GLUTAMATE TRANSPORTERS--ALTERATIONS IN SPINAL CORD INJUR

产品说明：脊髓损伤不仅产生对神经元和神经胶质的初始损伤，而且由于一连串事件而产生继发性组织损伤。次要机制之一涉及兴奋性氨基酸释放显著增加引起的兴奋性毒性。谷氨酸转运体被假设为有助于调节突触间隙中的细胞外谷氨酸浓度，在正常生理学和病理学条件（例如兴奋性毒性）中提供重要功能。兴奋性氨基酸的释放增加是对脊髓急性创伤的反应。本基金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体的影响。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脑中。Na依赖性转运蛋白的亚型已从大鼠、兔和人中克隆。虽然它们在脑中的分布已经确定，但它们在脊髓中的分布是有限的。在确定脊髓损伤对谷氨酸转运体的影响之前，必须描述它们在正常大鼠中的分布。我们假设钠依赖性和氯依赖性谷氨酸转运蛋白将显示不同的分布在脊髓，这些转运蛋白将被急性脊髓挤压损伤的差异改变。本基金申请的具体目的是：1）确定钠依赖性和氯依赖性谷氨酸转运体及其亚型在大鼠脊髓横截面中的分布。1a）通过用[3 H]-D-天冬氨酸、[3 H]-L-谷氨酸和[35 S]-胱氨酸的放射自显影术定量脊髓内不同的Na依赖性和Cl依赖性转运蛋白的功能解剖学存在和分布。1b）通过Na-依赖性转运的生物学表征的抑制剂置换[3 H]-D-天冬氨酸结合的能力来定量Na-依赖性转运蛋白的亚型分布。1c）Na依赖性亚型EAAC 1、GLT-1和GLAST的解剖学特异性将通过免疫组织化学用对每个亚型特异的一抗测定，并与Aim 1b中的结果进行比较。2)通过在T-10脊柱水平挤压伤确定急性脊髓损伤对转运蛋白的影响。在损伤后1、4和12小时，按照上述三个特定目的对组织进行评价。通过GFAP免疫组织化学，转运蛋白的改变也将与胶质反应相关。上述研究中获得的背景信息将导致未来的实验，以阐明改变谷氨酸转运蛋白的潜在机制和脊髓创伤的潜在干预措施。

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.