Defining Corneal Schwann cells in Injury

定义损伤中的角膜雪旺细胞

• 批准号: 10308502
• 负责人: ROYCE MOHAN
• 金额: $ 23.86万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308502
• 关键词: Affect; Antibodies; Axon; Blood Vessels; Caliber; Cell Differentiation process; Cell Proliferation; Cells; Confocal Microscopy; Cornea; Corneal Injury; Corneal Stroma; Data; Development; Doxycycline; Drug Targeting; Epithelial; Esthesia; Event; Extracellular Matrix; Extracellular Signal Regulated Kinases; Fibrosis; Foundations; Gene Expression; Genes; Genetic; Grant; Impairment; Injury; Investigation; Keratoplasty; Knowledge; Laboratories; Lead; Learning; Lesion; Mechanics; Mesenchymal; Mitogens; Modeling; Molecular; Mouse Protein; Mus; Myofibroblast; Nature; Nerve; Nerve Endings; Nervous System Trauma; Neuroglia; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Pathway interactions; Peripheral; Peripheral Nervous System; Pharmacology; Phenotype; Phosphotransferases; Play; Procedures; Process; Proteins; Proteolipids; RNA Sequence Analysis; Regulation; Reporter; Reporter Genes; Role; Schwann Cells; Sensory; Signal Pathway; Signal Transduction; Site; Smooth Muscle Actin Staining Method; Spinal Cord; Stains; Testing; Therapeutic; Time; Tissue Stains; Tissues; Topical application; Transgenic Mice; Transgenic Organisms; Validation; Vision; axon growth; axon regeneration; axonal degeneration; cell behavior; cell type; drug development; enhanced green fluorescent protein; functional outcomes; genetic approach; improved; in vivo; inhibitor; injury and repair; innovation; neovascularization; nerve damage; nerve injury; neurofibroma; novel; novel strategies; overexpression; prevent; programs; protein biomarkers; reinnervation; remyelination; repaired; restoration; sciatic nerve; small molecule; therapeutic target; transcriptome; vascular injury

The transparent cornea is a highly innervated tissue and sustains significant nerve damage during common procedures such as corneal transplantation and vision correction. Unfortunately, the restoration of corneal sensory function after damage is usually inadequate due to aberrant and poor regeneration of axons. In vascularized tissues, such as the sciatic nerve and spinal cord Schwann cells (SCs) are known to support axonal regeneration after injury. However, little is known how corneal SCs respond to injury or surgical procedures as this cell types has not previously been investigated. Studies performed in sciatic injury models reveal that both the genetic encoded factors of SCs, as well as the extracellular matrix components govern the axonal repair process. The dedifferentiation of SCs into a repair SCs – a transient cell type – that re-differentiates into a terminal SC is a hallmark of SC-driven mechanisms in axonal regeneration. In the cornea, the bulk of sensory axons are unmyelinated axons, except at the limbus where they are myelinated. It is presumed that lesions of nonmyelinating corneal axons also enlist the support of their respective SCs for axonal regeneration, mirroring similar activities of SCs of injured vascularized tissues. This idea has not been formally tested before, as experimental evidence to support or refute this paradigm is lacking. To learn what genes are expressed specifically in corneal SCs, we performed a single cell RNA sequence analysis of the rabbit cornea and identified the corneal SC transcriptome. With cross-species validation of several SC-specific target proteins in mouse corneas and validation of a transgenic mouse line expressing proteolipid protein 1-enhanced green fluorescent protein (Plp1-eGFP), we demonstrated SC-specific reporter gene expression in vivo. In this exploratory R21 grant, we propose two aims. In specific aim 1, we will exploit the Plp1- eGFP reporter transgenic line in a corneal stromal injury model causing nerve severance and investigate corneal SC to myofibroblast differentiation over the course of axonal degeneration and repair. These studies will help define whether corneal SCs differentiate into myofibroblasts and nature of injury that promotes this aberrant phenotype. In specific aim 2, we will investigate the wingless (Wnt) signaling pathway in corneal SCs, as molecular components of this pathway showed differential high expression in SCs compared to other corneal cells. We plan to investigate how modulation of the Wnt inhibitor Dickkopf-1 (Dkk1) governs corneal axonal regeneration after injury and effects on corneal mechanical sensation. Together, these objectives will help to lay down the foundation to identify novel targets for SC-therapeutics towards improvement of corneal axonal growth and sensory impairment.

透明角膜是高度受神经支配的组织，并且在手术期间维持显著的神经损伤。 常见的手术，如角膜移植和视力矫正。可惜 损伤后角膜感觉功能的恢复通常是不充分的， 轴突再生。在血管组织中，如坐骨神经和脊髓许旺氏 已知神经干细胞（SC）在损伤后支持轴突再生。然而，很少有人知道如何 角膜干细胞对损伤或外科手术有反应，因为这种细胞类型以前没有被 研究了在坐骨神经损伤模型中进行的研究表明， SC的因子以及细胞外基质组分控制轴突修复过程。 干细胞去分化为修复干细胞--一种瞬时细胞类型--再分化为 终末SC是轴突再生中SC驱动机制的标志。在角膜中，大部分 感觉轴突的大多数是无髓鞘轴突，除了在利姆布斯处它们是有髓鞘的。是 假设非髓鞘化角膜轴突的病变也获得了它们各自的支持， SC用于轴突再生，反映了损伤的血管化组织的SC的类似活性。 这一想法之前没有经过正式的测试，作为实验证据来支持或反驳这一点 缺乏范式。为了了解哪些基因在角膜干细胞中特异性表达，我们进行了 对兔角膜进行单细胞RNA序列分析，鉴定出角膜SC 转录组在小鼠中对几种SC特异性靶蛋白进行跨物种验证 角膜和表达蛋白脂质蛋白1-增强的绿色的转基因小鼠系的验证 荧光蛋白（Plp 1-eGFP），我们证明了SC特异性报告基因在体内的表达。 在这个探索性的R21赠款中，我们提出了两个目标。在具体目标1中，我们将利用Plp 1- eGFP报告基因转基因系在角膜基质损伤模型中引起神经切断， 研究角膜SC在轴突变性过程中向肌成纤维细胞的分化， 修复.这些研究将有助于确定角膜SC是否分化为肌成纤维细胞， 促进这种异常表型的损伤性质。在具体目标2中，我们将研究 角膜SC中的无翅（Wnt）信号通路，作为该通路的分子组分 与其他角膜细胞相比，在SC中显示差异性高表达。我们计划调查 Wnt抑制剂Dickkopf-1（Dkk 1）的调节如何控制角膜轴突再生， 损伤和对角膜机械感觉的影响。这些目标将有助于奠定 为确定SC治疗的新靶点奠定基础， 轴突生长和感觉障碍。