摘要
目的 建立脑无复流模型并对灌注减低进行多维度评价.方法 利用激光散斑对比成像和双光子活体成像,比较C57BL/6(n=16)和BALB/c小鼠(n=37)短暂性大脑中动脉闭塞,以及BALB/c小鼠缺血1h或1.5h灌注改变情况.结合激光散斑对比成像、低倍率和较高放大倍率的灌注脑片图像,以及双光子显微镜监测红细胞流速和流量对短暂性大脑中动脉闭塞后脑灌注下降进行活体动态以及全脑切片和微血管的评估.用微管相关蛋白2染色和行为学评分评估梗死面积和行为学缺损.结果 在C57BL/6小鼠中,大脑中动脉区域大多数毛细血管在缺血期间仍然流动,而在BALB/c小鼠中,大多数毛细血管被阻断.此外,BALB/c小鼠缺血1.5 h后,24 h后再通时的皮质灌注减少至基线76.1%(与BALB/c sham组89.0%相比减少,P = 0.046),而缺血1 h减少至79.9%,与BALB/c sham组无明显差异(P=0.299).切片全脑灌注评估BALB/c小鼠短暂性大脑中动脉闭塞1.5 h导致全脑灌注减少至75.1%(与BALB/c sham组100%相比降低,P＜0.001),双光子活体成像评估大脑中动脉流域皮质表面毛细血管血流在再通24h,红细胞流速降至基线水平的50.3%(与BALB/c sham组110.7%相比降低,P=0.010),红细胞流量降至基线水平的38.9%(与BALB/c sham组119.2%相比降低,P=0.010);高倍率切片成像评估毛细血管有灌注长度为241μm±33 μm(与BALB/c sham组997 μm±103 μm相比减少,P=0.001),有灌注的毛细血管占据的总体积分数为0.0128±0.0018(与BALB/c sham组0.0539±0.0055相比减少,P＜0.001).微管相关蛋白2染色提示BALB/c小鼠短暂性大脑中动脉闭塞1.5 h导致梗死面积占全脑面积约36%,行为学评分提示行为学缺损,评分升高至9分.结论BALB/c小鼠短暂性缺血1.5 h导致明显的脑灌注下降以及毛细血管无复流,适合建立无复流模型.激光散斑对比成像、低倍率和较高放大倍率的灌注脑片图像,以及双光子显微镜活体成像的结合,可以对灌注变化进行多维度评价.
Abstract
Objective The purpose of this study was to explore a suitable method to model no-reflow phenomenon following ischemic stroke and to evaluate perfusion decrease from multiple perspectives.Methods Laser scatter contrast imaging and two-photon live imaging were used to compare transient middle cerebral artery occlusion in C57BL/6 and BALB/c mice and perfusion alterations in BALB/c mice with 1 or 1.5 h of ischemia.Several imaging techniques including laser scatter contrast imaging,low and higher magnification images of perfused brain slices and two-photon microscopy to monitor erythrocyte flow rate and flux were used to assess in vivo dynamics as well as whole brain sections and microvasculature for decreased cerebral perfusion after transient middle cerebral artery occlusion.Infarct size and behavioral deficits were assessed with microtubule-associated protein 2 staining and behavioral scoring.Results In C57BL/6 mice,most capillaries in the middle cerebral artery region remained flowing during ischemia,whereas most capillaries were blocked in BALB/c mice.In addition,cortical perfusion at 24 h of recanalization was significantly reduced to 76.1%of baseline following 1.5 h of ischemia in BALB/c mice(P=0.046 compared with the sham group),whereas for it was reduced to 79.9%following 1h of ischemia which was not significantly different from the sham group(P=0.299).Transient middle cerebral artery occlusion in BALB/c mice for 1.5 h resulted in a reduction in whole-brain perfusion to 75.1%(P＜0.001 compared with the sham group),and erythrocyte flow rate assessed by two-photon live-imaging of erythrocyte flow on the cortical surface of the middle cerebral artery basin was reduced to 50.3%of baseline levels at 24 h of recanalization(P=0.010 compared with the sham group),and erythrocyte flux decreased to 38.9%of baseline levels(P= 0.010 compared with the sham group);high-magnification imaging of sections assessed an approximately 76%reduction in the length of capillaries with perfusion(P=0.0001 compared with the sham group),and a reduction in the fraction of the total volume occupied by perfused capillaries by an approximately 76%reduction(P＜0.001 compared with the sham-operated group).Microtubule-associated protein 2 staining suggested that transient middle cerebral artery occlusion for 1.5 h in BALB/c mice resulted in infarcts that accounted for approximately 36%of the total cerebral area and behavioral scores elevated to 9,suggesting behavioral deficits.Conclusion Transient ischemia in BALB/c mice for 1.5 h resulted in a significant decrease in cerebral perfusion as well as capillary no-reflow and thus can model the no-reflow phenomenon following ischemic stroke.The combination of laser scatter contrast imaging,low magnification and higher magnification images of perfused brain slices,and two-photon microscopy live imaging allows for a multifaceted assessment of perfusion changes.
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