Underpinnings of corneal innervation: anatomical, molecular, and functional studies of corneal sensory afferents in physiologic and pathologic states

项目摘要 眼表(OS)中丰富的感觉传入网络，由三叉神经的眼支供应 神经执行多种生理功能，包括感觉、调节各种反射和 营养和生长因子的分泌。在OS结构中，角膜是 身体。因此，角膜/OS神经功能障碍被证明是一系列疾病的基础， 包括创伤、感染、代谢失衡和屈光手术等治疗干预。 角膜疾病在美国仍然是一个主要的健康问题，角膜神经功能障碍导致 许多此类疾病，如神经营养性角膜炎、角膜神经性疼痛和干眼病(DED)。 仅DED一项就被证明影响高达75%的特定人口，导致质量显著下降 寿命的延长和视觉模糊风险的增加。因此，一个全面的表征 角膜感觉神经对于理解导致OS疾病的病理生理学至关重要。为了满足这一点 挑战和响应关于OS神经支配的FOA RFA-EY-21-004，我们组装了一个 具有很强的协作记录和互补专业知识的跨学科团队，包括Eyes 疾病模型和伤口愈合(Lee)；疼痛和哺乳动物的躯体感觉系统(Luo)；活体 角膜神经纤维成像和再生(Rompolas)；单细胞转录/表观基因组学和 相关计算分析(Wu)；以及神经生理学和数据分析/机器学习(Ding)。我们 提出将先进的成像方法、新颖的单细胞多组学和尖端鼠标相结合 遗传模型执行三个层次的分析：1)形态-使用单个神经元遗传标记，我们将 通过可视化解决角膜感觉神经元的精细形态、空间组织和连通性 角膜和脑干/脊髓中的轴突树枝。我们还将研究个人之间的互动 感觉传入和角膜上皮细胞。2)分子-我们将进行整合的单细胞转录 和表观基因组学来询问亚型特异性标记基因的表达和表观遗传格局 角膜感觉神经元。3)功能性-我们将对角膜感觉进行实时生理分析 采用体内双光子钙离子成像技术。进一步阐明感觉神经在OS中的作用 病理生理学，我们将确定它们的形态、分子特征和反应性是如何改变的 一种手术诱导的DED模型。此外，我们还将实现一种新颖的高速成像和机器学习 用于量化DED模型中对角膜感觉刺激和自发行为的诱发反应的平台。 综上所述，我们的研究将生成一个全面的数据集，准备进行整合，以填补关键的 该领域的知识差距，为未来的研究奠定坚实的基础，并为更多领域的发展提供信息 治疗OS疾病的精确治疗策略。

PROJECT ABSTRACT A rich network of sensory afferents in the ocular surface (OS), supplied by the ophthalmic branch of the trigeminal nerve, performs a multitude of physiological functions, including sensation, regulation of various reflexes, and secretion of trophic and growth factors. Of the OS structures, the cornea is the most highly innervated tissue in the body. As such, dysfunction of the corneal/OS nerves has been shown to underlie a wide range of diseases, including trauma, infections, metabolic imbalances, and therapeutic interventions such as refractive surgeries. Corneal diseases continue to be a major health problem in the US, and corneal nerve dysfunction contributes to many of these disorders, such as neurotrophic keratitis, corneal neuropathic pain, and dry eye disease (DED). DED alone has been shown to impact up to 75% of a given population, causing significant reduction in quality of life and increased risk for visually debilitating opacities. Therefore, a comprehensive characterization of corneal sensory nerves is critical for understanding the pathophysiology leading to OS diseases. To meet this challenge and in response to the FOA RFA-EY-21-004 on OS innervation, we have assembled an interdisciplinary team with a strong collaborative track record and complementary expertise, including eye disease models and wound healing (Lee); pain and the mammalian somatosensory system (Luo); intravital corneal nerve fiber imaging and regeneration (Rompolas); single-cell transcriptomics/epigenomics and related computational analyses (Wu); and neurophysiology and data analysis/machine learning (Ding). We propose to combine advanced imaging approaches, novel single-cell multi-omics, and cutting-edge mouse genetic models to perform three levels of analysis: 1) Morphology - using single neuron genetic labeling, we will resolve the fine morphology, spatial organization, and connectivity of corneal sensory neurons by visualizing axonal arborization in the cornea and brainstem/spinal cord. We will also examine interactions of individual sensory afferents and corneal epithelial cells. 2) Molecular - we will perform integrated single-cell transcriptomics and epigenomics to interrogate the subtype-specific marker gene expression and epigenetic landscape of the corneal sensory neurons. 3) Functional - we will perform real-time physiologic analyses of corneal sensory afferents using in vivo two-photon calcium imaging. To further elucidate the role of sensory nerves in OS pathophysiology, we will determine how their morphology, molecular profile, and responsiveness are altered in a surgically induced DED model. Moreover, we will implement a novel high-speed imaging and machine learning platform to quantify evoked responses to corneal sensory stimuli and spontaneous behavior in the DED model. Taken together, our study will generate a comprehensive data set poised for integration that will fill in critical knowledge gaps in the field, create a robust foundation for future studies, and inform the development of more precise therapeutic strategies to treat OS diseases.