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

• 批准号: 9248809
• 负责人: Dylan A. McCreedy
• 金额: $ 6.1万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 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/9248809
• 关键词: Acids; Acute; 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 Deficit; Oxidative Stress; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacology; Play; Population; Prostaglandins; Reactive Oxygen Species; Recovery; Recruitment Activity; 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 candidate; therapeutic target; tool; white matter

 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）后的继发性损伤中起关键作用。目前，没有广泛接受的FDA批准的用于减轻SCI后炎症的治疗剂。L-选择素是一种粘附受体，促进白细胞募集到炎症部位。Noble-Haeusslein实验室的初步数据显示，在用双氯芬酸（DFA）治疗的L-选择素敲除或野生型小鼠中，SCI后的保留和长期恢复得到改善，双氯芬酸（DFA）是一种非甾体抗炎药（NSAID），其通过在膜近端结构域处切割诱导L-选择素脱落。DFA在SCI后立即和3小时给药时有效，但在8小时时无效。因此，L-选择素是一个潜在的治疗靶点，以减少急性损伤的脊髓继发性损伤。然而，L-选择素脱落对特定白细胞亚群募集的影响仍不明确。该建议的假设是，L-选择素脱落，通过在膜近端结构域切割，减少SCI后白细胞促炎亚群的募集。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DFA通过L-选择素脱落实现其有益效果，并为未来的研究确定新的候选治疗剂。特异性目的1将检验L-选择素脱落减少特定白细胞亚群浸润到急性损伤脊髓中的假设。在损伤后3小时，在用DFA或溶剂对照处理的野生型（WT）和L-选择素敲除（KO）小鼠中，将在SCI后72小时内进行流式细胞术。将利用体内成像来观察急性损伤的脊髓中血管中免疫标记的白细胞群体的行为。特异性目的2将检验DFA的益处特异于L-选择素在膜近端结构域的脱落的假设。在L（E）-Same小鼠中，通过流式细胞术定量白细胞浸润，直至SCI后72小时，所述L（E）-Same小鼠在L-选择素的膜近端结构域中缺乏切割位点，使得白细胞对L-选择素脱落具有抗性。将使用Basso小鼠量表（BMS）和网格行走试验测量长期神经恢复，以确定L（E）-Same小鼠中DFA的作用是否消除。具体目标3将检验以下假设：与DFA相比，N-苯基邻氨基苯甲酸（一种安全性改善的NSAID）诱导L-选择素脱落并改善SCI后的长期恢复。WT小鼠将在SCI后3小时用N-苯基邻氨基苯甲酸处理。将通过流式细胞术和ELISA定量L-选择素脱落，直至损伤后72小时。将使用流式细胞术评估白细胞浸润，并基于BMS测量长期神经功能恢复。这些结果将有助于揭示L-选择素在SCI后招募特定白细胞群中的作用，并验证L-选择素脱落作为急性损伤脊髓的治疗策略。这项建议的发现可能适用于以破坏性炎症为标志的其他中枢神经系统疾病。