Immunohistochemical study of cytokine induction and endotherial cell damage on hypoxic brain injury

已有研究表明，细胞因子在缺氧缺血性脑损伤中起重要作用。为探讨细胞因子与一氧化氮(NO)在脑损伤中的关系。[材料与方法]取10只7日龄SD大鼠脑片，每片厚350μm。灌流切片只有一个损伤层，断头后3h进行代谢恢复，并在组织培养井中进行研究。在每项研究中，有30 mm的治疗周期，如低氧或NMDA，然后用含氧ACSF灌流4小时。冰冻切片厚度20μm，垂直切面切片，进行IL-1-β免疫组织化学染色。取相邻冰冻切片进行HE染色和TUNEL染色。此外，还观察了新的nNOS抑制剂7-硝基吲唑(7NI)对过氧亚硝酸根标志物硝基酪氨酸(NT)的影响。[结果]缺氧或NMDA处理后15~30min，与对照组相比，缺氧或NMDA处理均可诱导脑片IL1-β表达。7NI不能减少神经元损伤和NT阳性细胞，说明NO不能导致缺氧早期神经元死亡。[结论]缺氧后神经元组织中出现的细胞因子可能通过激活NMDA受体而导致NO的诱导，导致神经细胞的凋亡出现在晚期。

It is reported that Cytokines play important roles on hypoxic ischemic brain injury. In this study, the relationship between cytokines and nitric oxide (NO) in brain injury was investigated.[Material and Method]In each experiment forty cerebrocortical slices, each 350μm thick, were obtained from ten 7-day old Sprague-Dawley rats. Perfused slices had only one injury layer, were allowed 3 hrs to metabolically recover from decapitation, and were studied in a tissue culture well. In each study there was a 30 mm treatment period, such as hypoxia or NMDA, followed by 4 hours of perfusion with oxygenated ACSF. Frozen sections 20 μm thick were cut in the vertical plane of slices, and processed for Immunohistochemistry of IL1-beta. Adjacent frozen sections were used for HE staining and TUNEL staining. Expression of nitrotyrosine (NT), a marker of peroxynitrite was also investigated with/without 7-nitroindazole (7NI), a novel nNOS inhibitor. Apoptosis in slices was detected by TUNEL staining after etarabon (MCI-186) treatment before NMDA perfusion, a kind of free radical scavenger.[Results]Hypoxia or NMDA treatment caused IL1-beta expression at 15 to 30 min after the each insult, compared to control slices. 7NI could not decrease neuronal damage and NT positive cells, showing that NO did not cause neuronal death at early phase after hypoxia. Etarabon significantly decreased TUNEL positive cells on brain slices after NMDA treatment.[Conclusion]It is suggested that cytokine, which appears in neuronal tissue just after hypoxia, might cause NO induction by activation of the NMDA receptor, resulting in apoptosis of neuronal cells appeared at late phase.