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

缺氧性脑损伤中细胞因子诱导和内皮细胞损伤的免疫组织化学研究

基本信息

批准号：
13671141
负责人：
HASEGAWA Koh
金额：
$ 0.64万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671141/
关键词：
hypoxic brain injury cytokine nitric oxide nitrotyrosine free radical scavenger 血管内皮細胞

项目摘要

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.

近年来研究表明，细胞因子在缺氧缺血性脑损伤中发挥重要作用。本研究旨在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7日龄SD大鼠10只，取40个350μm厚的皮质脑片。灌注切片只有一个损伤层，允许3小时从断头代谢恢复，并在组织培养孔中进行研究。在每项研究中，有一个30 mm的治疗期，如缺氧或NMDA，然后用充氧ACSF灌注4小时。在切片的垂直平面上切割20 μm厚的冷冻切片，并进行IL 1-β的免疫组织化学处理。取相邻冰冻切片行HE染色和TUNEL染色。硝基酪氨酸（NT），过氧亚硝酸盐的标志物的表达也进行了研究与/无7-硝基吲唑（7 NI），一种新的nNOS抑制剂。在自由基清除剂NMDA灌流前，用依他拉邦（MCI-186）处理切片，TUNEL染色检测细胞凋亡。[结果]与对照组相比，缺氧或NMDA处理后15 ~ 30 min，脑片中IL 1-β表达增加。7 NI不能减少神经元损伤和NT阳性细胞，表明NO在缺氧后早期不引起神经元死亡。依他拉朋显著减少NMDA处理后脑片上TUNEL阳性细胞。[结论]缺氧后神经组织中出现的细胞因子可能通过激活NMDA受体诱导NO的产生，导致神经细胞凋亡出现在缺氧后期。