Abstract
Purpose :
Long-term exposure to benzalkonium chloride (BAK) causes toxicity reactions on the ocular surface. Sodium hyaluronate (SH) has been postulated as a potential neutralizing agent for BAK-induced toxicity. The goal of this work was to evaluate the protective effect of SH on BAK-induced toxicity using an in vitro model.
Methods :
The NAV14 cell line (SV40-immortalized murine conjunctival epithelium) was used. Cell monolayers were exposed to different concentrations of BAK (0.001%; 0.005%; 0.01%) and SH (0.2%; 0.3%; 0.4%) for 15 minutes; then, the cells were washed, and fresh culture media was added. Cell viability was evaluated after 2 h by resazurin reduction and lactate dehydrogenase (LDH) enzyme release. Also, cell migration and proliferation over 24 hours were determined by the scratch wound-healing assay. Data was analyzed by two-way ANOVA and is shown as mean±SD of two independent experiments with 4-6 replicates each. Statistical significance was defined as p<0.05.
Results :
BAK induced a concentration-dependent decrease on cell viability (BAK 0.001%: 91±14%, BAK 0.005%: 45±9% and BAK 0.01%: 22±10% of control cells, p<0.001) and an increase in LDH release (no BAK: 0.22±0.02, BAK 0.001%: 0.31±0.02, BAK 0.005%: 1.14±0.05 and BAK 0.01%: 1.21±0.05, p<0.001). Conversely, SH neutralized these effects also in a concentration-dependent manner (p<0.001). In the presence of SH 0.4% (highest effect), cell viability was BAK 0.001%: 104±22%, BAK 0.005%: 109±9% and BAK 0.01%: 75±13% of control cells (p<0.001 for BAK 0.005-0.01%) while LDH release was no BAK: 0.24±0.03, BAK 0.001%: 0.26±0.01, BAK 0.005%: 0.37±0.02 and BAK 0.01%: 0.49±0.22, (vs no SH: p<0.001 for BAK 0.005-0.01%). BAK also reduced wound closure in vitro (after 24 h, no BAK: 76±14%, BAK 0.001%: 42±12%, BAK 0.005%: 17±16% and BAK 0.01%: 0.0% wound closure, p<0.001 for all BAK). Conversely, SH neutralized this effect in a concentration-dependent fashion (p<0.001). In the presence of SH 0.4% (highest effect), wound closure at 24 h was: no BAK: 81±15%, BAK 0.001%: 58±6%, BAK 0.005%: 63±10%, BAK 0.01%: 60±8% (vs no SH: p<0.001 for BAK 0.005-0.01%).
Conclusions :
SH neutralized BAK toxicity on conjunctival epithelial cells in a concentration-dependent manner. SH 0.4% was even protective at the highest preservative concentration. These findings support the use of SH to mitigate BAK toxicity in patients, although more studies are needed.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.