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

角膜神经支配的基础：生理和病理状态下角膜感觉传入的解剖学、分子和功能研究

• 批准号: 10701843
• 负责人: Vivian Lee
• 金额: $ 113.99万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10701843
• 关键词: Address; Affective; Afferent Neurons; Alkaline Phosphatase; Anatomy; Axon; Behavior; Behavioral; Biology; Blinking; Brain Stem; Cells; Central Nervous System; Computer Analysis; Cornea; Corneal Diseases; Cre lox recombination system; Data; Data Analyses; Data Set; Databases; Development; Disease; Disease model; Dose; Dry Eye Syndromes; Epigenetic Process; Epithelial Cells; Epithelium; Esthesia; Eye; Eye diseases; Foundations; Functional disorder; Future; Gene Expression; Genetic; Genetic Models; Genetic Transcription; Genomics; Goals; Growth Factor; Health; Image; Individual; Infection; Knowledge; Label; Machine Learning; Measures; Mediating; Metabolic; Microscopy; Molecular; Molecular Profiling; Morphology; Mus; Natural regeneration; Nerve; Nerve Fibers; Neurons; Neuropathy; Neurotrophic Keratitis; Operative Surgical Procedures; Pain; Pathologic; Pathology; Peripheral; Physiological; Physiology; Play; Population; Productivity; Property; Quality of life; Recording of previous events; Reflex action; Regulation; Research Personnel; Resources; Risk; Role; Sensory; Sensory Nerve Endings; Specific qualifier value; Speed; Spinal Cord; Stimulus; Structure; Structure of trigeminal ganglion; System; Tamoxifen; Therapeutic; Therapeutic Intervention; Time; Tissues; Topical application; Transgenic Mice; Trauma; Trigeminal nerve structure; Vision; Visual; Visualization; afferent nerve; behavioral response; calcium indicator; cell type; corneal epithelium; density; epigenomics; experimental study; functional group; functional plasticity; ganglion cell; imaging approach; imaging modality; in vivo calcium imaging; individual variation; inducible Cre; innovation; insight; intravital imaging; molecular sequence database; mouse genetics; mouse model; multiple omics; nerve supply; neurophysiology; novel; ocular pain; ocular surface; ocular surface disease; painful neuropathy; parabrachial nucleus; response; sensory stimulus; single-cell RNA sequencing; somatosensory; transcriptomics; wound healing

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.

项目摘要 眼表 (OS) 中丰富的感觉传入网络，由三叉神经的眼科分支提供 神经，执行多种生理功能，包括感觉、各种反射的调节，以及 营养因子和生长因子的分泌。在操作系统结构中，角膜是受神经支配程度最高的组织 身体。因此，角膜/OS神经功能障碍已被证明是多种疾病的基础， 包括创伤、感染、代谢失衡以及屈光手术等治疗干预。 角膜疾病仍然是美国的一个主要健康问题，角膜神经功能障碍导致 其中许多疾病，例如神经营养性角膜炎、角膜神经性疼痛和干眼病 (DED)。 研究表明，仅 DED 就会影响高达 75% 的特定人群，导致质量显着下降 生命和视力衰弱混浊的风险增加。因此，综合表征 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。为了满足这个 挑战并响应关于操作系统神经支配的 FOA RFA-EY-21-004，我们组装了一个 跨学科团队，具有强大的协作记录和互补的专业知识，包括眼科 疾病模型和伤口愈合（Lee）；疼痛与哺乳动物体感系统（罗）；活体内的 角膜神经纤维成像和再生（Rompolas）；单细胞转录组学/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（丁）。我们 建议将先进的成像方法、新颖的单细胞多组学和尖端的小鼠结合起​​来 遗传模型执行三个级别的分析：1）形态学 - 使用单神经元遗传标记，我们将 通过可视化解决角膜感觉神经元的精细形态、空间组织和连接性 角膜和脑干/脊髓的轴突分枝。我们还将研究个体之间的相互作用 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组学 和表观基因组学来询问亚型特异性标记基因表达和表观遗传景观 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 使用体内双光子钙成像的传入。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、分子谱和反应性是如何改变的 手术诱导的 DED 模型。此外，我们将实现一种新颖的高速成像和机器学习 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。 总而言之，我们的研究将生成一个可供整合的全面数据集，该数据集将填补关键问题 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 治疗操作系统疾病的精确治疗策略。