Hypoxia Alters Corneal Circadian Rhythms and Disrupts Epithelial, Neural, and Immune Balance.

Purpose: The cornea operates under robust circadian control and is essential for ocular-surface homeostasis. Hypoxic stress-prevalent in many eye disorders and systemic conditions-can disturb these rhythms and compromise epithelial, neural, and immune balance. Here, we examine how environmental hypoxia (EH) and chemical hypoxia (CH) reshape corneal clock-gene expression and tissue integrity. Methods: Male C57BL/6J mice were exposed to normoxia (NC), EH (10% O2), or CH (CoCl2, 15 mg/kg/d intraperitoneally) for 14 days. Corneas were collected at 3-hour intervals across a 24-hour cycle for bulk RNA sequencing. Rhythmic genes were identified by Jonckheere-Terpstra-Kendall CYCLE (JTK_CYCLE), and pathway enrichment was assessed using Gene Set Enrichment Analysis (GSEA) and Phase Set Enrichment Analysis (PSEA). Immunofluorescence staining evaluated epithelial junction proteins (ZO-1 and occludin), neural markers (βIII-tubulin), and immune cells (Ly6G⁺ neutrophils, γδ T cells). Results: Hypoxia significantly increased rhythmic transcripts (by ∼43% in EH and ∼35% in CH), with over 65% of rhythmic genes showing phase shifts. CH specifically upregulated core circadian clock genes (Per1, Cry2, Nr1d2, Rora) and downregulated Per2, possibly via HIF-1α-mediated mechanisms. Both EH and CH impaired epithelial barrier integrity, reduced corneal nerve density, and altered immune cell infiltration, with peak disruption at Zeitgeber time 18 (ZT18). Additionally, CH uniquely induced barrier dysfunction and immune suppression at ZT3, indicating a model-specific vulnerability window. Conclusions: Hypoxia drives model-specific and circadian phase-dependent reprogramming of corneal transcriptomic rhythms, resulting in coordinated structural and immune dysfunction. Identifying ZT18 and ZT3 as critical phases highlights the potential for chronotherapeutic interventions in hypoxia-related ocular surface disorders.