Matrix Metalloproteinases and Regeneration after TBI

描述(申请人提供)：创伤性脑损伤(TBI)后长期功能恢复的程度明显与受损回路内自适应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)及其调节性金属蛋白酶(MMPs)在中枢神经系统损伤后轴突生长和突触重组中的作用。鉴于这些分子是在易受脑损伤影响的脑区发现的，我们已经开始研究损伤诱导突触可塑性过程中明胶酶和基质分解酶家族的成员。我们推测，MMPs与其ECM底物在突触重组过程中的相互作用决定了脑损伤后长期康复的成功。具体地说，我们假设MMPs在去掉退变终末的过程中控制ECM的解离，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嗅神经损伤(UEC)后MMP表达和功能活性的时空模式，UEC是一种在海马区诱导明确的反应性突触发生过程的侮辱。在该模型中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模式作为比较的基线，我们将使用大鼠颅脑创伤模型(TBI+BEC INSTULT)结合过度的神经兴奋和内嗅觉损伤(TBI+BEC INSTULT)的海马区定向去传入(TBI+BEC INSULT)来分析脑创伤后基质金属蛋白酶的表达和功能。我们已经证明，这个模型可靠地产生了持久的、适应不良的突触可塑性和严重的长期认知缺陷。首先，我们将检测损伤后部分MMPs(明胶酶A和B；基质分解素)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(光镜/电镜免疫组织化学、Western blots)和mRNA(RT-PCR、Northern印迹和/或原位杂交)的表达。其他实验将确定对蛋白质和信使核糖核酸的影响如何与基质金属蛋白酶活性相关(凝胶酶谱和发色酶分析)。接下来，我们将确定这些损伤诱导的MMPs/ECM的变化是否与突触可塑性的电生理指标(LTP、双脉冲易化、电流源密度分析)和认知结果的变化(Morris水迷宫操作)有关。最后，我们将通过以下方法测试MMPs与脑损伤后突触重组的关系：1)应用特定的基质金属蛋白酶抑制剂，评估突触可塑性产生的程度；2)以NMDA和多巴胺受体为靶点的化合物增强损伤诱导的可塑性，然后评估基质金属蛋白酶的表达和功能活性。总之，这些研究将确定MMPs是否在脑外伤引起的再生过程中发挥作用，并可能确定脑创伤患者的新疗法。

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.