Multicellular regulation of corneal wound healing

描述（由申请人提供）：角膜是体内神经支配最密集的组织。对上皮中角膜神经的损伤可源自创伤，包括手术、疾病、感染和化学损伤，并可导致干眼症（一种角膜病，表现为恐惧症和疼痛）、角膜溃疡，并最终导致失明。在所有这些情况下，信号分子都由角膜上皮和神经释放，从而产生自体刺激和交叉刺激的可能性。然而，对这种串扰的了解相对较少 在体内平衡或再生过程中。为了评估这些潜在的相互作用，我们开发了神经和上皮细胞的共培养。使用这个模型，我们证明了Ca 2+动员后发生的刺激任一细胞类型。上皮细胞和神经都会释放ATP来响应损伤或激动剂刺激，并激活一个在Ca 2+动员、细胞迁移和疼痛中发挥作用的嘌呤受体家族。动员分为两个阶段， 快速增加和减少，然后是第二组较慢的振荡。用腺苷三磷酸双磷酸酶（一种胞外酶）预处理，去除了最初的反应，而用N-甲基-D-天冬氨酸（NMDA）抑制剂预处理阻止了第二次反应。神经释放谷氨酸，上皮和神经都表达NMDA受体。初步数据表明，环境压力导致的Ca 2+振荡，在远离膜的NMDA受体的本地化的变化，并在细胞-细胞信号的变化减少。使用MATLAB的分析表明细胞间信号传导的不同模式。由于我们的共培养系统没有捕获感觉轴突和角膜上皮细胞之间存在的天然连接的复杂性，我们使用Thy-1-GFP纯合大鼠。初步原位成像研究离体显示，我们可以成像胞质钙动员和荧光感觉神经。我们的目标是确定感觉神经和角膜上皮细胞信号协调创伤诱导的反应及其对迁移的影响的机制。我们推测，神经和上皮细胞释放ATP和神经释放营养因子调节介导伤口修复的特异性嘌呤能和谷氨酸能受体的激活。

DESCRIPTION (provided by applicant): The cornea is the most densely innervated tissue in the body. Damage to corneal nerves in the epithelium can originate from trauma including surgery, disease, infection and chemical injury and can lead to dry eye, a keratopathy, that is exhibited by photophobia and pain, corneal ulcers, and, ultimately, blindness. In all of these conditions, signaling molecules are released by both corneal epithelium and nerves, creating potential for auto- and cross-stimulation. However, relatively little is known about this crosstalk during homeostasis or regeneration. To evaluate these potential interactions, we developed a co-culture of nerves and epithelia. Using this model, we demonstrated that Ca2+ mobilization occurs after stimulation of either cell type. Both epithelia and nerves release ATP in response to injury or agonist stimulation and activate a family of purinoreceptors that play a role in Ca2+ mobilization, cell migration, and pain. The mobilization is divided into two phases with an initial rapid increase and decrease followed by a second set of slower oscillations. Pretreatment with apyrase, an ectoenzyme, removed the initial response, whereas pretreatment with N-methyl-D-aspartate (NMDA) inhibitors prevented the second response. Nerves release glutamate, and both epithelia and nerves express NMDA receptors. Preliminary data indicate that environmental stress causes a decrease in the Ca2+ oscillations, a change in localization of the NMDA receptor away from the membrane, and a change in cell-cell signaling. Analyses using MATLAB demonstrate distinct patterns in cell-cell signaling. Since our co-culture system does not capture the complexity of the native connections that exist between sensory axons and corneal epithelial cells, we used the Thy-1-GFP homozygous rat. Preliminary in situ imaging studies performed ex vivo reveal that we can image cytosolic Ca2+ mobilization and fluorescent sensory nerves. Our goal is to determine mechanisms by which sensory nerves and corneal epithelial cells signal to coordinate the response induced by trauma and its impact on migration. We hypothesize that release of ATP by nerves and epithelium and release of trophic factors by nerves regulates the activation of specific purinergic and glutamatergic receptors that mediate wound repair.