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脑淋巴引流阻滞加重蛛网膜下腔出血后脑脊液所致海马神经元凋亡

孙保亮, 贾丽丽, 王轩, 杨明峰, 张颜波, 袁慧

山东省高校脑微循环重点实验室、泰山医学院附属医院神经内科，泰安271000； 济南市第六人民医院神经内科，济南 250200

摘要

本文旨在探讨脑淋巴引流系统在蛛网膜下腔出血(subarachnoid hemorrhage, SAH)后神经元损伤中的作用。分别建立 兔SAH、SAH+ 脑淋巴引流阻滞(cerebral lymphatic blockage, CLB)模型，于SAH 模型建立后5 d 抽取脑脊液，加入培养的 大鼠海马神经元中，随机将神经元分为空白对照组、正常脑脊液组、SAH 组、SAH+CLB 组，分别于培养0.5 h、1 h、 2 h、4 h 后，检测乳酸脱氢酶(lactate dehydrogenase, LDH)漏出率，流式细胞术检测神经元凋亡，免疫组织化学法检测细 胞凋亡蛋白Bax、Hsp70 的表达。结果显示：正常脑脊液对神经元LDH 漏出率无影响，SAH 组和SAH+CLB 组脑脊液使 神经元LDH 漏出率增加，以SAH+CLB 组更为显著；正常脑脊液没有引起神经元凋亡，SAH 组出现神经元凋亡，SAH+CLB 组神经元凋亡更为严重。在SAH 组和SAH+CLB 组均检测到Bax 及Hsp70 蛋白表达；Bax 蛋白表达在SAH+CLB 组大于SAH 组，且呈时间依赖性增强；在0.5 h 和1 h，SAH+CLB 组Hsp70 蛋白表达高于SAH 组，而在2 h 和4 h 则表达减弱，SAH+CLB 组表达高峰(1 h)早于SAH 组(2 h) 。以上结果表明，CLB 加重SAH 脑脊液对神经元的损伤，提示脑淋巴引流系统在SAH 后可能起到一定的内源性保护作用。

关键词： 蛛网膜下腔出血; 脑淋巴引流; 神经元; 凋亡; Bax; Hsp 7 0

分类号：R743.35

[Cerebral lymphatic blockage aggravates apoptosis of hippocampal neurons induced by cerebrospinal fluid from experimental subarachnoid hemorrhage.] [Ariticle in Chinese]

SUN Bao-Liang, JIA Li-Li, WANG Xuan, YANG Ming-Feng, ZHANG Yan-Bo, YUAN Hui

Key Laboratory of Cerebral Microcirculation in Universities of Shandong Province, Department of Neurology, Affiliated Hospital ofTaishan Medical College, Taian 271000, China； Department of Neurology, the Sixth People's Hospital of Jinan City, Jinan 250200, China

Abstract

This work was performed to determine the role of cerebral lymphatic drainage pathway in the development of neural injury following subarachnoid hemorrhage (SAH). SAH and cerebral lymphatic blockage (CLB) models in adult New Zealand rabbits were used. Cerebrospinal fluid (CSF) was obtained from experimental animals 5 d after modeling and was added into cultured rat hippocampal neurons. The neurons were randomly divided into blank control, normal CSF, SAH, and SAH+CLB groups. At different points of time, lactate dehydrogenase (LDH) leakage was detected by colorimetric method. Flow cytometry was used to detect the apoptosis of neurons. Expressions of Bax and heat-shock protein 70 (Hsp70) were determined by immunohistochemical staining. LDH leakage detection revealed that, compared with blank control group, CSF from normal rabbit did not damage the neurons, whereas the leakage of LDH increased in SAH group and SAH+CLB group. The increasing effect was more obvious in SAH+CLB group than that in SAH group. Normal CSF did not induce the apoptosis of neurons, whereas neuron apoptosis was found in SAH group and the apoptosis was even more severe in SAH+CLB group. Bax and Hsp70 protein expressions were found in both SAH and SAH+CLB groups. Expression of Bax protein in SAH+CLB group was stronger than that in SAH group in a time-dependent manner. At 0.5 h and 1 h, the expression of Hsp70 protein in SAH+CLB group was stronger than that in SAH group, whereas the expression became weaker at 2 h and 4 h. These results suggest that blockage of cerebral lymphatic drainage pathway deteriorates the damage of neurons treated with CSF from SAH, indicating this pathway may act as an endogenous protective role in SAH.

Key words: subarachnoid hemorrhage; cerebral lymphatic drainage; neurons; apoptosis; Bax; Hsp70

收稿日期：2009-03-31　　录用日期：2009-05-08

通讯作者：孙保亮　　E-mail: blsun88@163.com

引用本文：

孙保亮, 贾丽丽, 王轩, 杨明峰, 张颜波, 袁慧. 脑淋巴引流阻滞加重蛛网膜下腔出血后脑脊液所致海马神经元凋亡[J]. 生理学报 2009; 61 (4): 317-323.

SUN Bao-Liang, JIA Li-Li, WANG Xuan, YANG Ming-Feng, ZHANG Yan-Bo, YUAN Hui. [Cerebral lymphatic blockage aggravates apoptosis of hippocampal neurons induced by cerebrospinal fluid from experimental subarachnoid hemorrhage.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (4): 317-323 (in Chinese with English abstract).