Lipid Mediators in Corneal Nerve Regeneration

描述（由申请人提供）：角膜神经支配的改变导致角膜感觉受损，严重干眼和上皮损伤，进而可能导致角膜溃疡，融化和穿孔。这些改变通常发生在屈光手术、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减轻严重的干眼症，但没有治疗方法可以补偿神经支配的丧失。本研究项目建立在我们发现色素上皮衍生因子（PEDF）加二十二碳六烯酸（DHA）或二十二碳六烯酸衍生物神经保护素D1 （NPD1）诱导角膜手术损伤间质神经后神经再生的基础上。我们现在的重点是确定PEDF加DHA或NPD1在实验性屈光手术后角膜神经再生的分子机制。我们还在1型单纯疱疹病毒（HSV-1）动物模型中研究了这些介质的生物活性，重点关注受损的敏感性、神经再生和炎症。我们的中心假设是PEDF通过激活胞质磷脂酶A2/细胞外调节激酶（cPLA2/ERK1/2）信号传导释放DHA合成NPD1， NPD1刺激神经生长因子（NGF）表达，进而调节角膜神经再生。除了角膜手术外，1型单纯疱疹病毒感染还会导致敏感性丧失、干眼和角膜病变，从而导致基质和上皮损伤。我们提出验证NPD1诱导疱疹病毒感染后角膜神经敏感性恢复和再生的假设。我们将采用：1)与临床环境相关的屈光手术和HSV-1感染的体内模型；2)三叉神经节神经元与角膜上皮细胞和间质成纤维细胞的原代培养、细胞系培养和共培养，揭示神经突生长的分子机制；3) lc -串联质谱脂质组学分析表征和定量DHA及其衍生物NPD1；4)气体感观法评估角膜对神经再生后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定量角膜上皮、亚基底和间质神经。提出的研究目标是新的机制来理解和治疗角膜神经损伤引起的并发症。我们的创新方法将确定屈光手术和HSV-1感染后神经营养性角膜炎和干眼的潜在药物。

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.