Two months after capsaicin injection, rats were divided into two groups (five animals per group). the vehicle group received eye drops containing phosphate-buffered saline (PBS) alone, and the experimental group was treated with eye drops containing 1 mM SP (Sigma-Aldrich) and IGF-1 (1 μg/mL; BD Biosciences, Bedford, MA) in PBS. In general, concentrations 10- to 100-fold greater than those effective in vitro are needed to evaluate the effects of growth factors and peptides in vivo. The concentrations of SP and IGF-1 used in the present study were thus 50 and 100 times, respectively, those shown to be effective in our previous in vitro experiments.
7 We also previously confirmed the effectiveness of these doses in promoting wound closure in vivo.
8 Both eyes received 5 μL of solution six times a day for 2 weeks. Corneal epithelial barrier function was evaluated by measurement of fluorescein penetrance, for 3 days and 1 and 2 weeks after the onset of instillation of eye drops. The fluorophotometric method was based on that of a previous study
16 but was modified for rats. It is simple and practical and it provides a quantitative measure of corneal epithelial damage, especially of that associated with superficial punctate keratopathy. Furthermore, the obtained values correlate well with the grading of corneal epithelial damage by slit lamp observation.
16 In brief, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (35 mg/kg), and 5 μL of 0.5% fluorescein sodium was then instilled into the conjunctival sac. The eyes were maintained closed for 10 minutes, after which the excess fluorescein was washed out with saline, and the eyes were kept closed for an additional 20 minutes. Fluorescein penetrance into the central cornea was then measured with a slit lamp fluorophotometer (Anterior Fluorometer FL-500; Kowa, Nagoya, Japan) that had been modified for rats. The instrument was focused on the central cornea, with a measurement angle of 60°. Fluorescence intensity was measured five times in a 0.023-mm
2 area and then averaged, and fluorescein penetrance was expressed in photon counts per millisecond. Preliminary time course experiments revealed that fluorescein penetrance was substantial and stable 8 weeks after injection of rats with capsaicin, which is why we initiated treatment with SP and IGF-1 2 months after capsaicin injection.