Trauma to developing brain-injury and repair mechanisms

描述（由申请人提供）：儿童创伤性脑损伤（TBI）是死亡和残疾的主要原因。虽然临床研究表明，发育中的大脑特别容易受到伤害，但这种脆弱性的基础仍然不清楚。在这里，我们将确定是否基质金属蛋白酶（MMP）-9是早期组织损伤的关键启动子，其活性的调节将赋予早期神经保护，并建立一个有利于大脑发育和认知恢复的环境。明胶酶MMP-9是蛋白水解酶MMP家族的成员，其以非活性形式分泌并通过多种机制在细胞外基质（ECM）中活化。虽然MMPs对于正常的脑功能至关重要，但过度和不受控制的活性导致蛋白水解失调，最终导致细胞损伤/死亡。在这里，我们将确定MMP-9是否启动了一个早期的自我持续的损伤反应，这是耦合到中性粒细胞弹性蛋白酶和中性粒细胞浸润的活性。我们假设MMP-9通过灭活α 1蛋白酶抑制剂（中性粒细胞弹性蛋白酶的主要生理抑制剂）介导早期组织损伤，从而支持中性粒细胞弹性蛋白酶介导的神经元损伤并促进白细胞募集。使用免费的药理学和遗传学策略，再加上过继转移，我们将检查MMP-9和中性粒细胞弹性蛋白酶之间的协同作用，在信号中性粒细胞招聘和介导早期细胞损伤。目的1将检验MMP-9升高导致血脑屏障破坏、神经损伤和白色物质损伤的假设。目的2将检验MMP-9由浸润的中性粒细胞传递，促进血脑屏障破坏的假设。目的3将确定MMP-9是否使中性粒细胞弹性蛋白酶的抑制剂α 1-蛋白酶抑制剂失活，从而允许中性粒细胞弹性蛋白酶产生组织损伤和进一步的中性粒细胞募集。目的4将检验急性损伤脑中早期明胶酶活性的阻断将导致长期结构和行为恢复的假设。为了检验这些假设，我们将使用出生后第21天的TBI小鼠模型和几种策略来调节MMP-9和中性粒细胞弹性蛋白酶活性。我们将比较脑损伤野生型（WT）小鼠与MMP-9无效突变或过表达基质金属蛋白酶组织抑制剂-1的转基因小鼠中组织损伤（包括屏障功能障碍）的早期指标。将在WT和MMP-9和中性粒细胞弹性蛋白酶缺失动物中使用过继转移技术检查MMP-9和中性粒细胞弹性蛋白酶在启动自我持续性损伤反应中的相互依赖性。通过最先进的磁共振成像和一系列全面的行为测定，我们将进一步确定明胶酶活性的早期药物阻断是否支持结构恢复并改善长期认知结果。总之，这些研究为了解年轻大脑对TBI的独特脆弱性以及为脑损伤儿童开发最合适的治疗方法提供了重要基础。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) in children is the leading cause of death and disability. Although clinical studies have shown that the developing brain is particularly vulnerable to injury, the basis for this vulnerability remains unclear. Here we will determine if matrix metalloproteinase (MMP)-9 is a key initiator of early tissue damage and that modulation of its activity will confer early neuroprotection and establish an environment that is favorable to brain development and cognitive recovery. The gelatinase MMP-9 is a member of the MMP family of proteolytic enzymes that are secreted in an inactive form and are activated in the extracellular matrix (ECM) by a variety of mechanisms. While MMPs are critical for normal brain functioning, excessive and uncontrolled activity leads to dysregulated proteolysis, culminating in cell injury/death. Here we will determine if MMP-9 initiates an early self-perpetuating injury response that is coupled to activity of neutrophil elastase and neutrophil infiltration. We hypothesize that MMP-9 mediates early tissue damage by inactivating a1-protease inhibitor, the primary physiologic inhibitor of neutrophil elastase, thus supporting neutrophil elastase-mediated neuronal injury and furthering leukocyte recruitment. Using complimentary pharmacologic and genetic strategies, together with adoptive transfer, we will examine the cooperativity between MMP-9 and neutrophil elastase in signaling neutrophil recruitment and mediating early cell injury. Aim 1 will test the hypothesis that elevated MMP-9 contributes to disruption of the blood-brain barrier, neural injury, and white matter damage. Aim 2 will test the hypothesis that MMP-9, conveyed by infiltrating neutrophils, promotes disruption of the blood-brain barrier. Aim 3 will determine if MMP-9 inactivates a1-protease inhibitor, an inhibitor of neutrophil elastase, thus allowing neutrophil elastase to produce tissue injury and further neutrophil recruitment. Aim 4 will test the hypothesis that blockade of early gelatinase activity in the acutely injured brain will result in long-term structural and behavioral recovery. To test these hypotheses we will use a murine model of TBI at postnatal day 21 and several strategies to modulate MMP-9 and neutrophil elastase activity. We will compare early indices of tissue damage including barrier dysfunction in brain injured wildtype (WT) mice to transgenic mice with a null mutation in MMP-9 or overexpress tissue inhibitor of matrix metalloproteinase-1. The interdependency of MMP-9 and neutrophil elastase in initiating a self-perpetuating injury response will be examined using adoptive transfer techniques in WT and MMP-9 and neutrophil elastase null animals. With state-of-the-art magnetic resonance imaging and a comprehensive battery of behavioral assays, we will further determine if early pharmacologic blockage of gelatinase activity supports structural recovery and improves long-term cognitive outcomes. Together, these studies provide an important foundation for understanding the unique vulnerability of the young brain to TBI and for developing the most appropriate therapies for the brain-injured child.