Animals
All animal experiments were conducted following the guidelines of the Animal Experiment Committee of Iwate University, Japan. Wistar rats were obtained from CLEA Japan (Tokyo) and they were housed in a 12-h/12-h light/dark cycle with access to water ad libitum. Male 6–12 months old wistar rats (n = 16) were used in this study.
Induction Of Focal Photoreceptor Degeneration
N-Methyl-N-Nitrosourea (MNU: Sigma-Aldrich, Tokyo) solution was freshly prepared with dimethyl sulfoxide immediately before use and stored at 4 °C in the dark. The final concentration of MNU solution was adjusted to 0.3 g/ml. Rats were anesthetized by intramuscular injection of 45 mg/kg ketamine and 4.5 mg/kg xylazine. Then, with an operating microscope, an incision was made in the conjunctiva to expose the sclera. One microliter of NMU solution was subretinally injected through the sclera by using an automatic syringe (Neurosyringe AC, ACrux inc., Morioka) with a 32-gauge needle (Hamilton Company, Reno, NV). As a control, the same volume of vehicle was applied to the contralateral eye.
Multi-focal ERG
Seven days after the subretinal injection, multifocal ERGs were recorded. For recording multifocal ERGs, mREC system (ACrux Inc., Morioka) attached to PuREC (Mayo Corp, Aichi) was used (Fig. 1 and Supplementary video). Under anesthetized condition as the same with taking OCT, the rat was placed on the recording tray. The distance from the screen to the eye was set to 19 cm, corresponding to 45 degrees of the viewing angle. The electrodes were attached to the tail, under the tongue and cornea. The recording of mfERG was performed with the setting at 37 retinal divisions.
Optical Coherence Tomography (OCT)
Under the anesthetized condition, their pupils were dilated with tropicamide (Midrin-P, Santen Co., Ltd., Osaka, Japan). A purified sodium hyaluronate (Hyalein Mini ophthalmic solution Santen Co., Ltd., Osaka, Japan) was applied to the eye, to avoid dryness during the procedure of OCT. Image acquisition of 1.1 mm length of the rat retina including the optic disk was performed using the line scan mode on an OCT imaging device equipped with a special ordered lens (RS-3000, NIDEK Co., Ltd., Aichi, Japan).
WGA- And PNA-staining Of Retinal Wholemount And Cryo-section
To evaluate the loss of cones and rods, the staining with peanut agglutinin (PNA), preferentially binds to cone photoreceptors, and wheat germ agglutinin (WGA), binds to rods, were performed.16 After euthanasia with carbon dioxide, rat eyes were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C and retinal whole mounts were made following washing steps with PBS. The specimens were incubated with 3% Bovine serum albumin (BSA) solution including 0.3% TritonX-100 in PBS for 60 minutes at room temperature and then treated with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA; 1 µg/ml, Vector Laboratories, Tokyo) and Rhodamine-conjugated wheat germ agglutinin (WGA; 5 µg/ml, Vector Laboratories) overnight at 4 °C. After washing with PBS 3 times, the retinal specimen was mounted on a slide using a mounting medium (VECTASHIELD, Vector Laboratories). The retinal wholemount was observed with a fluorescence microscope (AxioVision; Zeiss, Tokyo).
After taking photographs, the retinal specimen was washed with PBS, immersed in 10%, 20%, and 30% sucrose in PBS sequentially, and then embedded in an OCT compound (Sakura Finetek, Tokyo). Ten micrometers of cryo-sections were obtained and observed using the fluorescence microscope.
Statistical analysis
Statistical analyses for in vitro experiments were performed using GraphPad Prism (MDF, Tokyo). The statistical method used was Tukey’s multiple comparison test, Dunnett's multiple comparison test, and Student’s t-test.