Chronic Restraint Stress Misaligns Corneal Clock via β-Adrenergic and Glucocorticoid Signaling, Altering Epithelial, Neural, Immune, Metabolic States.

Purpose: To determine whether chronic restraint stress (RS), a validated model of chronic psychological stress, disrupts the corneal circadian transcriptome and alters epithelial, neural, immune, and metabolic outputs, and to distinguish the relative contributions of β-adrenergic and glucocorticoid signaling. Methods: Male C57BL/6J mice were subjected to RS for 2 h/day at ZT3 (Zeitgeber Time 3) for 14 consecutive days. Cohorts received either propranolol, metyrapone, or no intervention; non-stressed littermates served as controls. Systemic stress activation was confirmed via plasma levels of adrenocorticotropic hormone, corticosterone, epinephrine, and norepinephrine, along with superior cervical ganglion and adrenal gene expression. Whole corneas were collected every three hours over a 24-hour cycle (ZT0-ZT21) and analyzed by bulk RNA sequencing. Rhythmic transcripts were identified using JTK_CYCLE; differential gene screening was performed using DESeq2. Epithelial mitoses, immune-cell counts, ZO-1/occludin expression, subbasal nerve architecture, and Cochet-Bonnet corneal sensitivity were assessed. Results: RS induced a broad phase advance and amplitude attenuation of rhythmic genes, with downstream reprogramming of immune, proliferative, and metabolic pathways. Phenotypically, RS increased corneal mechanical sensitivity, reduced neutrophil and γδ T-cell counts, elevated epithelial mitotic activity, and diminished ZO-1/occludin expression, whereas subbasal nerve length remained unaffected. Propranolol partially restored circadian phase alignment and improved epithelial and metabolic parameters; metyrapone preferentially enhanced immune-related gene activity and immune-cell abundance. Conclusions: RS disrupts corneal circadian output and impairs epithelial, immune, and metabolic homeostasis via β-adrenergic and glucocorticoid mechanisms. Temporal targeting of these neuroendocrine pathways may represent a strategy to mitigate stress-induced ocular-surface dysfunction.