Antioxidants Restore Lacrimal Gland Function Through A Calcium-Mediated Mechanism In Experimental Models Of Dry-eye Disease

Abstract

Purpose: : Dry-eye disease is a multifactorial ocular surface disorder resulting from increased tear evaporation or insufficient tear production. Tear components are produced by the 'lacrimal functional unit', consisting of lacrimal glands, ocular surface, lids, Meibomian glands and interconnecting neural reflex loops. Oxidative stress is a key contributor to dry-eye disease, ultimately resulting in ocular surface inflammation through dysregulated osmolarity of the tear film, and a potent modulator of intracellular calcium (Ca²⁺) channels, which are critical for gland exocrine activity. We here investigated the hypothesis that antioxidants can restore lacrimal gland function by preventing oxidative stress-induced dysfunction of intracellular Ca²⁺ channels.

Methods: : Cultures of lacrimal acinar cells were subjected to controlled conditions of oxidative stress to mimic dry-eye disease in vitro. Non-obese diabetic mice were used as an in vivo model for dry-eye disease. We tested the potency of the prototypic antioxidant curcumin to prevent oxidative stress-induced changes in lacrimal acinar cell function using Ca²⁺ imaging, planar lipid bilayer electrophysiology, immunocytochemistry, histology, and quantification of surrogate markers for lacrimal gland function.

Results: : Hydrogen peroxide (H₂O₂)-induced oxidative stress (2 μM, 16 h) resulted in a 63% reduction of primary lacrimal acinar cells responding to acetylcholine stimulation. Ryanodine receptors isolated from oxidative stress-treated (10 μM H₂O₂, 30 min) cultures of lacrimal acinar cells showed significantly increased channel activity and lost their regulation by cytosolic Ca²⁺, but retained their pharmacological profile including sensitivity to the blocker Ruthenium Red. Surrogate markers for lacrimal gland function were decreased after exposure to H₂O₂. For instance, lipocalin-1 release was reduced by 68±5.1%. Co-application of the antioxidant curcumin (0.002% w/v) fully prevented loss of exocrine activity, as assessed by surrogate markers for lacrimal gland function, including lipocalin-1 (89±8.6%). Male NOD mice (8 weeks old) showed immune cell infiltration in the lacrimal gland and a concomitant reduction in gland activity. One-week treatment with curcumin (0.002% w/v, administered i.p. or topically) almost completely restored lacrimal gland function. For instance, lipocalin-1 levels increased from 43±3.5% before to 95±9.5% of control after curcumin treatment.

Conclusions: : Antioxidants target disrupted Ca²⁺ signaling to restore lacrimal gland function, and thus provide a feasible approach for dry-eye disease pharmacotherapy.

Keywords: antioxidants • lacrimal gland • cornea: tears/tear film/dry eye