Lipid Mediators in Corneal Nerve Regeneration

角膜神经再生中的脂质介质

• 批准号: 9903357
• 负责人: Haydee E.P. Bazan
• 金额: $ 36.5万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903357
• 关键词: Affect; Aging; Antibodies; Axon; Biological Assay; Brain-Derived Neurotrophic Factor; CRISPR/Cas technology; Calcium; Chemical Burns; Clinical; Clinical Research; Coculture Techniques; Cornea; Corneal Diseases; Corneal Injury; Corneal Stroma; Corneal Ulcer; Dendritic Cells; Diabetes Mellitus; Diabetic mouse; Disease; Docosahexaenoic Acids; Epithelial; Epithelial Cells; Epithelium; Esthesia; Event; Fatty Acids; Female; Flow Cytometry; Future; Gender; Gene Expression; Gene Expression Regulation; Genes; Grant; Human; Immune; Impaired wound healing; Impairment; Incidence; Infection; Inflammatory Response; Injections; Injury; Keratoconus; Keratoplasty; Knockout Mice; Knowledge; Lead; Lecithin; Lipids; Lymphocyte; Mediator of activation protein; Membrane; Microfluidics; Modeling; Molecular; Molecular Biology Techniques; Molecular Mechanisms of Action; Multiple Sclerosis; Mus; Natural regeneration; Nerve; Nerve Regeneration; Neurites; Neurons; Neuropeptides; Neurotrophic Keratitis; Omega-3 Fatty Acids; Operative Surgical Procedures; Outcome; PLA2G6 gene; Pathology; Perforation; Phospholipase; Play; Procedures; Production; Publications; Research; Research Project Grants; Role; Semaphorins; Signal Pathway; Sjogren' s Syndrome; Structure of trigeminal ganglion; Surface; Testing; Therapeutic Agents; Tissues; afferent nerve; behavior measurement; blink reflexes; corneal epithelium; corneal regeneration; corneal surgery; density; diabetic; effective therapy; experimental study; eye dryness; functional restoration; immunoregulation; in vivo; in vivo Model; inflammatory modulation; injured; innovation; lipid mediator; macrophage; male; melting; mouse model; nerve supply; neuroprotectin D1; neurotransmission; neurotrophic factor; neutrophil; novel; ophthalmic nerve; pigment epithelium-derived factor; pigment epithelium-derived factor receptor; preservation; programs; protein expression; regenerative; response; restoration; selective expression; sex; tandem mass spectrometry

Abstract Alterations in corneal innervation result in impaired corneal sensation, severe dry eye and damage to the epithelium that may in turn lead to corneal ulcers, melting and perforation. These alterations frequently occur after refractive surgery, cornea transplant, herpetic infection, chemical burns, keratoconus, multiple sclerosis, Sjogren’s syndrome, aging and diabetes mellitus. Although there are treatments to alleviate severe dry eye, there are no therapies to compensate for the loss of innervation. This research project builds upon our finding that pigment epithelium-derived factor (PEDF) plus the w-3 fatty acid docosahexaenoic acid (DHA) or the docosanoid derivative neuroprotectin D1 (NPD1) stimulate nerve regeneration after corneal surgery that damages the stromal nerves. We have recently found that: 1) corneas stimulated with PEDF+DHA also synthesize other docosanoids, one of them identified as resolvin D6 (RvD6); 2) treatment with PEDF results in activation of a calcium independent phospholipase A2ζ (iPLA2ζ); and 3) treatment also stimulates the gene expression in the cornea of neurotrophins and Semaphorin 7A (SEMA7A), which are secreted into tears, and neuropeptides in the trigeminal ganglia (TG) of a mouse model. Our objective will be to define the cascade of molecular events that conduct the corneal nerve regeneration stimulated by PEDF+DHA. Our central hypothesis is that PEDF+DHA, through specific docosanoids, activates selective gene programs and modulates the inflammatory response that in turn, induces the nerve regeneration that leads to preservation of corneal integrity. We will employ: 1) PEDF-receptor (PEDF-R) knockout (KO) mice and in vivo models of corneal injury relevant to the clinical setting; 2) microfluidic chambers for co-culture of TG neurons and corneal epithelial cells to define the molecular mechanism of neurite outgrowth; 3) LC-tandem mass spectrometry lipidomic analysis to identify and quantify the incorporation of DHA in membrane phosphatidylcholine molecular species and DHA-derivatives NPD1, RvD6 and other docosanoids; 4) flow cytometry and our immunostaining assays to determine content of lymphocytes, dendritic cells, macrophages and neutrophils; 5) immunostaining to quantify corneal nerves; 6) behavioral measurements of ocular sensation to assess the functionality of the regenerated nerves; and 7) molecular biology techniques, including gene editing to study the role of the different genes involved in the signaling of nerve regeneration activated by PEDF+DHA and docosanoids. The proposed studies target new molecular mechanisms to understand and treat complications due to corneal nerve damage. Our innovative approach will define agents for neurotrophic keratitis and dry eye after refractive surgery.

摘要 角膜神经支配的改变导致角膜感觉受损、严重干眼症和角膜上皮细胞损伤。 角膜上皮的破坏，可能导致角膜溃疡、融化和穿孔。这些变化经常发生。 在屈光手术、角膜移植、疱疹感染、化学烧伤、圆锥角膜、多发性硬化症 干燥综合征、衰老与糖尿病。虽然有一些治疗方法可以缓解严重的干眼症， 没有治疗方法来补偿神经支配的损失。这个研究项目建立在我们的发现之上 色素上皮衍生因子（PEDF）加上w-3脂肪酸二十二碳六烯酸（DHA）或 二十二烷衍生物神经保护素D1（NPD 1）刺激角膜手术后的神经再生， 会损伤基质神经我们最近发现：1）用PEDF+DHA刺激的角膜也 合成其他二十二烷类化合物，其中一种被鉴定为消退素D 6（RvD 6）; 2）用PEDF处理导致， 钙非依赖性磷脂酶A2 β（iPLA 2 β）的激活;和3）治疗还刺激基因 - 分泌到泪液中的神经营养蛋白和脑信号蛋白7A（SEMA 7A）在角膜中的表达，和 图10显示了小鼠模型的三叉神经节（TG）中的神经肽。我们的目标将是确定级联的 PEDF+DHA刺激角膜神经再生的分子事件。我们的中央 一种假说认为，PEDF+DHA通过特定的二十二烷类化合物激活选择性基因程序， 调节炎症反应，进而诱导神经再生， 保持角膜的完整性。我们将采用：1）PEDF-受体（PEDF-R）敲除（KO）小鼠，并在小鼠中进行基因敲除。 与临床环境相关的角膜损伤的体内模型; 2）用于TG共培养的微流体室 神经元和角膜上皮细胞，以确定轴突生长的分子机制; 3）LC串联 质谱脂质组学分析以鉴定和定量DHA在膜中的掺入 磷脂酰胆碱分子种类和DHA衍生物NPD 1、RvD 6和其他二十二烷类; 4）流动 流式细胞术和我们的免疫染色测定来确定淋巴细胞、树突状细胞、巨噬细胞 和中性粒细胞; 5）免疫染色以量化角膜神经; 6）眼神经的行为测量; 感觉以评估再生神经的功能;以及7）分子生物学技术，包括 基因编辑，以研究参与神经再生信号传导的不同基因的作用， PEDF+DHA和二十二烷类。拟议的研究针对新的分子机制，以了解和 治疗角膜神经损伤引起的并发症。我们的创新方法将定义神经营养剂 屈光手术后角膜炎和干眼症。