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（由适用提供）：炎症在脊髓损伤（SCI）后的继发性损伤中起着至关重要的作用。目前，尚无广泛接受的，FDA批准的，用于减轻SCI后注射的治疗方法。 L-选择素是一种粘合剂受体，可促进白细胞募集到感染部位。在L-Haeusslein实验室中的初步数据显示，在L-甲基蛋白敲除或用双氯氟乙酰酸（DFA）治疗的L-甲基蛋白敲除或野生型小鼠SCI后，具有改善的保留率和长期恢复，这是一种非甾体类抗炎药（NSAID），可通过cleavage cleavage cleavage cleavage cleavage cleavage the Membrane Proxy dombrane Proxy dombrane Promxy dombrane domainsy dombrane domainsy dombrane domainsy dombrane domainsy。 DFA在SCI后立即和3小时时立即施用，但在8小时时不得施用。因此，L-选择素代表了降低急性损伤脊髓中继发损伤的潜在治疗靶标。但是，L-选择蛋白脱落对特定白细胞子集募集的影响仍然不确定。该提议的假设是，通过在膜代理结构域上的裂解L-选择素脱落，可以减少SCI后白细胞促炎子集的促进子集的募集。这些目的是确定L-选择蛋白脱落对特定白细胞亚群募集的影响，确认DFA通过L-选择素的脱落来实现它的有益作用，并确定一种新的候选疗法以供未来研究。具体目标1将检验以下假设：L-选择素脱落将白细胞的特定子集浸润到急性损伤的脊髓中。 SCI后SCI在野生型（WT）和L-选择蛋白基因敲除（KO）小鼠后3小时后3小时以野生型（WT）和L-选择蛋白敲除（KO）小鼠的身份进行流式细胞仪。体内成像将用于观察急性损伤脊髓中免疫标记的白细胞群体的行为。具体目标2将检验以下假设：DFA的益处是特定于在膜代理域中脱离L-选择素的假设。在L（E）-AME小鼠中，将通过流式细胞仪量化白细胞浸润，该流式细胞仪在L（e） - SAME小鼠中缺乏L-链电蛋白的膜代理结构域中缺乏裂解位点，从而使白细胞具有抗L-SELECTIN SEDDING的白细胞。长期的神经系统恢复将使用Basso小鼠量表（BMS）和网格步行测试测量，以确定在L（e） - Same小鼠中是否消除了DFA的效果。具体目标3将检验以下假设：与DFA相比，N-phenylanythranalic Acid是具有改善安全性的NSAID，可诱导L-选择素脱落并改善SCI后的长期恢复。 SCI后3小时，WT小鼠将用N-苯基义酸治疗。 L-选择素的脱落将通过流式细胞仪和损伤后72小时来量化。将使用流式细胞仪评估白细胞浸润，并根据BMS测量长期神经系统恢复。集体的结果将有助于发现L-选择素在SCI后募集特定白细胞种群中的作用，并验证L-选择蛋白脱落是急性损伤的脊髓中的治疗策略。该提案的发现可能适用于以破坏注射为标志的其他中枢神经系统疾病。