胞表面特异性标志抗原(Ectodermal dysplasia-1, ED-1)免疫荧光双标染色结果相对照,探讨SPIO增强MRI对缺血后适应所致脑缺血炎症反应改变的活体监测价值。方法：采用光化学反应诱导建立树鼩血栓性脑缺血模型,在造模成功4h后夹闭右侧颈总动脉5min,再灌注5min,共计交替进行3个循环实施缺血后适应。应用3.0T MRI,在缺血后4h、24h及48h注射SPIO,观察SPIO增强后MRI改变特点,测量不同时间点T2WI rSI值；通过普鲁士蓝染色显示缺血灶内铁粒子形态及部位；采用抗IL-1β与ED-1免疫荧光双标染色激光扫描共聚焦显微镜检查观察脑缺血组及缺血后适应组巨噬细胞浸润及IL-1β的表达情况。结果：假手术组各时间点MRI未见异常；缺血组于注射SPIO后24h及以后时间点扫描缺血灶内可见低信号,4h注射组24h、48h及72h低信号形态未见明显改变,T2WI rSI值未见变化(P>0.05),在24h、48h及72h时T2WI rSI低于缺血平扫组(P<0.05)；24h注射组48h及72h扫描可见低信号,低信号形态未见明显改变,T2WI rSI值未见变化(P>0.05),48h及72h T2WI rSI与缺血平扫组对比,无统计学差异(P>0.05)；48h注射组,72h时可见低信号,但T2WI rSI与缺血平扫组对比,无统计学差异(P>0.05)；缺血后适应组注射SPIO后24h及以后各时间点缺血灶内可见低信号,缺血后适应4h注射SPIO组24h、48h及72h时T2WI rSI高于缺血4h注射SPIO组(P<0.05),缺血后适应24h注射SPIO组48h及72h缺血灶T2WI rSI略低于缺血24h注射SPIO组,但经统计学分析,两组间T2WI rSl无统计学差异(P>0.05),缺血后适应48h注射SPIO组与缺血组72h T2WI rSI相比较,经统计学分析,无统计学差异(P>0.05)；普鲁士蓝染色显示缺血4h注射组24h时含铁粒子细胞主要位于血管腔内,随缺血时间延长,血管腔内含铁粒子细胞减少,于72h主要位于血管外组织内；24h及48h注射组含铁粒子细胞较4h注射组减少,缺血后适应组各时间点含铁粒子细胞均较缺血组减少；抗IL-1β与ED-1免疫荧光双标染色激光扫描共聚焦显微镜检查显示缺血后24h缺血灶可见大量ED-1阳性小胶质细胞/巨噬细胞浸润,IL-1β表达增强,随时间延长,48h及72h小胶质细胞/巨噬细胞数量减少,IL-1β表达减弱,但在72h病变周边出现了多个体积较大的巨噬细胞；IL-1β表达与ED-1共染。缺血组及缺血后适应组中24h时IL-1β与ED-1阳性表达细胞数均高于48h及72h(P<0.05),并且48h时IL-1β与ED-1阳性表达细胞数均高于72h(P<0.05),缺血组24h及48h时IL-1β与ED-1阳性表达细胞数均高于缺血后适应组同一时间点(P<0.05),缺血组72h时IL-1β与ED-1阳性表达细胞数与缺血后适应组无明显差别(P>0.05)。结论：小胶质/巨噬细胞参与了急性期血栓性脑梗塞动物模型的缺血性脑损伤的病理过程；应用SPIO增强MRI成像可用于活体监测脑缺血后急性期巨噬细胞的浸润情况：缺血后适应可以减轻脑缺血后小胶质/巨噬细胞的浸润及炎症介质的表达,SPIO增强MRI可以在活体内监测脑缺血后适应对脑缺血后急性期炎症反应的改变。更多还原

【Abstract】 Objective: To establish the models of thrombotic cerebral ischemia and ischemic postconditioning in tree shrews,and to observe the the MRI characteristics,including plain, diffusion weighted imaging(DWI) and gadolinium-diethylenetriaminepenta-acetate (Gd-DTPA) enhanced scan,and to explore the value of MRI for monitoring thrombotic cerebral ischemia and ischemic postconditioning, which may provide a theoretical basis for clinical application and experimental evidence.Methods:The thrombotic cerebral ischemia were induced by photochemical reaction in tree shrews to establish the ischemic models,and at4h after ischemia ischemic postconditioning were established by3repeated cycles of occlusion of the right carotid arteries5min,and reperfusion5min.3.0T MR imaging was performed after4h,24h,48h and72h after ischemia, to observe the change characteristics of thrombotic cerebral ischemia. The data were measured on the workstation including the volume of abnormal signal on the DWI and T2WI images, the values of relative signal intensity (rSI) on the T1WI and T2WI images, the relative apparent diffusion coefficient(rADC) on the ADC images and the signal intensity(SI) on the enhanced images of the lesion center and marginal area. The changes of the cells and cortical infarct size in the different regions were observed between cerebral ischemia group and ischemic postconditioning group through HE staining,2,3,5-triphenyltetrazolium chloride(TTC) staining, respectively. Results:There were no abnormalities in MRI and TTC in the sham group at all time points. In the ischemia group,the signal changes were observed at4h and the signal relative value of the central area at24h was the largest, which had a significant difference in comparison with4h,48h and72h(P<0.05). Moreover, the signal relative values were no significant difference in different time points between the ischemia group and the ischemic postconditioning group(P>0.05). In the ischemia group, the rADC value decreased in the different parts of the lesions and decreased to about57.63%in the central area at4h, remained relative low level in24h. However, the rADC value in the central area significantly increased at48h and72h, which had significant difference at4h,24h,48h and72h(P<0.05). The rADC values were no significant difference at each time point in the central area between the ischemia group and the ischemic postconditioning group (P>0.05). In the ischemia group, the rADC value in the marginal area decreased from4h to24h and decreased to about66.16%in24h. However, the rADC value increased after24h, which were significant difference in comparison with at4h,48h and72h(P<0.05). In the ischemic postconditioning group, the rADC values in the marginal area gradually increased from4h to72h. Compared with ischemic group, at each time point, the rADC values of the ischemic postconditioning group was statistically different at24h,48h (P<0.05) and no significant difference at4h,72h (P>0.05). In the ischemic group, the rADC value at4h had statistically significant between the lesion center and marginal area (P <0.05), however, there were no significant difference at24h,48h,72h (P>0.05).Compare the volume of infarct area measured by TTC and high signal on DWI, there was statistically significant at4h (P<0.05), however, there were no significant difference at24h,48h and72h(P>0.05). In the ischemia group, the largest volume(83.5±9.2mm3)of cerebral ischemic lesion showed at24h. The largest volume at4h had significant difference in comparison with24h,48h and72h (P<0.05). In the ischemic postconditioning group, the volume of cerebral ischemic lesion showed no significant increase from4h(64.9±6.8mm3) to24h(69.1±5.7mm3). There had no significant difference at the different time (P>0.05). The lesions of all the animal between the ischemia group and ischemic postconditioning group showed abnormal enhancement after injection Gd-DTPA at different time points. SI increase rate at24h in the ischemia group was higher than the other time points, which was significant difference compared with that of4h,48h and72h (P＜0.05). The volume on enhanced images was lower than on T2WI images, which had significant difference (P<0.05). Compared SI increase rate in the ischemia group with the ischemic postconditioning group, there were significant difference at24h,48h and72h (P<0.05), which the latter was lower than the former. Light microscopy showed neurons shrinkage in the central area after4hour-ischemia and swelling between ischemic tissue and normal tissue edema, and ischemic penumbra neurons swelling and pyknosis, parts of the endovascular platelet accumulation and thrombosis. After24hour-ischemia, ischemic core tissue dissolved, showing the loose network structure and the demyelination of neurons, pyknosis were obviously observed by light microscopy. Moreover, after48and72hour-ischemia, eosinophilic neurons appeared, which made the whole cells be stained red.Conclusion:MRI may be useful to reflect the pathological evolution process at different time points after ischemia of the thrombotic cerebral ischemia animal models and evaluate the effects of ischemic postconditioning. The infarcted volume of ischemic postconditioning group was smaller than ischemic group, combined with semi-quantitative parameter rADC value, through which the cause may be supposed to save the penumbra. In the ischemic postconditioning group, SI increase rate after enhance was lower than the ischemic group, which may be considered to decrease the damage to the blood-brain barrier. Objective:To establish the models of thrombotic cerebral ischemia and ischemic postconditioning in tree shrews,and to observe the SPIO-enhanced MRI characteristics, compared with Prussian blue staining and anti-IL-1β and anti-ED-1by double immunofluorescence staining,and to explore the value of the SPIO-enhanced MRI for monitoring thrombotic cerebral ischemia and ischemic postconditioning, which may provide a theoretical basis for clinical application and experimental evidence.Methods:The thrombotic cerebral ischemia were induced by photochemical reaction in tree shrews to establish the ischemic models,and at4h after ischemia ischemic postconditioning were established by3repeated cycles of occlusion of the right carotid arteries5min,and reperfusion5min. SPIO was injected at4h,24h and48h after ischemia and3.0T MR imaging was performed at24h after injection and to observe the characteristics of SPIO-enhanced MRI.The T2WI rSI were measured on the workstation on the T2WI images.By showed The morphology and location of iron particle in the lesion was detected by Prussian blue staining.The invasion of macrophages after cerebral ischemia in the lesion was observed with anti-IL-1β and anti-ED-1by double immunofluorescence staining laser scanning confocal microscopic examination. Results:There were no abnormalities in MRI in the sham group at all time points. In the ischemia group,the low signal changes in the ischemic lesions were observed at24h after injection and the patterns of the low signal were identical at24h,48h and72h after injected SPIO at4h.The T2WI rSI at24h,48h and72h after injected SPIO at4h had a significant difference in comparison with plain scan(P<0.05).The T2WI rSI had no significant difference among24h,48h and72h after injected SPIO at4h(P>0.05). The patterns of the low signal were identical at48h and72h after injected SPIO at24h.The T2WI rSI at48h and72h after injected SPIO at24h had no significant difference in comparison with plain scan(P>0.05).The T2WI rSI at72h after injected SPIO at48h had no significant difference in comparison with plain scan(P>0.05).In the ischemic postconditioning group,the T2WI rSI at24h,48h and72h after injected SPIO at4h were higher than in the ischemia group and there were a significant difference between the groups (P<0.05). The T2WI rSI at48h and72h in the ischemic postconditioning group after injected SPIO at24h had no significant difference in comparison with the ischemia group(P>0.05). The T2WI rSI at72h in the ischemic postconditioning group after injected SPIO at48h had no significant difference in comparison with the ischemia group(P>0.05). The iron particles were mainly located within the cells in the vascular lumens by Prussian blue staining at24h after ischemia. The iron particles were mainly located in the cells out of the vascular lumens at72h. The numbers of the iron particles at24h and48h after ischemia were lower than at4h.The numbers of the iron particles in the ischemic postconditioning group were lower than in the ischemia group.There were a large number of ED-1positive microglia/macrophages detected by double immuno-fluorescence staining laser scanning confocal microscopic examination in the surrounding area of the infarct at24h after ischemia. While the number of microglia/macrophages at48h and72h is less than at24h.But there were many large macrophages around the lesions at72h. The IL-1β expression can be seen at24h after ischemia which was stained with ED-1. The numbers of ED-1and IL-1B positive cells at24h after ischemia in the ischemic and postconditioning groups were more than at48h and72h (P<0.05). The numbers of ED-1and IL-1B positive cells at48h after ischemia in the ischemic and postconditioning groups were more than at72h (P<0.05). The numbers of ED-1and IL-1β positive cells at24h and48h after ischemia in the ischemic groups were more than and in the postconditioning group(P<0.05). The numbers of ED-1and IL-1β positive cells at72h after ischemia in the ischemic groups had no significant difference in comparison with the ischemic postconditioning group(P>0.05).Conclusion: Microglia/macrophages involved in the pathological process of ischemic brain injury in the acute phase of thrombotic infarction animal models.In vivo SPIO-Enhanced MRI imaging can be used for monitoring of macrophage invasion situation of acute thrombotic cerebral ischemia induced by photochemical reaction in tree shrews.Ischemic postconditioning can reduce cerebral ischemia microglia/macrophage invasion and expression of inflammatory mediators. SPIO-enhanced MRI can monitor the changes in the acute phase inflammatory response after cerebral ischemia postconditioning in vivo.更多还原