Lipid Mediators in Corneal Nerve Regeneration

摘要 角膜神经支配的改变会导致角膜知觉受损、严重干眼和角膜损伤。 可能导致角膜溃疡、融化和穿孔的上皮细胞。这些变化经常发生。 屈光手术后，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、衰老和糖尿病。虽然有一些治疗方法可以缓解严重的干眼症， 目前还没有治疗方法来弥补神经功能的丧失。这项研究项目是以我们的发现为基础的 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 二十二碳烷衍生物神经保护素D1(NPD1)促进角膜手术后神经再生 损害间质神经。我们最近发现：1)PEDF+DHA刺激的角膜也 合成其他二十二碳酸类化合物，其中一种被鉴定为解决素D6(RvD6)；2)用PEDF治疗导致 激活钙非依赖性磷脂酶A2ζ(iPLA2ζ)；以及3)治疗也刺激该基因 分泌到泪液中的神经营养因子和信号素7A(SEMA7A)在角膜中的表达，以及 小鼠三叉神经节(TG)的神经肽。我们的目标将是定义 引导PEDF+DHA刺激角膜神经再生的分子事件。我们的中央 假说是PEDF+DHA，通过特定的二十二碳类化合物，激活选择性基因程序和 调节炎症反应，进而诱导神经再生，从而导致 保持角膜的完整性。我们将使用：1)PEDF受体(PEDF-R)基因敲除(KO)小鼠和 与临床环境相关的角膜损伤活体模型；2)共培养TG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 质谱脂组学分析鉴定和定量DHA在膜中的掺入 磷脂酰胆碱分子种类和DHA-衍生物NPD1、RvD6和其他二十二烷类化合物；4)流动 流式细胞术和我们的免疫染色检测淋巴细胞、树突状细胞、巨噬细胞的含量 和中性粒细胞；5)免疫染色以量化角膜神经；6)眼部行为测量 用于评估再生神经功能的感觉；以及7)分子生物学技术，包括 基因编辑研究不同基因在神经再生信号转导中的作用 PEDF+DHA和二十二碳二烯类化合物。拟议的研究瞄准了新的分子机制，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制剂 屈光手术后的角膜炎和干眼。

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.