Investigation of Corneal Epithelial Tight Junction Disruption and Barrier Function Impairment Induced by Benzalkonium Chloride

Purpose: This study investigated the effects of benzalkonium chloride (BAK), a commonly used ophthalmic preservative, on corneal epithelial barrier function. Methods: Separate animals were assigned to the control and BAK groups. Rabbits received a topical instillation of 0.02% BAK solution every 5 min for a total of 5 doses (20 min). Corneal resistance (CR) was quantitatively measured at 30, 60, and 120 min after the final instillation using a corneal resistance device (CRD). Corneal epithelial damage was evaluated using fluorescein staining, histopathological analysis using hematoxylin and eosin (HE) staining, and immunofluorescence staining for the tight junction protein Zonula occludens-1 (ZO-1). The results were compared with those of the saline-treated control eyes. Results: In the BAK-treated group, CR measured using the CRD method was 82.50 ± 8.92%, 74.17 ± 10.19%, and 76.67 ± 8.35% ( n = 6) at 30, 60, and 120 min after the final instillation, respectively. These values were significantly lower than those of the control group at the corresponding time points (100.00 ± 3.34%, 99.17 ± 3.96%, and 101.67 ± 4.08%, respectively; n = 6) ( P < 0.0125). Fluorescein staining revealed mild superficial punctate keratopathy in most BAK-treated eyes, whereas the control eyes remained intact. HE staining revealed partial loss of superficial epithelial cells in the BAK group. Immunofluorescence analysis demonstrated that ZO-1 was continuously expressed along the corneal epithelial cell borders in control corneas, whereas ZO-1 expression was discontinuous and fragmented in corneas after BAK exposure. In addition, semiquantitative evaluation of the tissue sections indicated that BAK exposure altered ZO-1 localization. Conclusions: BAK disrupts the tight junctions of the corneal epithelium, thereby reducing barrier function. These findings emphasize the importance of evaluating preservative toxicity in ophthalmic formulations to ensure safety of the ocular surface. Although this is a pilot study, it provides important insights into the physiological mechanisms of BAK-induced corneal epithelial damage.