L-selectin shedding as a novel therapeutic strategy to mitigate acute secondary damage after spinal cord injury

项目摘要/摘要 炎症在脊髓损伤(SCI)后继发性组织损伤中起关键作用，然而，没有 被广泛接受的治疗方法，用于减轻损伤脊髓中的破坏性炎症事件。L--选择 是免疫细胞上的一种黏附和信号受体，最近被证明是一种关键的 脊髓损伤后的长期神经缺陷。FDA批准的双氯芬酸对L-选择素功能的干扰作用 非类固醇抗炎药可诱导L-选择素脱落，改善组织储备和 在脊髓损伤后3小时给药，可获得长期恢复。因此，L-选择素的脱落代表了一种 潜在的治疗策略，以减轻与急性炎症相关的损害。然而，具体的 L-选择素减轻继发性组织损伤的机制尚不清楚。L-选择素已经 显示促进中性粒细胞的破坏性效应功能，中性粒细胞是人类中最丰富的免疫细胞类型 血液首先大量侵入受伤的脊髓。这一提议的假设是，L-- 选择素脱落法降低中性粒细胞及相关次级组织的致病活性 脊髓损伤后的损害。本工作的目的是确定L-选择素脱落对激活的影响 进一步阐明中性粒细胞在脊髓损伤后继发性组织损伤中的作用， 并确定静脉注射是否延长了双氯芬酸的治疗窗口。《特定目标1》将测试 L-选择素脱落降低中性粒细胞毒性效应功能激活的假说 有髓鞘的存在。髓鞘可作为L-选择素的丰富配体，并可能加重细胞毒效应 中性粒细胞的功能。用不能释放L-选择素的小鼠(L(E)小鼠)和双氯芬酸治疗的WT小鼠， 体外研究髓鞘对L-选择素脱失对中性粒细胞效应功能的影响 以及在严重受伤的脊髓中。《特定目标2》将检验中性粒细胞 主要的免疫细胞类型，其破坏性功能可通过L-选择素的脱落而减轻。早期中性粒细胞 将在L(E)小鼠和用双氯芬酸治疗的WT小鼠上进行耗竭研究，以确定 L-选择素通过延缓致病作用减少继发性损伤和神经功能缺失 中性粒细胞活动。具体目标3将检验静脉注射双氯芬酸可导致 L-选择素对血液中中性粒细胞的快速释放，延长了机会之窗。长期的 神经恢复和组织保留将在延迟静脉注射后进行评估 WT小鼠双氯芬酸。双氯芬酸治疗也将在L(E)小鼠身上进行评估，以确认该治疗方法 作用机制主要是通过L-选择素的脱落。集体结果将有助于揭开L的角色-- 选择素脱失与中性粒细胞在脊髓损伤后继发性损伤中的作用及L-选择素脱失作为脊髓损伤后继发性损伤机制的验证 治疗的目标是改善长期的神经康复。这项提案的结果也将是 适用于减轻在其他中枢神经系统损伤或紊乱中观察到的破坏性炎症。

Project Summary/Abstract Inflammation plays a critical role in secondary tissue damage after spinal cord injury (SCI), however, there is no widely accepted therapeutic for mitigating destructive inflammatory events in the injured spinal cord. L-selectin is an adhesion and signaling receptor on immune cells that has been recently shown to be a critical mediator of long-term neurological deficits following SCI. Disrupting L-selectin function with diclofenac, an FDA-approved non-steroidal anti-inflammatory drug (NSAID) that induces L-selectin “shedding”, improves tissue sparing and long-term recovery when administered by 3 hours post-SCI. L-selectin shedding, therefore, represents a potential therapeutic strategy to mitigate damage associated with acute inflammation. However, the specific mechanisms through which L-selectin attenuates secondary tissue damage remain unclear. L-selectin has been shown to promote destructive effector functions in neutrophils, the most abundant immune cell type in human blood and first to invade the injured spinal cord in large numbers. The hypothesis of this proposal is that L- selectin shedding reduces the pathogenic activities of neutrophils and associated secondary tissue damage after SCI. The objectives of this work are to determine the effect of L-selectin shedding on the activation of neutrophil effector functions, further elucidate the role of neutrophils in secondary tissue damage after SCI, and determine if intravenous delivery extends the therapeutic window for diclofenac. Specific Aim 1 will test the hypothesis that L-selectin shedding reduces the activation of cytotoxic neutrophil effector functions in the presence of myelin. Myelin can serve as an abundant ligand for L-selectin and may exacerbate cytotoxic effector functions in neutrophils. Using mice that cannot shed L-selectin (L(E) mice) and WT mice treated with diclofenac, the effect of L-selectin shedding on neutrophil effector functions will be quantified in vitro in response to myelin exposure as well as in the acutely injured spinal cord. Specific Aim 2 will test the hypothesis that neutrophils are the primary immune cell type whose destructive functions are mitigated by L-selectin shedding. Early neutrophil depletion will be investigated in L(E) mice and in WT mice treated with diclofenac to determine the extent to which L-selectin shedding reduces secondary damage and neurological deficits by attenuating pathogenic neutrophil activities. Specific Aim 3 will test the hypothesis that intravenous delivery of diclofenac can induce rapid shedding of L-selectin on neutrophils in the blood and extend the window of opportunity. Long-term neurological recovery and tissue sparing will be assessed following delayed intravenous administration of diclofenac in WT mice. Diclofenac treatment will also be assessed in L(E) mice to confirm that the therapeutic mechanisms of action is through L-selectin shedding. The collective results will help uncover the roles of L- selectin shedding and neutrophils in secondary damage after SCI and validate L-selectin shedding as a therapeutic target to improve long-term neurological recovery. The findings from this proposal will also be applicable to attenuating damaging inflammation observed in other central nervous system injuries or disorders.