Blink, Lacrimation, and Nociception: Precision Mapping and Integrated Atlas Generation of Corneal Trigeminal Afferents - ABSTRACT Corneal nerves that mediate pain, blink reflexes and tear production are indispensable in the proper maintenance of ocular surface homeostasis. However, the complexity of these neurons, both at the axon level in the cornea, and cell bodies in the trigeminal ganglion, has made it increasingly difficult to grasp their full nature, resulting in key knowledge gaps in the field. Consistent with the call for the current U01, we will address this barrier via comprehensive analyses of corneal nerves at the morphologic, molecular, and functional level. We have assembled a multidisciplinary team with complementary expertise, enabling integrated analyses of spatial, electrophysiologic, genomic and behavioral profiles in mice; and AI-assisted structure-function studies in human subjects, to open new inroads in the field. In Aim 1, we will anatomically and functionally map corneal-projecting trigeminal afferents in mice. Genetic approaches will be applied to rigorously profile the spatial arrangement of nociceptors in the cornea and trigeminal ganglion using seven- color immunolabeling. This spatial information will be directly linked with the electrophysiological profiling of these respective populations. In Aim 2, genomic analyses of corneal-projecting trigeminal afferents will be conducted applying a new platform for spatial RNA-seq at cellular resolution. We will use mouse strains to parse out the transcriptomes and behavioral outputs at the genetic level. Moreover, we apply two novel approaches to selectively target corneal nerves involved in tear production versus blinking. In Aim 3, we will discover morphological patterns of corneal nerves that predict blinking, lacrimation, and nociception in humans. We will image corneal nerves with in vivo confocal microscopy of subjects with differential blink, tear, and nociceptive behavioral outputs, thereby capturing functional analogs of the mouse experiments in Aims 1 and 2. With the AI-based auto-segmentation algorithm that we are developing, we will be able to apply machine learning for multidimensional profiling of nerve patterns, and then compare these with respective behavioral outputs. These AI-guided efforts will provide critical clues for understanding corneal afferent structure-function in humans. In summary, our collective studies will lead to an unprecedented cartography of corneal afferents in blink, lacrimation, and nociception. The advancements from this work will be poised to facilitate a deeper understanding of related pathobiology including neuropathic ocular pain and dry eye disease that will lay the foundation for future translational and clinical research. All genomic, imaging and electrophysiologic datasets produced will be made publicly available, and all software products for corneal nerve image segmentation will be made freely available online as open-source and easy-to-use software packages.
