The role of L-selectin in leukocyte recruitment and longer term recovery after spinal cord injury

 描述(由申请人提供)：炎症在脊髓损伤(SCI)后的继发性损伤中起着关键作用。目前，还没有被广泛接受的、FDA批准的减轻脊髓损伤后炎症的治疗药物。L-选择素是一种黏附受体，促进白细胞重新聚集到炎症部位。诺布尔-豪斯林实验室的初步数据显示，L-选择素基因敲除小鼠或接受双氯芬酸治疗的野生型小鼠脊髓损伤后的保存和长期恢复得到改善。双氯芬酸是一种非类固醇抗炎药，通过裂解膜近端区域诱导L-选择素脱落。DFA在脊髓损伤后即刻和3h给药有效，但在8h无效。因此，L-选择素是减少急性脊髓损伤继发性损伤的潜在治疗靶点。然而，L-选择素脱落对特定白细胞亚群募集的影响仍不清楚。这一建议的假设是，L-选择素的脱落，通过膜近端区域的切割，减少了脊髓损伤后促炎症亚群的募集。本研究的目的是确定L-选择素脱落对特定白细胞亚群募集的影响，证实其通过L-选择素脱落实现其有益作用，并为进一步的研究寻找新的候选治疗方法。特定目标1将验证这样的假设，即L-选择素的脱落减少了特定的白细胞亚群对急性损伤脊髓的渗透。在脊髓损伤后72小时进行流式细胞仪检测，野生型(WT)和L-选择素基因敲除(KO)小鼠在伤后3小时接受DFA治疗或空白对照。体内成像将被用来观察急性损伤脊髓血管中免疫标记的白细胞群体的行为。特异靶2将验证DFA的益处是由于L-选择素在膜近端区域的脱落所特有的假说。流式细胞仪将对脊髓损伤后72小时内的L(E)-同一小鼠的白细胞渗透进行定量-在L-选择素的膜近端区域缺乏切割位点，使白细胞对L-选择素脱落具有抵抗力。长期神经功能恢复将通过巴索小鼠评分和栅格行走测试来确定是否在L(E)同一只小鼠中取消了DFA的作用。具体目标3将验证N-苯基邻氨基苯甲酸的假设，与DFA相比，N-苯基邻氨基苯甲酸是一种安全性更好的非甾体抗炎药，可以诱导L-选择素的脱落，并改善脊髓损伤后的长期恢复。WT小鼠在脊髓损伤后3小时给予N-苯基邻氨基苯甲酸治疗。用流式细胞仪和酶联免疫吸附试验对伤后72小时内L-选择素的脱落进行定量。将使用流式细胞仪评估白细胞的渗透，并基于BMS测量长期神经恢复情况。这些共同的结果将有助于揭示L-选择素在脊髓损伤后特定白细胞群招募中的作用，并验证L-选择素在急性脊髓损伤中的治疗策略。这一建议的发现可能适用于其他以破坏性炎症为标志的中枢神经系统疾病。

 DESCRIPTION (provided by applicant): Inflammation plays a critical role in secondary damage after spinal cord injury (SCI). Currently, there is no widely accepted, FDA approved, therapeutic for mitigating inflammation following SCI. L-selectin is an adhesion receptor that facilitates recruitment of leukocytes into sites of inflammation. Preliminary data in the Noble-Haeusslein lab show improved sparing and long-term recovery after SCI in L-selectin knockout or wild- type mice treated with diclofenac acid (DFA), a non-steroidal anti-inflammatory drug (NSAID) that induces L- selectin shedding via cleavage at the membrane proximal domain. DFA was effective when administered immediately and at 3 hours, but not at 8 hours, post-SCI. L-selectin, therefore, represents a potential therapeutic target to reduce secondary damage in the acutely injured spinal cord. However, the effect of L- selectin shedding on the recruitment of specific leukocyte subsets remains undefined. The hypothesis of this proposal is that L-selectin shedding, through cleavage at the membrane proximal domain, reduces the recruitment of pro-inflammatory subsets of leukocytes following SCI. The objectives are to determine the effect of L-selectin shedding on recruitment of specific leukocyte subsets, confirm that DFA achieves it beneficial effects via L-selectin shedding, and identify a new candidate therapeutic for future studies. Specific Aim 1 will test the hypothesis that L-selectin shedding reduces infiltration of specific subsets of leukocytes into the acutely injured spinal cord. Flow cytometry will be performed up to 72 hours post-SCI in wild-type (WT) and L-selectin knockout (KO) mice treated with DFA or a vehicle control at 3 hours post-injury. In vivo imaging will be utilized to observe the behavior of immunolabeled leukocyte populations in vessels in the acutely injured spinal cord. Specific Aim 2 will test the hypothesis that benefit of DFA is specific to shedding of L-selectin at the membrane proximal domain. Leukocyte infiltration will be quantified by flow cytometry up to 72 hours post-SCI in L(E)-Same mice that lack the cleavage site in the membrane proximal domain of L-selectin, rendering leukocytes resistant to L-selectin shedding. Long-term neurological recovery will be measured using the Basso Mouse Scale (BMS) and grid walk tests to determine if the effect of DFA is abolished in L(E)-Same mice. Specific Aim 3 will test the hypothesis that N-phenylanthranalic acid, an NSAID with an improved safety profile compared to DFA, induces L-selectin shedding and improves long-term recovery after SCI. WT mice will be treated with N-phenylanthranalic acid at 3 hours post-SCI. L-selectin shedding will be quantified by flow cytometry and ELISA up to 72 hours post-injury. Leukocyte infiltration will be assessed using flow cytometry and long-term neurological recovery will be measured based on the BMS. The collective results will help uncover the role of L-selectin in recruitment of specific leukocyte populations after SCI and validate L- selectin shedding as a therapeutic strategy in the acutely injured spinal cord. The findings from this proposal may be applicable to other central nervous system disorders marked by damaging inflammation.