Fenofibrate

Two kinds of ophthalmic solutions, a vehicle solution and a 0.05% fenofibrate hydrochloride solution, were compounded and used to compare the effect of the PPARα agonist. Ophthalmic vehicle solution was prepared using 0.1 mL polyoxyethylene sorbitan monooleate (Wako Pure Chemical Industries, Osaka, Japan) and 100 mL NaCl-based PBS (0.01 M; pH 7.4), which was prepared with disodium hydrogen phosphate dodecahydrate (232 g), sodium dihydrogen phosphate dihydrate (23.7 g), and distilled water (4000 mL). To prepare the 0.05% fenofibrate hydrochloride ophthalmic solution, we added 10 mg fenofibrate (Wako Pure Chemical Industries) to 20 mL vehicle solution. fenofibrate is poorly soluble in aqueous solutions, therefore, fenofibrate hydrochloride ophthalmic solution was stirred for 70 min. All ophthalmic solutions were recognized pH 3.7 to pH 3.75 and stored at 4 °C in a refrigerator, and used within a month of the initial compounding without sterile filtration.

HT and cortisol treatments were performed by rearing the fish at 33 °C and at 26 °C with hydrocortisone (5 × 10^–6 M; Sigma-Aldrich, Gillingham, UK), respectively, as previously described^{15 (link),16 (link)}. PPARα agonist treatment was performed with fenofibrate (Wako, Tokyo, Japan) or GW-7647 (Tocris Bioscience, Glasgow, UK) at a concentration of 1 × 10^–6 or 5 × 10^–6 M from 0 dpf to 5 dph. Control was treated with 0.05% Dimethyl sulfoxide (DMSO; Sigma-Aldrich), similar to DMSO concentrations in PPARα agonist treatment, because the concentrations less than 1% do not have toxic effects for medaka embryos^{43 (link)}. After treatments, fish were maintained up to adulthood (2 mph) at 26 °C. The survival rates were shown in Supplementary information Table S2.