Lipid Mediators in Corneal Nerve Regeneration

描述(申请人提供)：角膜神经的改变导致角膜知觉受损，严重干眼和上皮损伤，继而可能导致角膜溃疡、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、干燥综合征和糖尿病之后。虽然有治疗方法可以缓解严重的干眼，但没有治疗方法来弥补神经功能的丧失。这个研究项目建立在我们发现色素上皮衍生因子(PEDF)和二十二碳六烯酸(DHA)或二十二碳六烯酸衍生物神经保护素D1(NPD1)联合诱导角膜手术后神经再生的基础上，这会损害基质神经。我们现在专注于确定PEDF加DHA或NPD1在实验性屈光手术后再生角膜神经的分子机制。此外，我们还在单纯疱疹病毒1型(HSV-1)的动物模型中研究了这些介体的生物活性，重点是敏感性受损、神经再生和炎症。我们的中心假设是PEDF通过激活胞浆磷脂酶A2/细胞外调节的激酶(cPLA2/ERK1/2)信号来释放DHA以合成NPD1，从而刺激神经生长因子(NGF)的表达，进而调节角膜神经再生。除了角膜手术，HSV-1感染还会导致敏感度下降、干眼和角膜病变，这些都可能导致基质和角膜上皮损伤。我们建议验证NPD1在疱疹病毒感染后诱导敏感度恢复和角膜神经再生的假设。我们将采用：1)与临床相关的屈光手术和HSV-1感染的活体模型；2)三叉神经节神经元的原代和细胞系培养以及与角膜上皮细胞和基质成纤维细胞的联合培养，以揭示轴突生长的分子机制；3)LC-串联质谱仪脂体分析以表征和定量DHA及其衍生物NPD1；4)气体感受仪以评估神经再生后角膜对不同刺激方式的敏感性；5)分子生物学技术和我们的免疫染色方法对角膜上皮、基底层和基质神经进行定量。拟议的研究目标是了解和治疗角膜神经损伤引起的并发症的新机制。我们的创新方法将确定神经营养性角膜炎和屈光手术后干眼和HSV-1感染的潜在药物。

DESCRIPTION (provided by applicant): 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, corneal transplant, herpetic infection, chemical burns, keratoconus, multiple sclerosis, Sjogren's syndrome 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 docosahexaenoic acid (DHA) or the docosanoid derivative neuroprotectin D1 (NPD1) induces nerve regeneration after corneal surgery that damages stromal nerves. We now focus on defining the molecular mechanism by which PEDF plus DHA or NPD1 regenerates corneal nerves after experimental refractive surgery. We additionally investigate the bioactivity of these mediators in an animal model of herpes simplex virus type-1 (HSV-1), focusing on compromised sensitivity, nerve regeneration and inflammation. Our central hypothesis is that PEDF acts through a mediated mechanism that activates cytosolic phospholipase A2/extracellular-regulated kinase (cPLA2/ERK1/2) signaling to release DHA for the synthesis of NPD1, which stimulates nerve growth factor (NGF) expression that, in turn, modulates corneal nerve regeneration. In addition to corneal surgery, HSV-1 infections cause lost sensitivity, dry eye and corneal lesions that could lead to stromal and epithelial damage. We propose to test the hypothesis that NPD1 induces recovery of sensitivity and regenerates corneal nerves after herpes viral infection. We will employ: 1) in vivo models of refractive surgery and HSV-1 infection relevant to the clinical setting; 2) primary and cell line cultures and co-cultures of trigeminal ganglion neurons with corneal epithelial cells and stromal fibroblasts to uncover the molecular mechanisms of neurite outgrowth; 3) LC-tandem mass- spectrometry lipidomic analysis to characterize and quantify DHA and its derivative, NPD1; 4) gas esthesiometry to assess corneal sensitivity to different modalities of stimulus after nerve regeneration; and 5) molecular biology techniques and our immunostaining method to quantify the epithelial, sub-basal and stromal corneal nerves. The proposed studies target new mechanisms to understand and treat complications due to corneal nerve damage. Our innovative approach will define potential agents for neurotrophic keratitis and dry eye after refractive surgery and HSV-1 infection.