Abstract
Purpose :
To investigate how the duration of contact lens wear affects the recovery of ocular biomarkers, such as protein, total RNA, and Mucin-5AC, from rabbit tears. This would further inform about the viability of contact lenses to be used as a non-invasive tear collection method.
Methods :
New Zealand White rabbits (4 months of age; 3-5kg) were fitted with a 1-Day Acuvue Moist (J&J) contact lens (CL), unstimulated and without anesthesia. The CL modalities were 1 min., 4 and 8 hours (n=4/time point) with the order of wear randomized. On removal, the CL was placed in dry vials, then refrigerated at -20°C within 15 minutes. Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific) and Synergy H1 Spectrophotometer (BioTek) set at 562nm. RNA was isolated using a E.Z.N.A Total RNA Extraction Kit and quantified with NanoDrop 2000 Spectrophotometer (Thermo Scientific) for concentration and purity (260/280nm ratio). Dot blots were used for Mucin-5AC (MUC5AC) quantification (1µl of each sample and using a MUC5AC antibody) and processed using ImageJ software. One-way ANOVA and Bonferroni post hoc tests were done in Microsoft Excel (Microsoft Corp.). The values are means±SD; p<0.05 indicated statistical significance.
Results :
The level of recovered protein after 1 min. (104.7±23.29µg/ml) and 8 hours (108.2±37.43µg/ml) of CL wear was similar, and lowest after 4 hours (78.1±23.53µg/ml). Total RNA results followed the same pattern with the highest levels after 1 min. (12±1.63ng/µl) and 8 hours (9.5±1.71ng/µl) of CL wear, and lowest after 4 hours (6.7±0.61ng/µl). The level variations in both recovered protein (p=0.173) and isolated total RNA (p=0.239) were not statistically significant. All extracted RNA was highly purified across CL wear with 260/280nm ratios of 2 (1 min.), 1.9 (4 hours), and 1.8 (8 hours). MUC5AC concentrations were significantly higher after 4 hours (1.2±0.25) than 1 min. (0.4±0.20) and 8 hours (0.9±0.47), specifically between 1 min. and 4 hours of CL wear (p=0.002), after post hoc analysis.
Conclusions :
CL wear of only 1 min. is needed to recover tear fluid protein and total RNA, but longer durations of 4 hours may be useful for the collection of MUC5AC. Future directions will be geared towards modifying these CL collection methodologies at each tear sampling step to achieve universal application as a validated standardized protocol in the ocular field.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.