Dry Eye (DE) is a prevalent disorder of the ocular surface, causing ocular pain and discomfort, affecting millions of people worldwide. The DE diagnosis includes signs and symptoms of ocular dryness that may be caused by a range of underlying conditions. However, many symptoms of DE correlate poorly with clinical signs, and a subset of patients exhibit severe symptoms with few clinical signs. In these patients, corneal neuropathic pain (NP) may be a factor. The neurobiological events leading to NP in DE are poorly understood. Immune-neural interactions occurring after injury to a peripheral tissue, where the immune response modulates neural responsiveness and nociception, are essential in the development of NP. In NP conditions outside of the eye, Cathepsin S (CTSS), a protease with critical functions in inflammation, is crucial regulator of NP. In these conditions, CTSS acts both at the site of injury and in the CNS. It is our hypothesis that CTSS modulates corneal NP, acting at multiple sites, peripherally at the ocular surface, the trigeminal nerve ganglion (TG) and in the CNS, constituting a viable therapeutic target for the treatment of corneal NP. Three specific aims are proposed: Aim 1. To determine the role of CTSS and its effectors in peripheral and central neuroinflammatory processes in a mouse model of DE with corneal nerve injury. Ocular surface inflammation and nerve injury will be induced in C57BL/6 wild type (WT) mice by topical administration of Benzalkonium chloride (BAC). The involvement of CTSS and known downstream mediators of neuropathic pain at the ocular surface, the TG and trigeminal brain stem complex (TBSC) will be explored through analysis of changes in gene and protein expression. Aim 2. To validate the involvement of CTSS in development of mechanical hyperalgesia and allodynia in WT and CTSS knockout mice with induction of DE and NP, and to evaluate the therapeutic potential of topical and systemic CTSS inhibition. The response to BAC injury in WT mice will be compared to CTSS knockout mice (CTSS-/-), assessing corneal nerve integrity using in vivo confocal microscopy and pain sensation with a series of behavioral tests. The therapeutic potential of topical or systemic CTSS inhibition will be assessed utilizing the CTSS inhibitor, Z-Phe-Leu-COCHO (Z-FL). Aim 3. To conduct a discovery analysis of alterations in the transcriptome of the TG and TBSC after corneal nerve injury, utilizing spatial transcriptomics and NextGen mRNA sequencing. The 10x Genomics Visium Gene Expression Solution platform, allowing near single-cell sequencing while retaining cell localization information, will enable more specific information about the transcriptome in distinct areas of the TG and TBSC with induction of DE and NP. At the conclusion of this project, we will have determined the role of CTSS in corneal and central neuroinflammation and pain sensation and evaluated whether its inhibition has a therapeutic effect on NP. We will also ha...