Trauma to developing brain-injury and repair mechanisms

描述(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仍不清楚。在这里，我们将确定基质金属蛋白酶-9是否是早期组织损伤的关键启动者，其活性的调节将提供早期的神经保护，并建立有利于大脑发育和认知恢复的环境。明胶酶MMP9是明胶酶家族中的一员，以非活性形式分泌，通过多种机制在细胞外基质(ECM)中被激活。虽然MMPs对正常的大脑功能至关重要，但过度和不受控制的活动会导致蛋白分解的失调，最终导致细胞损伤/死亡。在这里，我们将确定基质金属蛋白酶-9是否启动了一种早期的自持性损伤反应，这种反应与中性粒细胞弹性蛋白酶的活性和中性粒细胞的渗透有关。我们推测，基质金属蛋白酶-9通过失活中性粒细胞弹性蛋白酶的主要生理抑制因子A1-蛋白酶抑制物，从而支持中性粒细胞弹性蛋白酶介导的神经元损伤和进一步的白细胞募集，从而介导早期的组织损伤。利用互补的药理学和遗传学策略，结合过继转移，我们将检验基质金属蛋白酶-9和中性粒细胞弹性蛋白酶在信号转导中性粒细胞募集和介导早期细胞损伤方面的协同性。目的1将验证这一假设，即升高的基质金属蛋白酶-9有助于血脑屏障的破坏、神经损伤和脑白质损伤。目的2将验证一种假设，即由渗入的中性粒细胞传递的基质金属蛋白酶-9促进血脑屏障的破坏。目的3将确定基质金属蛋白酶-9是否使中性粒细胞弹性蛋白酶的抑制物A1-蛋白酶抑制物失活，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将验证这样的假设，即阻断急性损伤大脑中早期明胶酶活性将导致长期的结构和行为恢复。为了验证这些假设，我们将使用出生后第21天的小鼠脑损伤模型和几种策略来调节基质金属蛋白酶-9和中性粒细胞弹性蛋白酶的活性。我们将比较包括脑损伤野生型(WT)小鼠的屏障功能障碍在内的组织损伤的早期指标与基质金属蛋白酶-9零突变或过度表达基质金属蛋白酶组织抑制物-1的转基因小鼠。在WT、MMP9和中性粒细胞弹性蛋白酶缺失的动物中，将使用过继转移技术来研究MMP9和中性粒细胞弹性蛋白酶在启动自持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分析，我们将进一步确定早期药物阻断明胶酶活性是否支持结构恢复和改善长期认知结果。总而言之，这些研究为了解年轻大脑对脑外伤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。 公共卫生相关性：虽然创伤性脑损伤(TBI)是儿童后天性脑损伤和发病率最常见的原因，并对认知发展产生不利影响，但在了解如何最好地支持康复过程方面进展甚微。在脑外伤的小鼠模型中，利用遗传学和药理学方法，我们将确定未受抑制的基质金属蛋白酶-9指导的蛋白分解如何建立不利于恢复的环境，以及早期阻断这一活动是否支持认知恢复。这些研究为开发针对脑损伤儿童的基质金属蛋白酶靶向治疗奠定了基础。

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. PUBLIC HEALTH RELEVANCE: Although traumatic brain injury (TBI) is the most frequent cause of acquired brain injury and morbidity in children and adversely impacts cognitive development, there has been little progress toward understanding how best to support recovery processes. Using genetic and pharmacologic approaches in a murine model of TBI, we will determine how unchecked, matrix metalloproteinase (MMP)-9 directed proteolysis establishes an environment that is unfavorable to recovery and if early blockade of this activity supports cognitive recovery. These studies establish the basis for developing a MMP-targeted therapeutic for the brain-injured child.