蛛网膜下腔出血大鼠脑内蛋白质经淋巴引流的荧光示踪

目的 探讨蛛网膜下腔出血(SAH)大鼠脑实质内大分子物质经淋巴引流的变化.方法 将健康成年雄性Wistar大鼠分为生理盐水组、伊文思蓝标记白蛋白(EBA)组、SAH+EBA组.采用枕大池两次注入自体动脉血法建立SAH模型,应用改良的脑实质微量注射技术将EBA注入大鼠左侧尾壳核,生理盐水组用生理盐水代替EBA.于注射后0.5、1、2、3、5 d处死动物,观察并比较各组不同时间点EBA在脑内、颈总动脉壁、颈部淋巴结等部位的分布.结果 EBA组于注射后1 d荧光信号首先出现在左侧脑实质、侧脑室及其血管周围,并逐步到达对侧;双侧颈总动脉外膜有密集的荧光信号,颈部淋巴结可见荧光信号.2 d后脑内荧光信号明显减弱,嗅球内荧光信号逐渐增强,腹主动脉旁淋巴结内有点状荧光信号.各淋巴结内荧光均于3 d时最强.SAH后脑内EBA引流至嗅球、颈部淋巴结和腹主动脉旁淋巴结的量减少且速度减慢.于0.5、1、2、3、5 d,EBA组颈深淋巴结EBA荧光密度分别为14.5±3.2、27.5±7.4、60.3±12.3、138.0±12.0和108.1±13.4,SAH+EBA组分别为8.9±2.0、11.9±2.5、17.4±3.7、26.7±4.5和59.0±8.1,后组各时间点密度均低于前组(F=13.17、24.04、66.81、302.77、59.36,P＜0.01);2、3、5 d,EBA组腹主动脉旁淋巴结EBA荧光密度分别为26.3±5.9、47.5±9.6和41.0±9.3,SAH+EBA组分别为11.0±1.5、12.5±2.8、23.6±3.2,后组各时间点均低于前组(F=38.17、72.52、19.01,P＜0.01).结论 SAH可导致脑内大分子物质经淋巴系统引流的功能障碍.

Identifying lymphatic drainage pathway of protein from subarachnoid hemorrhage rat brain using fluorescence Evans blue-labeled albumin injected into caudate-putamen

Objective To investigate the pathway of lymphatic drainage of proteins from cerebral parenchyma in subarachnoid hemorrhage rat models. Methods Healthy adult male Wistar rats were divided into Saline group, Evans blue-labeled albumin (EBA) group, and SAH + EBA group. SAH models were produced by double injection of autologous arterial blood into cisterna magna. Using a modified microinjection method, EBA was injected into left candate-putamen of the EBA group and EBA + SAH group rats. In Saline control group, saline was injected. After injection, at 12 hours, 1 day, 2 days, 3 days and 5 days, the animals were sacrificed and the fluorescence signals of EBA were imagined and analyzed along the possible lymphatic drainage pathway, e.g. the brain tissue, the wall of common carotid artery, and cervical lymphatic nodes. Results One day after injection, in EBA group, the fluorescence of EBA initially appeared on the left of the brain, the wall of common carotid artery, left lateral cerebral ventricle, and the perivascular spaces of cerebral vessels. The fluorescence signals gradually expanded to the opposite side.Large amount of fluorescence granules accumulated in the outer layer of common carotid artery. Fluorescence was also found in cervical lymphatic nodes. Two days after injection in this group, the density of fluorescencein the brain became weaker while the density of fluorescence in rhinencephalon became stronger. The fluorescence of EBA was found in lymphatic nodes adjacent to abdominal aorta. In SAH + EBA group,reduced amount and velocity of the drainage of EBA from left caudate-putamen to rhinencephalon, cervical lymphatic nodes, and lymphatic nodes adjacent to abdominal aorta were observed. From 12 hours to 5 days after injection, fluorescence intensity of EBA in deep cervical lymphatic nodes in SAH + EBA group(8.9 ±2. 0, 11.9 ± 2. 5, 17.4 ± 3.7, 26.7 ± 4. 5 and 59.0 ± 8. 1 ) were lower than those in EBA group ( 14. 5 ±3.2, 27.5 ±7.4, 60.3 ±12.3, 138.0±12.0 and 108. 1 ±13.4, F=13. 17, 24.04, 66.81, 302.77 and 59.36, P ＜ 0. 01 ). From 2 to 5 days, fluorescence intensity of EBA in lymphatic nodes adjacent to abdominal aorta was also lower in SAH + EBA group( 11.0 ± 1.5, 12. 5 ±2. 8, 23.6 ±3. 2) than those in EBA group(26. 3 ±5.9, 47.5 ±9.6, 41.0 ±9.3; F =38. 17, 72.52, 19.01, P ＜0.01). Conclusion SAH can result in reduced drainage of macromolecular substances, e.g. protein, from the brain via lymphatic pathway.