目的:探讨脑淋巴引流阻滞(CLB)后蛛网膜下腔出血(SAH)兔脑脊液对大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)的损伤作用。方法:建立兔SAH及CLB模型,于SAH模型建立后5d抽取脑脊液加入PC12细胞培养基中,随机将PC12细胞分为空白对照组(无血清F12培养基)、正常脑脊液组、SAH脑脊液组、SAH+CLB脑脊液组,分别于培养0.5h、1h、2h、4h后,通过四甲基偶氮唑盐法(MTT)检测细胞存活率并测定乳酸脱氢酶(LDH)释放率,采用免疫组化法检测细胞凋亡蛋白Bax及热休克蛋白70(HSP70)的表达。结果:MTT、LDH结果显示,与正常脑脊液组比较,SAH脑脊液组和SAH+CLB脑脊液组脑脊液使PC12细胞存活率降低,LDH释放率增加,以后组更为显著;正常脑脊液对PC12细胞无明显损伤作用。在SAH脑脊液组和SAH+CLB脑脊液组均发现PC12细胞Bax及HSP70蛋白表达;Bax蛋白表达后组大于前组,且呈时间依赖性增强;在0.5h和1h,SAH+CLB脑脊液组HSP70蛋白表达强于SAH脑脊液组,而在2h和4h则表达减弱。结论:脑淋巴引流阻滞可加重SAH兔脑脊液对PC12细胞的损伤,表明脑淋巴引流通路在SAH后可能起到内源性保护作用。
AIM: To determine the injured effect of cerebrospinal fluid(CSF) from subarachnoid hemorrhage (SAH) after cerebral lymphatic blockage (CLB) on PC12 cells. METHODS: SAH and CLB models of adult New Zealand rabbits were used. CSF was obtained from experimental animals after 5 d of modeling and was added into cultured PC12 cells. The cells were randomly divided into blank control(F12 Ham' s), normal CSF, SAH CSF, and SAH + CLB CSF groups. At different time points, the survival rate of PCI2 cells was measured by MTT assay. LDH leakage was detected. Expression of Bax and heat - shock protein 70 (HSP70) was determined by immunohistochemical staining. RESULTS : MTr assay and detection of LDH leakage revealed that the survival rate of PC12 cells was obviously inhibited and the leakage of LDH increased in SAH CSF group and SAH + CLB CSF group. CSF from normal rabbit did not damage the PC12, as compared to blank controls. Above effects were more obvious in SAH + CLB CSF group than those in SAH CSF group. Bax and HSP70 protein expression was found in both SAH CSF group and SAH + CLB CSF group. Expression of Bax protein in SAH + CLB CSF group was stronger than that in SAH CSF group in a time dependent manner. At 0.5 h and 1 h, the expression of HSP70 protein in SAH + CLB CSF group was stronger than that in SAH CSF group, whereas the expression became weaker at 2 h and 4 h in that group. CONCLUSION : Blockage of cerebral lymphatic drainage pathway deteriorates the damage of CSF from SAH on PC12 ceils, indicating this pathway may acts as an endogenous protective mechanism in SAH.