Proparacaine hydrochloride

Except for the 12 mice used for monitoring and photographic analysis during alkali burns, the remaining mice were randomly assigned to 2 groups: a control group without any intervention and an NaOH group subjected to alkali burn treatment. Each experiment involved 18 mice, with 9 mice per group. On the seventh day after alkali burns, corneal tissues from the right eyes of three individuals were pooled to create one sample, and each group comprised three biological replicates for subsequent experiments. Mice were anesthetized with 5% isoflurane and maintained under anesthesia with 2% isoflurane. Before causing corneal alkali damage, a topical anesthetic was applied using 0.5% proparacaine hydrochloride (Alcon, Fort Worth, TX, USA). A circular filter paper (2.0 mm × 2.0 mm) soaked with NaOH (1 mol/L) was attached to the central cornea of the right eye for 40 seconds to induce an alkali burn. Afterward, the paper was quickly removed, and the conjunctival sac was washed entirely with a 0.9% sterile saline solution for 1 minute. After alkali burns, the mice in the experimental and control groups were both treated with ofloxacin eye drops twice daily for 3 days to prevent infection.

ERGs were recorded using an Espion E2 system (Diagnosys). Mice were anesthetized as described above after dark adaptation overnight. Pupils were dilated, and a drop of proparacaine hydrochloride (0.5%; Alcon) was applied on the cornea for topical anesthesia. Flash ERG recordings were obtained simultaneously from both eyes with gold wire loop electrodes, with the reference electrode placed in the mouth and the ground subdermal electrode at the tail. ERG responses were obtained at increasing light intensities over the ranges of 1 × 10⁻⁴ to 10 cd⋅s/m² under dark-adapted conditions and 0.3 to 100 cd⋅s/m² under a rod-saturating background light. The stimulus interval between flashes varied from 5 s at the lowest stimulus to 60 s at the highest ones. 2 to 10 responses were averaged depending on flash intensity. ERG signals were recorded with 0.3-Hz low-frequency and 300-Hz high-frequency cutoffs sampled at 1 kHz. Analysis of a-wave and b-wave amplitudes was performed using Espion ERG Data Analyzer software (version 6.0.54). The a-wave amplitude was measured from the baseline to the negative peak, and the b-wave was measured from the a-wave trough to the maximum positive peak. Statistical comparisons between ERG amplitudes between animals of different genotypes were analyzed using a two-way ANOVA.

The mice were studied while under general anesthesia induced by intraperitoneal injection of 0.3 ml/kg chloral hydrate, followed by topical anesthesia of the eye by drops of 0.5% proparacaine hydrochloride (Alcon, Inc., Fort Worth, TX, USA). A sheet of filter paper (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was cut to size in order to provide disk-shaped pieces with diameters of 2 mm. These filter paper disks were presoaked in 1 N NaOH, and then applied to the center of the anesthetized cornea of the right eye of each mouse for 10 sec, followed by immediate eye irrigation with balanced salt solution (Alcon, Inc.), as previously described (15 (link)), and the animals were then randomly allocated to 1 of 3 different treatment groups: Levofloxacin eye drops (KB2), levofloxacin and FK506 eye drops (SY1), or levofloxacin and tobramycin dexamethasone eye drops (SY2).