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Benzalkonium chloride (BAK), the most commonly used preservative in ophthalmic solutions, is known to cause toxicity in the corneal epithelium. In this study, we investigated the effects of 20% human serum in cultures of BAK-damaged human corneal epithelial cells (hCECs) and in patients with toxic corneal epitheliopathy induced by BAK-containing eye drops.hCECs were exposed to various concentrations of BAK (0%, 0.002%, 0.02%, and 0.2%) in the presence or absence of 20% human serum. After 24 hours, the metabolic activity, proliferation, apoptosis, and proinflammatory cytokine expression were evaluated in the cells. Also, cell migration was assessed using a scratch test. In the clinical study, 24 patients with toxic corneal epitheliopathy secondary to BAK-containing antiglaucoma eye drops were treated with topical application of 20% autologous serum, and corneal epithelial integrity was evaluated.BAK induced cytotoxicity in hCECs by inhibiting the metabolic activity, proliferation, and migration and by increasing apoptosis in a concentration-dependent manner. The level of proinflammatory cytokine IL-8 was elevated in BAK-treated cells. Addition of 20% human serum to the cultures significantly promoted the cell metabolic activity, proliferation, and migration while markedly reducing apoptosis. In line with the in vitro results, corneal punctate epithelial erosions were decreased from a National Eye Institute scale score of 4.2 ± 2.1 to 1.3 ± 1.7 in 20 of 24 patients (84%) after treatment with 20% autologous serum.Data demonstrate that 20% human serum is effective in treating BAK-induced cytotoxicity in hCECs and provides a basis for using 20% autologous serum for patients with preservative-induced corneal epitheliopathy.