Sodium fluorescein

Rapid clearance occurs following topical ocular application, due in part to test agent drainage, blinking (every 5 minutes in mice), tear film, and tear film turnover.²⁸ Therefore, in consideration of in vitro to in vivo dosing translation, low, mid, and high doses of 2, 64, and 640 μg/mL were evaluated in a murine model of corneal wound healing. Corneal epithelial scrape wounds (2-mm diameter) were made with an Algerbrush under a dissecting microscope in the right eye.^{29 (link),30 (link)} Immediately following wound creation, 5 μL RP444 (2, 64, or 640 μg/mL) or vehicle (PBS) was applied. Topical application of the drops was repeated 5 minutes later, and then again at 6, 12, and 18 hours post wound. Corneal wound areas were assessed by staining with 1.5 μL 1% sodium fluorescein (Sigma-Aldrich Corp., St. Louis, MO, USA) every 6 hours until wound closure (24 hours) and images captured with an Olympus SZX16 stereomicroscope (Center Valley, PA, USA). Wound areas were outlined and measured with ImageJ software (https://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA), and expressed as a percentage of the original wound area, at 0 hours.^{30 (link)} Data were analyzed using 2-way repeated measures ANOVA with Bonferroni's test for multiple comparisons, with P < 0.05 considered significant.

Corneal epithelial integrity was evaluated using SD-OCT (Heidelberg Engineering, Heidelberg, Germany), as previously described.^{13 (link)} Briefly, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (75 mg/7.5 mg/kg body weight; Vedco, Inc., St. Joseph, MO, USA) and 1.5 μl of 1% sodium fluorescein (Sigma-Aldrich, Springfield, MO, USA) was instilled into each eye. This was followed by a 400 μl PBS wash to remove pooled fluorescein and debris. Eyes were immediately imaged using SD-OCT with 488-nm wavelength blue light illumination. For corneal staining image analysis, pixel intensity was measured in a 1.5-mm circular area in the central cornea using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).

Corneal epithelial integrity was evaluated using SD-OCT (Heidelberg Engineering, Heidelberg, Germany), as previously described.^{2 (link)} Briefly, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (75 mg/7.5 mg/kg body weight) (Vedco, Inc., St. Joseph, MO, USA) and 1.5 μL 1% sodium fluorescein (Sigma-Aldrich Corp., Springfield, MO, USA) was instilled into each eye. This was followed by a 400-μL PBS wash to remove pooled fluorescein and debris. Eyes were immediately imaged using SD-OCT with 488 nm wavelength blue light illumination. For corneal staining image analysis, pixel intensity was measured in a 1.5-mm circular area in the central cornea using ImageJ software (National Institutes of Health, Bethesda, MD, USA).