GLUTAMATE TRANSPORTERS--ALTERATIONS IN SPINAL CORD INJUR

谷氨酸转运蛋白——脊髓损伤的改变

• 批准号: 6322809
• 负责人: Susan A Queen
• 金额: $ 9.16万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-20 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6322809
• 关键词: aspartate; autoradiography; chloride ion; glia; glutamate transporter; glutamates; immunocytochemistry; laboratory rat; neuropathology; protein isoforms; protein localization; sodium ion; spinal cord injury; tissue /cell preparation

DESCRIPTION: (Verbatim from the Applicant's Abstract) Spinal cord injury produces not only the initial damage to neurons and glia, but also secondary tissue damage due to a cascade of events. One of the secondary mechanisms involves excitotoxicity due to marked increases in release of excitatory amino acids. The glutamate transporters are hypothesized to contribute to regulation of the extracellular glutamate concentration in the synaptic cleft, providing important function both in normal physiology and in pathological conditions, such as excitotoxicity. Increased release of excitatory amino acids occurs in response to acute trauma to spinal cord. The focus of this grant is to determine the effect of acute spinal cord crush injury in rats on the high affinity glutamate transporters. Pharmacologically distinct sodium (Na)-dependent and chloride (Cl)- dependent glutamate transporters are present throughout the brain. Subtypes of Na-dependent transporters have been cloned from rat, rabbit and humans. While their distribution with in brain has been determined, their distribution within the spinal cord is limited. Prior to determining the effects of spinal cord injury of the glutamate transporters, their distribution in normal rats must be delineated. We hypothesize the Na-dependent and Cl-dependent glutamate transporters will show distinct distributions in the spinal cord and that these transporters will be differentially altered by acute spinal cord crush injury. The Specific Aims of this grant application are to: 1) Determine the distribution of the Na-dependent and Cl-dependent glutamate transporters and subtypes in cross-sections of rat spinal cord. 1a) Quantify the functional anatomical presence and distribution within the spinal cord of both the pharmacologically distinct Na- dependent and Cl-dependent transporters by autoradiography with [3H]-D-aspartate, [3H]-L-glutamate and [35S]-cystine. 1b) Quantify the subtype distribution of the Na-dependent transporters by the ability of pharmacologically characterized inhibitors of Na-dependent transport to displace [3H]-D-aspartate binding. 1c) The anatomical specificity of the Na-dependent subtypes EAAC1,GLT-1 and GLAST will be determined by immunohistochemistry with primary antibodies specific to each subtype and compared to the results in Aim1b. 2) Determine the effect of acute spinal cord injury on the transporters by crush injury at the T-10 spinal column level. Tissue will be evaluated as in the above three Specific Aims at 1,4 and 12 hours after injury. Transporter alterations will also be correlated to glial responses by GFAP immunohistochemistry. The background information obtained in these above studies will lead to future experiments to elucidate the underlying mechanisms which alter the glutamate transporters and to potential interventions in spinal cord trauma.

描述：（逐字摘自申请人的摘要）脊髓损伤不仅对神经元和神经胶质细胞产生最初的损伤，而且由于一系列事件导致继发性组织损伤。次要机制之一涉及由于兴奋性氨基酸释放显着增加而导致的兴奋性毒性。据推测，谷氨酸转运蛋白有助于调节突触间隙中的细胞外谷氨酸浓度，在正常生理和病理条件（例如兴奋性毒性）中提供重要功能。脊髓急性创伤导致兴奋性氨基酸释放增加。该资助的重点是确定大鼠急性脊髓挤压损伤对高亲和力谷氨酸转运蛋白的影响。药理学上不同的钠 (Na) 依赖性和氯 (Cl) 依赖性谷氨酸转运蛋白存在于整个大脑中。钠依赖性转运蛋白的亚型已从大鼠、兔子和人类身上克隆出来。虽然它们在大脑中的分布已经确定，但它们在脊髓中的分布是有限的。在确定谷氨酸转运蛋白对脊髓损伤的影响之前，必须先描述它们在正常大鼠中的分布。我们假设 Na 依赖性和 Cl 依赖性谷氨酸转运蛋白将在脊髓中表现出不同的分布，并且这些转运蛋白将因急性脊髓挤压损伤而发生差异性改变。本次拨款申请的具体目标是： 1) 确定 Na 依赖性和 Cl 依赖性谷氨酸转运蛋白及其亚型在大鼠脊髓横截面中的分布。 1a) 通过使用 [3H]-D-天冬氨酸、[3H]-L-谷氨酸和 [35S]-胱氨酸进行放射自显影，量化药理学上不同的 Na 依赖性和 Cl 依赖性转运蛋白在脊髓内的功能解剖存在和分布。 1b) 通过 Na 依赖性转运的药理学特征抑制剂取代 [3H]-D-天冬氨酸结合的能力来量化 Na 依赖性转运蛋白的亚型分布。 1c) Na依赖性亚型 EAAC1、GLT-1 和 GLAST 的解剖特异性将通过免疫组织化学测定，使用对每种亚型具有特异性的一抗，并与 Aim1b 中的结果进行比较。 2) 确定 T-10 脊柱水平挤压损伤对急性脊髓损伤对转运蛋白的影响。将在受伤后 1.4 和 12 小时按照上述三个具体目标对组织进行评估。通过 GFAP 免疫组织化学检测，转运蛋白的改变也与神经胶质反应相关。上述研究中获得的背景信息将导致未来的实验阐明改变谷氨酸转运蛋白的潜在机制以及脊髓损伤的潜在干预措施。