SIGNALS INVOLVED IN REACTIVE GLIOSIS

参与反应性胶质细胞增生的信号

基本信息

批准号：
2750914
负责人：
TAE H OH
金额：
$ 20.85万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-15 至 2001-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2750914
关键词：
Mammalia acetylcholine acidity /alkalinity alternative medicine brain injury calcium flux epilepsy glial fibrillary acidic protein gliosis histopathology lactates messenger RNA microdialysis posttranslational modifications regulatory gene tissue /cell culture

项目摘要

DESCRIPTION (Investigator's Abstract): Reactive gliosis (RG), whose hallmark is increased immunoreactivity (IR) for GFAP, is induced by CNS injury including seizures. Cholinolytic seizures produce a marked increase in GFAP-IR in piriform cortex within 1 hr of seizure, however, the extracellular signals initiating RG are largely unknown. Our working hypothesis is that signals such as acidic pH initiate RG as measured by increased astrocytic GFAP in response to CNS injury or seizure. To investigate the potential signals for RG, we developed an in vitro system ideally suited to this purpose a non-reactive, "mature" astrocyte culture. Decreasing the pH of such cultures to 5.8-6.0 provokes a rapid increase in GFAP-IR within hours. This increase is diminished by Ca++ channel blockers. The time course of this reactivity mimics that of our in vivo animal model. We propose to test three specific hypotheses using an in vitro culture system in parallel with an in vivo animal model. Hypothesis A: In seizures, hyperactive neurons increase glycolytic flux resulting in the production of lactic acid and decreased extracellular pH. The decreased pH stimulates RG both in vivo and in vitro. Aim 1. Correlate lactic acid levels /extracellular pH during cholinolytic seizures in vivo with increases in GFAP-IR. 2. Investigate the effects of acidic pH on GFAP-IR in cultured non- reactive, "mature' astrocytes. 3. Determine if infusion of lactic acid into a focal brain area increases GFAP in normal animals. A second experiment will determine if infusion of buffers into the brain (piriform cortex) of animals experiencing cholinolytic seizures prevents increased GFAP. Hypothesis B: GFAP-IR increases as the result of a cascade of signals initiated by reduced pH. Aim 4. Investigate the effect of acidic pH on Cab translocation in cultured, "mature' astrocytes. Hypothesis C: GFAP responds in a biphasic manner. First, there is a transient increase in IR that is independent of de novo protein synthesis. Second, there is synthesis of GFAP mRNA and protein; this process may persist for weeks. 5 Determine if acidic pH causes posttranslational modifications (e.g., phosphorylation) of GFAP or structural changes in filaments in vivo and in vitro. a. Aim 6. Investigate the expression of immediate early genes (IEGs), GFAP mRNA and GFAP protein during induced seizures, and, in parallel, in acid-treated mature" astrocyte cultures. The significance of this proposal is that it will allow us to characterize the signals involved in RG and thus, provide us with a pharmacological strategy to modify the degree and the time course of the RG response to injury. This would be important since reactive astrocytes form a "glial scar which has long been thought to a form barrier to regenerating CNS axons.

描述（研究者摘要）：反应性神经胶质增生（RG），其标志是 GFAP 免疫反应性 (IR) 增加，由 CNS 诱导受伤，包括癫痫发作。胆碱溶解性癫痫发作会产生明显的然而，癫痫发作后 1 小时内梨状皮质中的 GFAP-IR 增加，启动 RG 的细胞外信号在很大程度上是未知的。我们的工作假设是酸性 pH 等信号启动 RG 通过中枢神经系统损伤引起的星形胶质细胞 GFAP 增加来测量或发作。为了研究 RG 的潜在信号，我们开发了一种非常适合此目的的体外系统，无反应性，“成熟” 星形胶质细胞培养。将此类培养物的 pH 值降低至 5.8-6.0 数小时内引起 GFAP-IR 快速增加。这个增加是 Ca++ 通道阻滞剂可减弱该作用。该反应的时间过程模仿我们的体内动物模型。我们建议测试三个使用体外培养系统的具体假设与体内动物模型。假设 A：癫痫发作时，神经元过度活跃增加糖酵解通量，从而产生乳酸和细胞外pH值降低。 pH 值降低会刺激体内 RG 和体外。目标 1. 关联乳酸水平/细胞外 pH 值体内胆碱溶解性癫痫发作期间 GFAP-IR 增加。 2. 研究酸性 pH 值对非培养物中 GFAP-IR 的影响反应性“成熟”星形胶质细胞。 3. 确定是否输注乳酸进入局灶性脑区会增加正常动物的 GFAP。一秒钟实验将确定是否将缓冲液注入大脑经历胆碱溶解性癫痫发作的动物（梨状皮层）可预防 GFAP 增加。假设 B：GFAP-IR 的增加是由于 pH 降低引发的信号级联。目标 4. 调查酸性 pH 值对“成熟”培养物中 Cab 易位的影响星形胶质细胞。假设 C：GFAP 以双相方式响应。第一的， IR 会短暂增加，与 de novo 无关蛋白质合成。其次，GFAP mRNA和蛋白质的合成；这个过程可能会持续数周。 5 确定酸性 pH 值是否会导致 GFAP 的翻译后修饰（例如磷酸化）或体内和体外细丝的结构变化。一个。目标6。研究立即早期基因 (IEG)、GFAP mRNA 和诱导癫痫发作过程中的 GFAP 蛋白，以及同时在酸处理过程中的 GFAP 蛋白成熟的“星形胶质细胞培养物。该提案的意义在于它将使我们能够表征 RG 中涉及的信号，因此，为我们提供了一种药理学策略来改变程度和 RG 对损伤做出反应的时间过程。这很重要，因为反应性星形胶质细胞形成“神经胶质疤痕”，长期以来一直被认为是形成中枢神经系统轴突再生的障碍。