Matrix Metalloproteinases and Regeneration after TBI

描述（由申请人提供）：创伤性脑损伤（TBI）后的长期功能恢复程度显然与损伤回路中适应性突触可塑性的能力有关。最近的证据支持细胞外基质蛋白（ecm）及其调节金属蛋白酶（MMPs）在中枢神经系统创伤后的神经突生长和突触重组中的作用。考虑到这些分子存在于易受创伤性脑损伤的大脑区域，我们已经开始研究明胶酶和基质溶酶MMP家族成员在损伤诱导的突触可塑性中的作用。我们假设MMPs和它们的ECM底物在突触重组过程中的相互作用决定了脑外伤后长期恢复的成功。具体来说，我们假设MMPs在去除退化末端过程中控制ECM解离，并随后调节参与突触发生的ECM相关分子的分布。我们的方法将首先确定单侧内腔损伤（UEC）后MMP表达和功能活动的时空模式，UEC是一种损伤，在海马内诱导反应性突触发生的明确过程。该模型诱导的神经元可塑性导致突触结构和功能的适应性恢复。以UEC模式为比较基线，我们将使用大鼠TBI模型，该模型结合了撞击损伤的过度神经兴奋和内鼻病变的靶向海马神经传递障碍（TBI+BEC损伤），分析脑外伤后MMP的表达和功能。我们已经证明，这种模式可靠地产生了持续的、不适应的突触可塑性和严重的长期认知缺陷。首先，我们将检测损伤后选择的MMPs（明胶酶A和B、基质溶解素）及其相关的ECM底物（胶原酶IV、硫酸软骨素蛋白多糖、enascin）的蛋白（LM/EM免疫组化、Western blots）和mRNA （RT-PCR、Northern blot和/或原位杂交）表达。其他实验将确定对蛋白质和mRNA的影响如何与MMP酶活性相关（凝胶酶谱和显色酶测定）。接下来，我们将确定这些损伤引起的MMPs/ ecm变化是否与突触可塑性电生理测量（LTP、成对脉冲促进、电流源密度分析）和认知结果（Morris水迷宫表现）的变化有关。最后，我们将通过：1)应用特异性的MMP抑制剂并评估突触可塑性的产生程度，以及2)使用靶向NMDA和多巴胺受体的化合物增强损伤诱导的可塑性，然后评估MMP的表达和功能活性，来测试MMP与脑外伤后突触重组之间的关系。总之，这些研究将确定MMPs是否在TBI引起的再生过程中发挥作用，并可能为脑外伤患者确定新的治疗方法。

DESCRIPTION (provided by applicant): The extent of long-term functional recovery following traumatic brain injury (TBI) is clearly associated with the capacity for adaptive synaptic plasticity within injured circuitry. Recent evidence supports a role for extracellular matrix proteins (ECMs) and their regulatory metalloproteinases (MMPs) in neurite growth and synaptic reorganization after CNS trauma. Given that these molecules are found within brain regions vulnerable to TBI, we have begun to examine members of the gelatinase and stromelysin MMP families during injury-induced synaptic plasticity. We hypothesize that the interaction of MMPs and their ECM substrates during synaptic reorganization determines the success of long-term recovery following TBI. Specifically, we posit that MMPs control ECM dissociation during removal of degenerating terminals, and subsequently regulate distribution of ECM associated molecules involved with synaptogenesis. Our approach will first identify the spatio-temporal pattern of MMP expression and functional activity after unilateral entorhinal lesion (UEC), an insult which induces the well-defined process of reactive synaptogenesis within the hippocampus. The neuronal plasticity induced in this model results in adaptive restoration of synaptic structure and function. With the UEC pattern as a baseline for comparison, we will profile MMP expression and function after brain trauma using the rat TBI model which combines excessive neuroexcitation of percussive injury with targeted hippocampal deafferentation of entorhinal lesion (TBI+BEC insult). We have shown that this model reliably produces a persistent, maladaptive synaptic plasticity and severe long-term cognitive deficits. Initially, we will examine both protein (LM/EM immunohistochemistry, Western blots) and mRNA (RT-PCR, Northern blot and/or in situ hybridization) expression for select MMPs (gelatinases A and B; stromelysin) and their associated ECM substrates (collagenase IV, chondroitin sulfated proteoglycan, enascin) after injury. Additional experiments will determine how effects on protein and mRNA are correlated with MMP enzyme activity (gel zymography and chromogenic enzyme assay). Next, we will establish whether these injury-induced changes in MMPs/ECMs are associated with alterations in electrophysiological measures of synaptic plasticity (LTP, paired-pulse facilitation, current-source-density analysis) and changes in cognitive outcome (Morris Water Maze performance). Finally we will test the association between MMPs and synaptic reorganization following TBI by: 1) applying specific MMP inhibitors and assessing the extent of synaptic plasticity generated, and 2) enhancing injury-induced plasticity with compounds targeting NMDA and dopamine receptors and then assessing MMP expression and functional activity. Together, these studies will establish whether or not MMPs play a role in regenerative processes evoked by TBI and potentially identify novel therapies for brain trauma victims.