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）是导致死亡和残疾的主要原因。尽管临床研究表明，发育中的大脑特别容易受到伤害，但这种脆弱性的基础尚不清楚。在这里，我们将确定基质金属蛋白酶(MMP)-9是否是早期组织损伤的关键发起者，其活性的调节将赋予早期神经保护并建立有利于大脑发育和认知恢复的环境。明胶酶MMP-9是MMP蛋白水解酶家族的一员，该家族以非活性形式分泌，并通过多种机制在细胞外基质（ECM）中激活。虽然MMPs对正常脑功能至关重要，但过度和不受控制的活动会导致蛋白质水解失调，最终导致细胞损伤/死亡。在这里，我们将确定MMP-9是否启动与中性粒细胞弹性酶活性和中性粒细胞浸润相关的早期自我延续损伤反应。我们假设MMP-9通过使中性粒细胞弹性酶的主要生理抑制剂a1-蛋白酶抑制剂失活来介导早期组织损伤，从而支持中性粒细胞弹性酶介导的神经元损伤和进一步的白细胞募集。利用互补的药理学和遗传学策略，以及过继性转移，我们将研究MMP-9和中性粒细胞弹性酶在中性粒细胞募集信号和介导早期细胞损伤中的协同作用。目的1将验证MMP-9升高导致血脑屏障破坏、神经损伤和白质损伤的假设。目的2将验证MMP-9通过浸润性中性粒细胞传递，促进血脑屏障破坏的假设。Aim 3将确定MMP-9是否使中性粒细胞弹性酶抑制剂a1-蛋白酶抑制剂失活，从而使中性粒细胞弹性酶产生组织损伤和进一步的中性粒细胞募集。目的4将验证急性脑损伤早期明胶酶活性阻断将导致长期结构和行为恢复的假设。为了验证这些假设，我们将使用出生后21天的TBI小鼠模型和几种调节MMP-9和中性粒细胞弹性酶活性的策略。我们将比较脑损伤野生型（WT）小鼠与MMP-9零突变或过表达基质金属蛋白酶-1组织抑制剂的转基因小鼠的早期组织损伤指标，包括屏障功能障碍。MMP-9和中性粒细胞弹性酶在启动自我延续的损伤反应中的相互依赖性将在WT和MMP-9和中性粒细胞弹性酶缺失的动物中使用过继转移技术进行检查。通过最先进的核磁共振成像和全面的行为分析，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，这些研究为理解年轻大脑对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的基础。

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.