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

描述（由申请人提供）：目前美国有超过25万人因脊髓损伤（SCI）而残疾，每年约有12,000例新损伤报告。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家族酶长期上调，可能通过产生活性氧（reactive oxygen species， ROS）参与小胶质/巨噬细胞活化、炎症和组织损伤。这种表达谱与巨噬细胞的侵袭和小胶质细胞的激活一致，形成促炎或M1表型，但与M2或抗炎表型不一致。敲除NOX2异构体可以减少ROS的产生。此外，NOX2的急性药理抑制可减少大鼠脊髓损伤后的病变体积。然而，该酶家族的表达谱目前尚不清楚，NOX2抑制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尚不清楚。因此，我们假设NOX2是脊髓损伤后M1小胶质细胞/巨噬细胞中表达的主要NOX亚型，抑制NOX2将减少慢性炎症并改善恢复，包括轴突萌芽/保留以及运动和自主神经功能。为了验证这一假设，我们提出了三个具体目标。在目的1中，我们将确定脊髓损伤后NOX异构体的表达谱及其与M1和M2表型的相关性。在这个目的中，我们将使用免疫组织化学来鉴定NOX异构体的表达谱及其与M1和M2激活标记的共定位。在目的2中，我们将证明NOX2抑制在小胶质细胞和巨噬细胞中诱导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）和药物抑制方法在急性、亚急性和慢性时间点评估中度脊髓损伤后的炎症。最后，在目的3中，我们将证明延迟给药NOX2抑制剂可改善脊髓损伤后轴突的发芽和保留，并恢复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，以及轴突保留和发芽。提出的研究将阐明氮氧化物在脊髓损伤后的作用，并展示氮氧化物抑制的治疗应用。这些研究的数据将有助于深入了解炎症（包括慢性炎症）在脊髓损伤恢复中的作用。

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. PUBLIC HEALTH RELEVANCE: Over 250,000 people in the United States are currently living with a SCI, and approximately 12,000 new injuries are reported each year. The proposed research study will clarify the role of the NADPH oxidase enzyme (NOX) in the injured spinal cord and demonstrate the therapeutic applications of NOX inhibition. Further, these studies will explore the utility of a delayed treatment approach, capitalizing on the delayed and chronic expression pattern of NOX after SCI, providing the possibility for a more clinically relevant therapeutic approach for SCI treatment.