Matrix Metalloproteinases and Regeneration after TBI

描述（由申请人提供）：创伤性脑损伤（TBI）后长期功能恢复的程度显然与受损回路内适应性突触可塑性的能力相关。 最近的证据支持细胞外基质蛋白（ECM）和它们的调节性金属蛋白酶（MMPs）在CNS创伤后轴突生长和突触重组中的作用。 鉴于这些分子被发现在脑内区域易受创伤性脑损伤，我们已经开始检查明胶酶和基质溶解素MMP家族的成员在损伤诱导的突触可塑性。 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TBI后长期恢复的成功。 具体来说，我们认为，基质金属蛋白酶控制ECM的解离过程中，退化的终端删除，并随后调节ECM相关分子参与突触的分布。 我们的方法将首先确定MMP的表达和功能活动的时空模式后，单侧内嗅病变（UEC），一种侮辱，诱导海马内反应性突触发生的明确过程。 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TBI模型描述脑创伤后MMP的表达和功能，该模型将过度神经兴奋的肿胀损伤与靶向海马传入神经阻滞的内嗅损伤（TBI+BEC损伤）相结合。 我们已经证明，这种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，我们将检查蛋白质（LM/EM免疫组织化学，Western印迹）和mRNA（RT-PCR，北方印迹和/或原位杂交）表达的选择基质金属蛋白酶（明胶酶A和B;基质溶解素）和相关的ECM底物（胶原酶IV，硫酸软骨素蛋白多糖，enascin）损伤后。 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（凝胶酶谱法和显色酶测定法）。 接下来，我们将确定这些损伤诱导的MMPs/ECM变化是否与突触可塑性的电生理测量（LTP，成对脉冲易化，电流源密度分析）和认知结果（Morris水迷宫性能）的变化相关。 最后，我们将通过以下方式测试MMPs与TBI后突触重组之间的关联：1）应用特异性MMP抑制剂并评估所产生的突触可塑性的程度，以及2）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表达和功能活性。 总之，这些研究将确定MMPs是否在TBI诱发的再生过程中发挥作用，并可能为脑创伤受害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.