双光子活体成像技术检测缺血性脑卒中超早期血脑屏障损伤

Two-photon in vivo imaging of blood brain barrier injury in the ultra-early stage of cerebral ischemic stroke

目的·评估双光子活体成像技术在缺血性脑卒中超早期血脑屏障(blood brain barrier,BBB)损伤的检测效果。方法·取8~12周龄的清洁级C57BL/6健康雄性小鼠12只,随机分为假手术组(Sham组)和大脑中动脉阻塞(middl ecerebral artery occlusion,MCAO)组,每组6只。MCAO组小鼠采用微线栓阻塞大脑中动脉60min,引发小鼠发生缺血性脑卒中(即建立MCAO模型);Sham组小鼠进行与MCAO组小鼠相同的所有手术操作,但不置入微线栓。当MCAO组小鼠缺血60min后,拔除线栓给予再灌注30~60min。随后,2组小鼠均行静脉注射荧光素488偶联的右旋糖苷(Alexa-Fluora-488conjugated dextran)和罗丹明6G(rhodamine 6G),分别标记血管和中性粒细胞,并采用四甲基罗丹明-5-马来酰亚胺(tetramethylrhodamine-5-maleimide,TMR)检测BBB的完整性。采用双光子荧光显微镜观察MCAO组小鼠脑卒中前、后脑血管内外荧光强度的实时变化。结果·与发生脑卒中前相比,MCAO组小鼠脑卒中发生后30~60min即可观察到脑血管外TMR荧光强度显著增强(P=0.000),提示发生了示踪剂渗漏。与Sham组相比,MCAO组小鼠发生脑卒中后中性粒细胞在血管内运动速度明显减慢(P=0.000)。结论·双光子活体成像技术可对缺血性脑卒中超早期的BBB损伤进行检测,为临床应用提供了一定的参考价值。

Objective · To evaluate the effect of two-photon in vivo imaging on detecting the blood brain barrier (BBB) injury in the ultra-early stage of cerebral ischemic stroke. Methods · Twelve clean grade C57BL/6 healthy male mice aged 8-12 weeks were randomly divided into Sham group and middle cerebral artery occlusion (MCAO) group, which were sham operated or middle cerebral artery occluded, respectively. After 60 min of ischemia, MCAO mice were treated with reperfusion for 30-60 min after the suture being removed. The vessels and the neutrophils of mice in the two groups were labeled intravenously with Alexa-Fluora-488 conjugated dextran and rhodamine 6G, respectively. The integrity of BBB was detected by intravenous injection of tetramethylrhodamine-5-maleimide (TMR). Before and after the stroke, the real-time changes of the fluorescence intensity of the inside and outside cerebral vessels of mice in the MCAO group were observed by two-photon fluorescence microscopy. Results · The fluorescence intensity of TMR in the external cerebrovascular of mice in the MACO group was significantly increased within 30-60 min after stroke (P=0.000), suggesting there existed tracer leakage. Compared with the Sham group, the movement of neutrophils in the blood vessels of mice in the MACO group was significantly slowed down (P=0.000). Conclusion · Two-photon in vivo imaging can be used to detect the BBB injury in the ultra-early stage of cerebral ischemic stroke, which provides a certain reference value for clinical application.