Understanding neural control of the ocular surface

项目摘要 目前，我们对神经系统如何维持眼表面动态平衡的理解是极其深刻的 有限的。需要新的技术、方法和模式来推动我们对科学的理解和解决 知识鸿沟。眼表和泪膜分泌腺(包括泪腺和眉板腺， 以及高脚杯单元)被仔细控制，以提供光学平滑、低散射的表面 适当的免疫和损伤反应。感官反馈以保持结构和功能的完整性 由角膜神经提供眼表，角膜神经从刺激(化学的，热的， 机械)至神经节(如三叉神经节)和脑区域(如丘脑腹侧后内侧)驱动 泪膜成分的产生以及眨眼反射。神经控制的这种微妙的平衡被打破了 损伤，周围神经病变，炎症，以及一系列广泛的免疫反应进一步复杂化 对各种疾病的影响。这种反馈回路的功能失调可能会导致进一步失调的下行螺旋。 眼表异常的神经控制会导致异常的感觉和疼痛，在最糟糕的情况下 可以禁用。要找到治疗方法，首先必须了解潜在的神经控制系统和 它是如何适应环境的。 在这个提案中，我们的目标是引入新的工具和模型来研究分子、细胞和功能的相互作用 跨负责眼表神经控制的系统，并检查它们在不同的 炎症和疼痛状况。我们已经组建了一支在多个领域拥有专业知识的优秀团队 包括先进的3D显微镜、神经科学、电生理学、疼痛、眼免疫学、眼脂 新陈代谢、眼表疾病、空间统计学和机器/深度学习。在这里，我们将利用切割 边缘技术和技术包括光学清理、轨迹跟踪、行为学上有效的行为分析， 机器/深度学习、空间统计学、基因编码钙成像、光片显微镜、 多路3D荧光原位杂交(FISH)成像和多阵列电极植入 大脑。这些工具将帮助我们评估跨器官的分子、细胞和功能相互作用，并开始 了解生物体水平上的眼表控制。我们还将使用几个相关的动物模型来 评估不同炎症和疼痛条件下的眼表控制情况。型号包括AWAT2缺陷 模拟蒸发性干眼病(DED)的小鼠，糖尿病小鼠，具有 模拟细菌性角膜炎的铜绿假单胞菌和人类供体眼睛。所有的老鼠模型都有 在角膜神经中表达gCaMP6f，从而可以对钙瞬变进行功能成像。有了这些型号，我们 将研究先天免疫和获得性免疫以及伤害性和神经病理性疼痛反应。此外, 我们将神经生长因子(NGF)应用到我们的模型中，以研究潜在的治疗方案如何改变眼睛 地面控制系统。

Project Summary Currently our understanding of how the nervous system maintains ocular surface homeostasis is extremely limited. New technologies, methods and models are needed to advance our scientific understanding and address knowledge gaps. The ocular surface and tear film-secreting glands (including the lacrimal and meibomian glands, as well as the goblet cells) are carefully controlled to provide an optically smooth, low-scattering surface with appropriate immune and injury responses. Sensory feedback to maintain the structural and functional integrity of the ocular surface is provided by the corneal nerves, which send feedback from stimuli (chemical, thermal, mechanical) to ganglia (e.g., trigeminal) and brain regions (e.g., ventral posteromedial thalamus) to drive production of tear film components as well as the blink reflex. This delicate balance of neural control is disrupted by damage, peripheral neuropathies, inflammation and further complicated by a wide array of immune responses to various diseases. Dysfunction of this feedback loop can lead to a downward spiral of further dysregulation. Aberrant neural control of the ocular surface can lead to abnormal sensation and pain, which in the worst cases can be disabling. To find remedies, it is first essential to understand the underlying neural control system and how it adapts to its environment. In this proposal, we aim to bring new tools and models to study molecular, cellular, and functional interactions across systems responsible for neural control of the ocular surface and examine how they change under different inflammatory and pain conditions. We have assembled an excellent team with expertise across multiple fields including advanced 3D microscopy, neuroscience, electrophysiology, pain, ocular immunology, ocular lipid metabolism, ocular surface disorders, spatial statistics, and machine/deep learning. Here, we will utilize cutting edge techniques and technologies including optical clearing, tract tracing, ethologically-valid behavior analysis, machine/deep learning, spatial statistics, genetically encoded calcium imaging, light-sheet microscopy, multiplexed 3D fluorescence in situ hybridization (FISH) imaging, and multi-array electrodes implanted in the brain. These tools will help us assess molecular, cellular, and functional interactions across organs and begin to understand ocular surface control at the organism level. We will also employ several relevant animal models to assess ocular surface control under different inflammatory and pain conditions. Models include AWAT2 deficient mice that mimic evaporative dry eye disease (DED), diabetic mice, an epithelial debridement model with Pseudomonas aeruginosa that mimics bacterial keratitis, and human donor eyes. The mouse models all have gCaMP6f expressed in corneal nerves allowing functional imaging of calcium transients. With these models we will study both innate and adaptive immunity as well as nociceptive and neuropathic pain responses. In addition, we will apply nerve growth factor (NGF) to our models to study how a potential treatment option alters the ocular surface control system.