Impact of benzalkonium chloride on corneal epithelial wound healing and corneal nerves

Abstract

Purpose : To investigate the impact of benzalkonium chloride (BAC) on corneal epithelial wound healing in mice and on the subepithelial and stromal corneal nerve plexus during wound healing.

Methods : C57BL/6 mice (8-10 week old female ) were anesthetized with isoflurane, followed by topical administration of 20% ethanol for 30 seconds, and a 2 mm diameter epithelial defect was created on the central cornea using a blade. As pretreatment a local single dose of 10 µl 0.1% BAC was administered for 10 seconds. The cornea was imaged with fluorescein staining every 6 hours, up to 24 hours. The images were analyzed using Photoshop software (Adobe Inc., CA, USA) and wound closure rate (%) was calculated and compared between groups with or without BAC administration. To observe the impact of topical BAC administration on corneal nerves, images of corneal nerves in calcitonin gene-related peptide (CGRP)-GFP Tg mice were obtained using in-vivo multiphoton confocal microscopy (SP8 DIVE, Leica Microsystems Inc., Wetzlar, Germany). After creating a corneal epithelial defect, corneal nerves were imaged 24 hours later and compared between the group with/without 10sec of 01.% BAC administration using Imaris software (Bitplane AG., Belfast, UK). Nerve volume was measured by Imaris software, and ratio of post-treatment to pre-treatment nerve volume was calculated. Additionally, corneal mechano-sensation was evaluated using Cochet-Bonnet esthesiometry before treatment and at 24 hours.

Results : Eyes treated with BAC (n=10) showed significantly delayed wound healing at 24 hours compared those without BAC (n=5) (wound closure rate, %, mean ± SD; 51.2 ± 13.3 vs 96.9 ± 6.0) (p=0.007). The ratio of post-treatment to pre-treatment corneal stromal nerve volume determined by multi photon microscopy and Imaris software was 28.3% in the BAC group and 53.2% in the group without BAC, showing a greater decrease in the BAC group. On the other hand, the decrease in subepithelial nerve volume was similar in both groups (14.1% and 7.1%, respectively). Furthermore, eyes treated with BAC also had reduced corneal mechano-sensation compared to eyes without BAC treatment.

Conclusions : Topical administration of BAC following corneal abrasion in mice delayed corneal epithelial wound healing and impairs corneal stromal nerves, representing a model of neurotrophic persistent corneal epithelial defect.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.