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

L-选择素在脊髓损伤后白细胞募集和长期恢复中的作用

基本信息

批准号：
9124572
负责人：
Dylan A. McCreedy
金额：
$ 5.8万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9124572
关键词：
Acids Adhesions Animals Anti-Inflammatory Agents Anti-inflammatory Area Attenuated Behavior Blood Bolus Infusion Cell Adhesion Molecules Cell surface Central Nervous System Diseases Data Diclofenac Dose Enzyme-Linked Immunosorbent Assay Euthanasia Event FDA approved Filament Flow Cytometry Future Harvest Hour Infiltration Inflammation Inflammatory Injury Knock-in Mouse Knock-out Knockout Mice L-Selectin Lectin Lesion Leukocyte Rolling Leukocyte Trafficking Leukocytes Measures Membrane Metalloproteases Methods Mus Mutant Strains Mice Nervous System Physiology Neurologic Oxidative Stress Patients Peptide Hydrolases Pharmaceutical Preparations Play Population Prostaglandins Reactive Oxygen Species Recovery Rehabilitation therapy Reporting Research Resistance Role Safety Site Spinal Cord Spinal cord injury Testing Therapeutic Time Toxic effect Walking Wild Type Mouse Work adhesion receptor antibody conjugate base effective therapy fluorophore improved in vivo imaging injured migration monocyte neurological recovery neutrophil novel novel therapeutics painful neuropathy public health relevance receptor therapeutic target tool white matter

项目摘要

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.

描述（由申请人提供）：炎症在脊髓损伤（SCI）后的继发性损伤中发挥着关键作用，目前，尚无广泛接受的 FDA 批准的用于减轻 SCI 后炎症的治疗方法，L-选择素是一种促进粘附的受体。 Noble-Haeusslein 实验室的初步数据显示，L-选择素敲除或野生型脊髓损伤后，白细胞募集到炎症部位的情况有所改善。双氯芬酸（DFA）是一种非甾体类抗炎药（NSAID），通过近膜结构域的裂解诱导 L-选择素脱落，DFA 型小鼠在立即给药和 3 小时后有效，但在 8 小时后无效。因此，L-选择素代表了减少急性损伤脊髓继发性损伤的潜在治疗靶标。然而，L-选择素脱落对特异性募集的影响。该提议的假设是，L-选择素脱落，通过近膜域的裂解，在 SCI 后募集促炎性白细胞亚群。招募特定的白细胞亚群，确认 DFA 通过 L-选择素脱落实现有益效果，并为未来的研究确定一种新的候选治疗方法，该假设将检验以下假设： L-选择素脱落减少了特定白细胞亚群对急性损伤脊髓的浸润，将在 SCI 后 72 小时内对用 DFA 或 L-选择素治疗的野生型 (WT) 和 L-选择素敲除 (KO) 小鼠进行流式细胞术。损伤后 3 小时的载体对照将用于观察急性损伤脊髓血管中免疫标记白细胞群的行为。假设 DFA 的益处特定于膜近端区域的 L-选择素脱落，将在 SCI 后 72 小时内通过流式细胞术对膜上缺乏裂解位点的 L(E)-Same 小鼠进行定量。 L-选择素的近端结构域，使白细胞对 L-选择素脱落具有抵抗力，将使用 Basso 小鼠量表 (BMS) 和网格来测量长期神经恢复。步行测试以确定 DFA 的作用是否在 L(E)-Same 小鼠中被消除，Specific Aim 3 将测试 N-苯邻蒽酸（一种与 DFA 相比安全性更高的 NSAID）会诱导 L-选择素脱落的假设。 SCI 后 3 小时用 N-苯邻氨基苯甲酸治疗，可改善 SCI 后的长期恢复，通过流式细胞术对 L-选择素脱落进行定量。将使用流式细胞术评估损伤后 72 小时内的白细胞浸润，并根据 BMS 测量长期神经功能恢复情况，这些结果将有助于揭示 L-选择素在招募特定白细胞群中的作用。 SCI 后并验证 L-选择素脱落作为急性损伤脊髓的治疗策略。该提案的发现可能适用于以破坏性炎症为特征的其他中枢神经系统疾病。