The NADPH oxidase enzyme as a therapeutic target after Spinal Cord Injury

描述(申请人提供)：美国目前有超过250,000人因脊髓损伤(SCI)而致残，每年约有12,000人报告新的伤害。炎症，包括小胶质细胞的激活和巨噬细胞的侵袭，在脊髓损伤后的继发性损伤中起着核心作用。脊髓损伤后，NOX家族的酶被慢性上调，并可能通过产生活性氧(ROS)而促进小胶质/巨噬细胞的激活、炎症和组织损伤。这一表达谱与巨噬细胞的入侵和小胶质细胞的激活进入致炎表型或M1表型一致，但与M2表型或抗炎表型无关。NOX2亚型的敲除会减少ROS的产生。此外，急性药物抑制NOX2减少了大鼠脊髓损伤后的损伤体积。然而，该酶家族的表达谱目前尚不清楚，抑制NOX2对脊髓损伤后慢性炎症、轴突保留或运动或自主神经功能的影响也不清楚。因此，我们假设NOX2是脊髓损伤后M1小胶质细胞/巨噬细胞表达的主要NOX亚型，抑制NOX2将减少慢性炎症，促进恢复，包括轴突发芽/保留以及运动和自主神经功能。为了检验这一假设，我们提出了三个具体目标。在目标1中，我们将确定脊髓损伤后NOX亚型的表达谱及其与M1和M2表型的相关性。为此，我们将使用免疫组织化学方法来确定NOX亚型的表达谱，以及它们与M1和M2激活标记的共存。在目标2中，我们将证明抑制NOX2诱导小胶质细胞和巨噬细胞的M2表型，并减少损伤脊髓的炎症。为此，我们将使用基因敲除(Gp91PHOX)和药物抑制方法来评估急性、亚急性和慢性中度脊髓损伤后的炎症反应。最后，在目标3中，我们将展示延迟给予NOX2抑制剂可以改善轴突萌发和保护，并恢复脊髓损伤后的运动和自主神经功能。利用NOX2特异性抑制剂，我们将评估运动和自主神经功能，以及轴突备用和发芽。这项拟议的研究将阐明脊髓损伤后NOX的作用，并展示NOX抑制的治疗应用。这些研究的数据将提供对炎症，包括慢性炎症，在脊髓损伤恢复中的作用的洞察力。

DESCRIPTION (provided by applicant): Over 250,000 people in the United States are currently living with disabilities caused by spinal cord injury (SCI), and approximately 12,000 new injuries are reported each year. Inflammation, including activation of microglia and invasion of macrophages, plays a central role in the secondary injury observed after SCI. After SCI, the NOX family of enzymes is chronically up-regulated and may contribute to microglial/macrophage activation, inflammation and tissue damage through the production of reactive oxygen species (ROS). This expression profile coincides with the invasion of macrophages and activation of microglia into a pro-inflammatory, or M1, phenotype, but not with the M2, or anti-inflammatory, phenotype. Knockout of the NOX2 isoform can reduce ROS production. Further, acute pharmacological inhibition of NOX2 reduces lesion volume following SCI in rats. However, the expression profile of the family of enzymes is currently unclear and the effect of NOX2 inhibition on chronic inflammation, axonal sparing, or motor or autonomic function after SCI is unknown. Therefore, we hypothesize that NOX2 is the primary NOX isoform expressed in M1 microglia/macrophages after spinal cord injury and that inhibition of NOX2 will reduce chronic inflammation and improve recovery, including axonal sprouting/sparing and motor and autonomic function. To test this hypothesis, we propose three specific aims. In aim 1, we will identify the NOX isoform expression profile and correlation with M1 and M2 phenotype after spinal cord injury. In this aim, we will use immunohistochemistry to identify the expression profile of the NOX isoforms and their co-localization with M1 and M2 activation markers. In aim 2, we will demonstrate that NOX2 inhibition induces an M2 phenotype in microglia and macrophages and reduces inflammation in the injured spinal cord. In this aim, we will assess inflammation after moderate SCI at acute, sub-acute and chronic time points using both genetic knockout (gp91PHOX) and pharmacological inhibition approaches. Finally, in aim 3 we will show that delayed administration of a NOX2 inhibitor improves axonal sprouting and sparing and restores motor and autonomic function after spinal cord injury. Utilizing a NOX2 specific inhibitor, we will assess motor and autonomic function, as well as axonal sparing and sprouting. The proposed research study will clarify the role of NOX after SCI and demonstrate the therapeutic applications of NOX inhibition. The data from these studies will provide insight into the role of inflammation, including chronic inflammation, in recovery from SCI.