颈淋巴阻断加重SAH脑脊液对PC12细胞的损伤

Effects of cerebrospinal fluid from experimental subarachnoid hemorrhage of cervical lymphatic blockage on LDH leakage and cystoskeleton of PC12 cells

目的探讨脑淋巴引流途径在蛛网膜下腔出血(SAH)后神经元损伤中的作用。方法建立兔SAH及脑淋巴引流阻滞(CLB)模型,于SAH模型建立后5d抽取脑脊液加入培养的PC12细胞中,随机将PC12细胞分为空白对照组、正常对照组(正常脑脊液)、SAH脑脊液组,SAH+CLB脑脊液组,分别于培养0.5、1、2、4h后,用比色法测定乳酸脱氢酶(LDH)释放率,采用免疫荧光染色法激光共聚焦显微镜观察PC12细胞细胞骨架。结果与正常对照组比较,SAH脑脊液组PC12细胞LDH释放率增加;SAH+CLB脑脊液组LDH释放率明显高于SAH脑脊液组。正常脑脊液不引起PC12细胞细胞骨架改变,SAH脑脊液组及SAH+CLB脑脊液组PC12细胞胞质着色较弱,突起变短甚至回缩,胞核呈现凋亡特征,上述表现以SAH+CLB组更为明显。结论脑淋巴引流阻滞加重SAH脑脊液对PC12细胞的损伤,提示通过改善脑淋巴引流途径可减轻SAH继发性脑损伤。

Aim To determine the role of cerebral lymphatic drainage pathway in the development of neural injury following subarachnoid hemorrhage （SAH）.Methods SAH and cerebral lymphatic blockage （CLB） models in adult New Zealand rabbits were used.Cerebrospinal fluid （CSF） was obtained from experimental animals after 5 days of modeling and was added into cultured PC12 cells.The PC12 cells were randomly divided into blank control,normal control （with normal CSF）,SAH with CSF,and SAH＋CLB with CSF groups.After half an hour,1 hour,2 hours and 4 hours,the LDH leakage of PC12 cells into media was detected by chromatometry.Cystoskeletons of PC12 cells were observed by laser confocal microscope after immunofluorescence staining.Results Compared with normal control group,an increased LDH leakage was found in SAH with CSF group and SAH＋CLB with CSF group.The increase was more obvious in SAH＋CLB with CSF group than that in SAH with CSF group.Cystoskeleton did not alter with normal CSF.In SAH with CSF group and SAH＋CLB with CSF group,cystoskeleton staining became weak in cytoplasm.Shortened axons and dendrons were found in the above groups.Apoptotic appearance of nucleus was also observed.The above changes in SAH＋CLB with CSF group were severer than those in SAH with CSF group.Conclusion The results suggest that blockage of cerebral lymphatic drainage pathway deteriorates the damage of PC12 cells with CSF from SAH,indicating cerebral ischemic injury following SAH may be attenuated by improving this pathway.