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目的应用伊文思蓝(EB)灌注法并结合脑组织EB含量测定,检测大鼠血脑屏障(blood-brain barrier,BBB)通透性的变化,为研究微波辐射致大鼠BBB结构与功能的影响提供简便、快速、精确和灵敏的技术方法。方法采用10、30和100 mW/cm2微波辐射55只雄性Wistar大鼠,分别于辐射后1、3和7 d活杀前0.5 h尾静脉注入2%EB,脑组织冰冻切片应用共聚焦激光扫描显微镜下观察EB在大鼠海马BBB通透性变化,并采用化学比色法对脑组织中EB含量进行定量分析。结果共聚焦激光扫描显微镜观察EB在405 nm波长激发状态下呈鲜艳明亮的红色荧光,正常脑组织中,EB仅局限于血管腔内,未见透过血管壁渗出至管腔外。10~100 mW/cm2微波辐射后3~7 d大鼠海马脑组织BBB通透性增加,脑组织中可见大小不一的光斑,边界模糊,呈片状,血管周围可见弥散的红色荧光;与假辐射组相比,辐射组于3~7 d内海马组织EB含量均明显升高。结论应用伊文思蓝灌注后结合共聚焦显微镜观察和脑组织中EB含量的测定可准确检测微波辐射后BBB通透性的改变,值得推广应用。
Objective To detect the changes of permeability of blood-brain barrier (BBB) in rats by Evans Blue (EB) perfusion combined with the content of EB in brain tissue. To study the effects of microwave irradiation on the structure and function of BBB in rats Impact Provides easy, fast, accurate and responsive technical methods. Methods Fifty-five male Wistar rats were irradiated with 10, 30 and 100 mW / cm2 microwaves respectively, and 2% EBs were injected into tail vein at 0.5 h before and 1, 3 and 7 d after irradiation. Frozen sections of brain tissue were examined by confocal laser scanning The changes of BBB permeability in the hippocampus of rats were observed under a microscope, and the content of EB in brain tissue was quantitatively analyzed by chemical colorimetry. Results Confocal Laser Scanning Microscopy showed that EB was bright red fluorescence at excitation wavelength of 405 nm. In normal brain tissue, EB was confined to the lumen of the blood vessel and no exudation to the lumen outside the lumen was observed. BBB permeability of hippocampal brain tissue increased from 10 to 100 mW / cm2 in rats exposed to microwave for 3 to 7 days. The light spots of different sizes were visible in the brain tissue. The boundary was fuzzy and flaky. The diffuse red fluorescence was seen around the blood vessels. Compared with sham radiation group, the content of EB in hippocampus in radiation group increased significantly within 3-7 d. Conclusion Evans Blue staining combined with confocal microscopy and determination of EB content in brain tissue can accurately detect the change of BBB permeability after microwave irradiation, which is worth popularizing and applying.