Animals
C57BL/6 male and female mice (8–10 weeks old) and Sprague-Dawley (SD) male rats (8–10 weeks old) were purchased from SLC (Shizuoka, Japan) and maintained at animal facilities in Tohoku University Graduate School of Medicine (Sendai, Japan) under a 12-h light/dark cycle. Mice were mated to obtain pups for primary Müller cell preparation. Male mice were used for retinal dissociation culture experiments. All experimental procedures conformed to "Regulations for Animal Experiments and Related Activities at Tohoku University" and were reviewed by the Institutional Laboratory Animal Care and Use Committee of Tohoku University, and finally approved by the President of University. This study was performed in compliance with the ARRIVE guidelines.
Preparation of mouse primary Müller cells
Preparation and culture of Müller cells have been described previously25,61. Briefly, eyes dissected from postnatal day (P)5 to P8 mouse pups were incubated in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at room temperature overnight. After washing with Dulbecco's phosphate buffered saline, eyes were treated with 0.25% trypsin solution for 15 mins. Retinas were isolated from eyes using sharp forceps and were broken into small pieces by pipetting several times. Prepared retinal explants were cultured in DMEM containing 10% FBS (DMEM10) in a CO2 incubator at 37 °C.
Conditioned media preparation of rotenone-treated Müller cells and microglia
Mouse primary Müller cells, prepared as described previously, and mouse brain-derived microglia (BV-2) were maintained in DMEM10. Müller and BV-2 cells were seeded at 3.0 × 105 and 6.5 × 105 cells in 60 mm dishes, respectively, and cultured in media containing various concentrations of rotenone (Tokyo chemical industry, Tokyo, Japan) for 24 h. Cells were washed with DMEM10 twice to remove rotenone and were then cultured in fresh media for an additional 24 h. Culture media were collected and passed through a 0.22 µm filter (Merck Millipore, Burlington, MA, USA). To obtain double-conditioned media, Müller and BV-2 cells (1.4 × 105 and 3.0 × 105 cells, respectively) were cultured with rotenone-stimulated BV-2 and Müller cell-conditioned media (3 mL) in 35 mm dishes for 24 h, respectively.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Primary Müller cells and BV-2 cells were seeded at 0.5 × 104 and 5 × 104 cells/well in 96-well cell culture plates, respectively, and were treated with various concentrations of rotenone for 24 h. In some cases, these cells were treated with conditioned media instead of rotenone containing media for 6 h. After cell lysis in each well of the 96-well cell culture plates, RNA was reverse transcribed into cDNA using the SuperPrepII cell lysis & RT kit (Toyobo, Osaka, Japan) according to the manufacturer’s instruction. qRT-PCR was performed in a 7500 fast real-time PCR system (Thermo Fisher Scientific) using TaqMan fast universal PCR master mix (Thermo Fisher Scientific) and a mixture of predesigned TaqMan primers and probes [Thermo Fisher Scientific or Integrated DNA Technologies (Coralville, IA, USA)] (see Table).
Retinal dissociation culture
Retinal dissociation cultures were performed as described previously62. Briefly, retinas were dissected from male mice and dissociated into single cells using the neural tissue dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Dissociated retinal cells were resuspended in Neurobasal-A medium (Thermo Fisher Scientific) containing B-27 supplement without antioxidants (Thermo Fisher Scientific) and were seeded at 1 × 105 cells/well (50 µL) in 96-well cell culture plates. The same volume of Müller and/or BV-2 cell-conditioned media was added to retinal cell mixtures and cultured for 24 h.
Cell viability assay
Primary Müller cells, BV-2 cells, and dissociated retinal cells were seeded at 0.5 × 104, 5 × 104, and 1 × 105 cells/well in 96-well cell culture plates, respectively, and were incubated in DMEM10 containing various concentrations of rotenone or Müller and/or BV-2 cell-conditioned media for 24 h. Culture media were removed, and cells were then incubated in DMEM10 containing 10% of the alamarBlue cell viability reagent (Thermo Fisher Scientific) for 3 h. In the case of retinal dissociated cells, alamarBlue was added to cultures one hour after seeding. Fluorescence intensity was measured at 590 nm (excitation: 560 nm) using a SpectraMax M2e microplate reader (Molecular Devices, San Jose, CA, USA).
Intravitreal administration
SD rats were anesthetized using intraperitoneal injection of 8 mg/kg xylazine (Bayer Yakuhin, Osaka, Japan) and 80 mg/kg ketamine (Daiichi Sankyo, Tokyo Japan). A mixture of 2.5 µL of rotenone dissolved in dimethyl sulfoxide (30 nmol) and/or the same volume of neurotrophin (NTF)5 recombinant proteins dissolved in phosphate buffer (0.15 or 1.5 µg) were administered intravitreally using a micro syringe with a 32G sharp needle (Ito corporation, Shizuoka, Japan).
Histology
Eyes were dissected from SD rats seven days after intravitreal injection of rotenone alone or together with Ntf5 recombinant proteins and were fixed with 4% paraformaldehyde in PBS overnight at 4 °C. Fixed eyes were embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan) and were cut into 10 µm-thick sections using a cryostat CM3050S (Leica Biosystems, Wetzlar, Germany). Immunostaining of retinal sections was performed as reported before63. Briefly, retinal sections were treated with blocking solution (10% normal donkey serum in PBS containing 0.01% Tween 20), followed by treatment with rabbit primary antibody (Abcam, Cambridge, UK; 1:500 dilutions with blocking solution) for an RGC marker, RNA-binding protein with multiple splicing (RBPMS). After extensive washing, retinal sections were incubated with Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:500 dilutions with blocking solution) and 4’,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan). To visualize the inner nuclear layer (INL) cells, retinal sections were stained with hematoxylin and eosin (HE; Muto Pure Chemicals, Tokyo, Japan).
Quantification of the retinal ganglion cell number and the INL thickness
Photos were acquired using a fluorescent microscope BZ-X810 (Keyence, Osaka, Japan) with a 20× objective lens on microscopic fields 200 µm far from the optic nerve head of at least three retinal sections stained with RBPMS antibodies or HE per eye. The number of RBPMS+ RGCs and INL thickness in each image were quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA). The average was calculated from five individual eyes per each experimental group.
Statistical analyses
Quantitative data were analyzed using the Tukey–Kramer and Dunnett’s tests with JMP Pro 14 software (SAS Institute, Cary, NC, USA). A P-value of <0.05 was considered statistically significant.