Role of Cathepsin S in Dry Eye Associated Neuropathic Pain

组织蛋白酶 S 在干眼相关神经性疼痛中的作用

• 批准号: 10527158
• 负责人: Maria C Edman
• 金额: $ 24.75万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527158
• 关键词: Affect; Afferent Neurons; Antigen Presentation; Area; Behavior; Benzalkonium Chloride; Binding; Brain Stem; C57BL/6 Mouse; CX3CL1 gene; Caspase; Categories; Cathepsins; Cells; Clinical; Complex; Cornea; Development; Diagnosis; Disease; Dry Eye Syndromes; Dryness; Environmental Risk Factor; Environmental Wind; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Etiology; Event; Exhibits; Exposure to; Eye; Fractalkine; Functional disorder; Ganglia; Gene Expression; Genomics; Hyperalgesia; Immune; Immune response; Immune system; Immunofluorescence Immunologic; Inflammation; Inflammatory; Injury; Knock-out; Knockout Mice; Lacrimal Apparatus; Location; MHC Class II Genes; Measurement; Mechanics; Mediating; Mediator of activation protein; Microglia; Modeling; Molecular; Mus; Nerve; Neuraxis; Neurobiology; Neurons; Nociception; PAR-2 Receptor; Pain management; Pathway interactions; Patients; Peptide Hydrolases; Peripheral; Peripheral Nerves; Peripheral nerve injury; Persons; Play; Process; Quality of life; Quantitative Reverse Transcriptase PCR; Regulation; Role; Series; Severities; Signs and Symptoms; Site; Spinal Cord; Symptoms; T-Cell Activation; Temperature; Therapeutic; Therapeutic Effect; Tissues; Topical application; Trigeminal System; Trigeminal nerve structure; Tumor-infiltrating immune cells; Vanilloid; Visual Acuity; Wild Type Mouse; allodynia; behavior test; carboxypeptidase C; chemokine; comorbidity; corneal epithelium; cytokine; eye dryness; ganglion cell; inhibitor; insight; mRNA sequencing; meibomian gland; mouse model; nerve injury; nerve supply; neuroinflammation; neurosensory; novel; ocular pain; ocular surface; pain reduction; pain sensation; painful neuropathy; patient subsets; phenylalanylleucine; preclinical study; prevent; protein expression; receptor; reflectance confocal microscopy; relating to nervous system; response; single cell sequencing; somatosensory; therapeutic target; transcriptome; transcriptomics

Dry Eye (DE) is a prevalent disorder of the ocular surface, causing ocular pain and discomfort, affecting millions of people worldwide. The DE diagnosis includes signs and symptoms of ocular dryness that may be caused by a range of underlying conditions. However, many symptoms of DE correlate poorly with clinical signs, and a subset of patients exhibit severe symptoms with few clinical signs. In these patients, corneal neuropathic pain (NP) may be a factor. The neurobiological events leading to NP in DE are poorly understood. Immune-neural interactions occurring after injury to a peripheral tissue, where the immune response modulates neural responsiveness and nociception, are essential in the development of NP. In NP conditions outside of the eye, Cathepsin S (CTSS), a protease with critical functions in inflammation, is crucial regulator of NP. In these conditions, CTSS acts both at the site of injury and in the CNS. It is our hypothesis that CTSS modulates corneal NP, acting at multiple sites, peripherally at the ocular surface, the trigeminal nerve ganglion (TG) and in the CNS, constituting a viable therapeutic target for the treatment of corneal NP. Three specific aims are proposed: Aim 1. To determine the role of CTSS and its effectors in peripheral and central neuroinflammatory processes in a mouse model of DE with corneal nerve injury. Ocular surface inflammation and nerve injury will be induced in C57BL/6 wild type (WT) mice by topical administration of Benzalkonium chloride (BAC). The involvement of CTSS and known downstream mediators of neuropathic pain at the ocular surface, the TG and trigeminal brain stem complex (TBSC) will be explored through analysis of changes in gene and protein expression. Aim 2. To validate the involvement of CTSS in development of mechanical hyperalgesia and allodynia in WT and CTSS knockout mice with induction of DE and NP, and to evaluate the therapeutic potential of topical and systemic CTSS inhibition. The response to BAC injury in WT mice will be compared to CTSS knockout mice (CTSS-/-), assessing corneal nerve integrity using in vivo confocal microscopy and pain sensation with a series of behavioral tests. The therapeutic potential of topical or systemic CTSS inhibition will be assessed utilizing the CTSS inhibitor, Z-Phe-Leu-COCHO (Z-FL). Aim 3. To conduct a discovery analysis of alterations in the transcriptome of the TG and TBSC after corneal nerve injury, utilizing spatial transcriptomics and NextGen mRNA sequencing. The 10x Genomics Visium Gene Expression Solution platform, allowing near single-cell sequencing while retaining cell localization information, will enable more specific information about the transcriptome in distinct areas of the TG and TBSC with induction of DE and NP. At the conclusion of this project, we will have determined the role of CTSS in corneal and central neuroinflammation and pain sensation and evaluated whether its inhibition has a therapeutic effect on NP. We will also have completed an unbiased analysis of transcriptome changes in TG and TBSC after corneal nerve injury, promoting discovery of new potential therapeutic targets for the treatment of pain and discomfort in DE.

干眼症是一种常见的眼表疾病，会引起眼部疼痛和不适，影响数百万人 全世界。DE的诊断包括眼干的体征和症状，这可能是由一系列潜在的 条件。然而，DE的许多症状与临床体征的相关性很差，部分患者表现出严重的 症状少，临床体征少。在这些患者中，角膜神经性疼痛(NP)可能是一个因素。神经生物学 导致DE中NP的事件知之甚少。周围组织损伤后发生的免疫-神经相互作用， 其中免疫反应调节神经反应和伤害性，在NP的发展中是必不可少的。 在眼球以外的NP条件下，组织蛋白酶S(Cts)是一种在炎症中具有关键功能的蛋白酶，是至关重要的。 NP的调节器。在这种情况下，CTSS既在损伤部位起作用，又在中枢神经系统起作用。我们的假设是CTSS 调节角膜NP，作用于多个部位，外周作用于眼表、三叉神经节(TG)和 在中枢神经系统，构成了治疗角膜NP的一个可行的治疗靶点。提出了三个具体目标：目标 1.确定CTSS及其效应物在小鼠外周和中枢神经炎性过程中的作用 DE合并角膜神经损伤模型的建立。C57BL/6野生型将诱发眼表炎症和神经损伤 (WT)小鼠局部给予苯扎氯铵(BAC)。CTSS和已知下游的参与 本课程将探讨眼表、三叉神经节和三叉神经脑干复合体(TBSC)的神经病理性疼痛的介体。 通过分析基因和蛋白质表达的变化。目的2.验证CTSS参与开发的情况 用DE和NP诱导WT和CTSS基因敲除小鼠的机械性痛觉过敏和痛觉过敏，并评价 局部和全身CTSS抑制的治疗潜力。将比较WT小鼠对BAC损伤的反应 对CTSS基因敲除小鼠(CTSS-/-)，用体内共聚焦显微镜和痛觉检测角膜神经的完整性 进行了一系列的行为测试。局部或全身性CTSS抑制的治疗潜力将通过 CTSS抑制剂Z-Phe-Leu-COCHO(Z-FL)。目的3.进行转录组改变的发现分析 利用空间转录和NextGen基因测序技术，对角膜神经损伤后的TG和TBSC进行分析。10倍 基因组学维西姆基因表达解决方案平台，允许近单细胞测序，同时保持细胞定位 信息，将使关于转录组的更具体的信息在TG和TBSC的不同区域 诱发DE和NP。在本项目结束时，我们将确定CTSS在角膜和中央的作用 并评价其抑制对神经性神经炎的治疗作用。我们还将 完成了对角膜神经损伤后TG和TBSC转录组变化的无偏分析，促进了 发现治疗DE疼痛和不适的新潜在治疗靶点。