All animals were used after receiving institutional approval and were handled in a humane manner. The procedures used complied strictly with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Before transplantation, the rats were anesthetized with ketamine hydrochloride (90 mg/kg body weight) and xylazine hydrochloride (10 mg/kg). The indicated cells or vehicle were injected into the superior subretinal space of the right eye of Sprague-Dawley (SD) male rats with a 30-gauge needle and syringe (Hamilton, Reno, NV). Two microliters containing 4 × 10
4 cells were injected.
10 For control, the same surgical procedures were performed, but the cells were not injected.
A total of 178 rats were used; 120 were used for electroretinography and histologic examinations. For transplantation control, the same volume of Hank’s balanced salt solution (HBSS) was injected. All the rats were observed without immunosuppression.
After 24 hours of rearing in standard cyclic room light (light-dark, 14-10 hours), the rats were placed under continuous 2500-lux light for either 2 (group A) or 7 (group B) days. Some of the rats in these groups were also observed without exposure to light (group A′, B′, and B″, respectively). Other rats were kept in standard cyclic room light for 1 month and were placed under continuous 2500-lux light for either 2 (group C) or 7 (group D) days. Some of the rats were also observed without light exposure (group D′). The details of each group of rats is shown in
Table 1 . A total of 38 rats were in group A: 5 received Tet-BDNF1-RPE and were treated with 0 mg/mL DOX eye drops, 5 received 2 mg/mL DOX, 5 received 5 mg/mL DOX, 6 received 10 mg/mL DOX, 4 had only the RPE transplantation, 6 had only an HBSS injection, 4 were sham-operation control subjects, and 3 were untreated control subjects. Forty-four rats were in group B: 5 rats received 0 mg/mL DOX eye drops, 5 received 2 mg/mL DOX, 6 received 5 mg/mL DOX, 6 received 10 mg/mL DOX, 4 received an RPE transplant only, 6 had only an HBSS injection, 4 were sham-operation control subjects, and 3 were untreated control subjects. There were 13 rats in group C: 5 rats received 0 mg/mL DOX, 5 received 10 mg/mL DOX, and 3 were the untreated control. There were 25 rats in group D: 5 rats received 0 mg/mL DOX eye drops, 5 received 10 mg/mL DOX, 4 had RPE only, 4 received only HBSS injection, 4 were sham-operation control subjects, and 3 were untreated control subjects. After exposure to light, the rats were killed by carbon dioxide, and the eyes were prepared for histologic analyses.
Fifty-eight rats were used for real-time PCR or ELISA: There were 15 rats in group A′ for PCR 3 days after transplantation, 32 rats in group B′ for PCR and B″ for ELISA 8 days after transplantation, and 11 rats in group D′ for PCR 38 days after transplantation.