GLUTAMATE TRANSPORTERS--ALTERATIONS IN SPINAL CORD INJUR

描述：(逐字摘自申请者摘要)脊髓损伤不仅对神经元和神经胶质细胞造成最初的损伤，而且由于一系列事件而导致继发性组织损伤。第二种机制之一是由于兴奋性氨基酸的释放显著增加而引起的兴奋性毒性。谷氨酸转运体被认为参与了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具有重要的功能，如兴奋性毒性。脊髓急性损伤时，兴奋性氨基酸的释放增加。这项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体的影响。药理上不同的钠(Na)依赖和氯(Cl)依赖谷氨酸转运体存在于大脑各处。已经从大鼠、兔和人中克隆了钠依赖转运蛋白的亚型。虽然它们在脑中的分布已经确定，但它们在脊髓中的分布有限。在确定谷氨酸转运体对脊髓损伤的影响之前，必须了解它们在正常大鼠体内的分布情况。我们假设依赖钠和依赖氯的谷氨酸转运体将在脊髓中显示出不同的分布，并且这些转运体将因急性脊髓挤压损伤而发生不同的改变。这项拨款申请的具体目的是：1)确定依赖钠和依赖氯的谷氨酸转运体及其亚型在大鼠脊髓横断中的分布。1)用[~3H]-D-天冬氨酸、[~3H]-L-谷氨酸和[~(35)S]-半胱氨酸放射自显影技术定量研究钠依赖和氯依赖转运蛋白在脊髓内的功能解剖分布。1b)通过药理学特征的钠依赖转运抑制剂取代[~3H]-D-天冬氨酸结合的能力来量化钠依赖转运体的亚型分布。1c)钠依赖亚型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脊柱水平确定急性脊髓损伤对转运体的影响。于伤后1、4、12小时按上述三项指标对组织进行评价。通过GFAP免疫组织化学，转运蛋白的改变也将与神经胶质细胞的反应相关。在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体的潜在机制，并可能对脊髓损伤进行干预。

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.