GLUTAMATE TRANSPORTERS--ALTERATIONS IN SPINAL CORD INJUR

描述：（摘自申请人摘要）脊髓损伤不仅会对神经元和神经胶质细胞产生初始损伤，还会由于一系列事件而产生继发性组织损伤。其中一个次要机制涉及由于兴奋性氨基酸释放的显著增加而引起的兴奋性毒性。谷氨酸转运体被认为参与突触间隙细胞外谷氨酸浓度的调节，在正常生理和病理条件下都发挥重要作用，如兴奋毒性。兴奋性氨基酸的释放增加是对脊髓急性损伤的反应。本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）依赖性谷氨酸转运体存在于整个大脑中。na依赖性转运蛋白的亚型已经从大鼠、兔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。在确定谷氨酸转运体对脊髓损伤的影响之前，必须确定其在正常大鼠中的分布。我们假设na依赖和cl依赖的谷氨酸转运蛋白在脊髓中表现出不同的分布，并且这些转运蛋白会因急性脊髓挤压损伤而发生不同的改变。本资助申请的具体目的是：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断面中的分布。1a)用[3H]- d -天冬氨酸、[3H]- l-谷氨酸和[35S]-胱氨酸进行放射自显影，定量测定药理学上不同的Na-依赖性和cl -依赖性转运体在脊髓内的功能解剖存在和分布。1b)通过药理学表征的na依赖转运抑制剂取代[3H]- d -天冬氨酸结合的能力，量化na依赖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方法确定，并与Aim1b的结果进行比较。2)在T-10脊柱水平通过挤压损伤确定急性脊髓损伤对转运蛋白的影响。在受伤后1、4和12小时，组织将按照上述三个特定目标进行评估。通过GFAP免疫组化，转运蛋白的改变也与神经胶质反应相关。上述研究获得的背景信息将引导未来的实验来阐明改变谷氨酸转运蛋白的潜在机制和脊髓损伤的潜在干预措施。

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.