Toxicity of amphotericin B on corneal epithelial cells stored in Optisol-GS: corneal epithelial cell morphology and migration

Abstract

Purpose : To evaluate the toxicity of amphotericin B in Optisol^™-GS corneal storage media (Bausch & Lomb) on corneal epithelial cells (CECs).

Methods : Cornea specimens were dissected from Japanese white rabbit eyes, and then refrigerated at 4°C in Optisol^™-GS, with or without amphotericin B. Storage medium was divided into the following 5 groups (4 corneas per group): Group 1 (control) without amphotericin B (Optisol^™-GS only), and Groups 2, 3, 4, and 5, which included amphotericin B at the concentration of 2.5, 5, 25, and 50 µg/ml, respectively. After 7-days storage, CEC morphology and migration were evaluated. Hematoxylin and eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay were performed using 7 µm cryosections obtained from the preserved corneas. For the purpose of migration assay, 3 corneal blocks (8 x 3 mm each) from 1 preserved cornea were cultured with Dulbecco's modified eagle medium and Ham's F12 nutrient mixture for 24 hours in a 12-well plate, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured.

Results : In Groups 3, 4, and 5, CEC deformity and vacuolation was observed in all preserved specimens. However, CEC morphology was maintained in Groups 1 and 2. The expression of ZO-1 was significantly decreased in the preserved corneal specimens in Groups 4 and 5. TUNEL labelling index (mean±SD) revealed increased CEC apoptosis in Group 3 (48.6±8.7%), which was further aggravated in Groups 4 (59.9±9.0%) and 5 (60.0±7.3%), compared with the control group (33.4±5.8%). The mean CEC migration was inhibited in Group 3 (0.93±0.21mm²), and also in Groups 4 (0.73±0.27mm²) and 5 (0.66±0.12mm²) in a dose-dependent manner, compared with the control group (1.16±0.18mm²).

Conclusions : When added to Optisol^™-GS, amphotericin B was found to be toxic to CECs and inhibit CEC migration. However, our findings revealed that at a concentration of under 5 µg/ml, amphotericin B can be considered safe for use in corneal transplant surgery.

This is a 2021 ARVO Annual Meeting abstract.