Fig 1.
Characterization of Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice.
(A) Magnified CX3CR1 single-positive cells observed in retinal flat-mounts from 4-week-old Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice and (B) CX3CR1-GFP and CCR2-RFP double-positive cells in RPE flat-mounts from 6-week-old Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice (scale bar = 20 μm). (C-E) Vertical sections from 4-week-, 3-month-, and 1.5-year-old Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice (scale bar = 50 μm). Inserts are the magnified images. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.
Fig 2.
Minocycline administration reduced CCR2-positive cells in retinal flat-mounts.
An intraperitoneal injection of minocycline was administered daily for 14 days to Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice (age: 4–6 weeks). The minocycline group was divided into two subgroups: the Mino50 (50 mg/kg) and Mino100 (100 mg/kg) groups. Phosphate-buffered saline (PBS) was administered to the control group. (A) The retinal flat-mounts of each group were prepared after treatment at 6 weeks of age and observed by laser confocal microscopy. (B) and (C) 3D images of the control and Mino100 groups. The numbers of (F) CX3CR1-GFP positive (GFP+RFP− and GFP+RFP+) cells, (G) CCR2-RFP positive (GFP-RFP+ and GFP+RFP+) cells, and (H) CX3CR1-GFP and CCR2-RFP double-positive (GFP+RFP+) cells in each group (n ≥ 5 per group). * indicates P < 0.05. OS, outer segment. GFP, green fluorescent protein; RFP, red fluorescent protein.
Fig 3.
Minocycline administration reduced CCR2-positive cells in RPE flat-mount.
An intraperitoneal injection of minocycline was administered daily for 14 days to Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice (age: 4–6 weeks). (A) RPE flat-mounts from control, Mino50, and Mino100 were prepared after treatment at 6 weeks of age. (B) and (C) 3D images of the control and Mino100. The numbers of (D) CX3CR1-GFP positive (GFP+RFP− and GFP+RFP+) cells, (E) CCR2-positive (GFP−RFP+ and GFP+RFP+) cells, and (F) CX3CR1-GFP and CCR2-RFP double-positive (GFP+RFP+) cells in each group (n ≥ 5 per group). * indicates P < 0.05. GFP, green fluorescent protein; RFP, red fluorescent protein.
Fig 4.
Diaminobenzidine staining for CCR2-RFP positive cells.
(A) and (B) Frozen sections of retinas derived from 3-month-old control and minocycline (50 and 100 mg/kg)-treated Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice were immunostained with anti-RFP antibody. Signals were visualized using the peroxidase-diaminobenzidine method. Positive signals are indicated by black arrows (scale bar = 50 μm). The number of positive cells in 1-mm wide immunostained sections of control and minocycline (50 and 100 mg/kg)-treated retinas were counted (n ≥ 5 per group) (C) * indicates P < 0.05. RFP, red fluorescent protein.
Fig 5.
Minocycline administration ameliorates photoreceptor cell death in Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice.
Minocycline (50 mg/kg [Mino50] and 100 mg/kg [Mino100]) or phosphate-buffered saline (PBS; control) was administered once daily to Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice (age: 6–8 weeks). (A) Retinas were frozen and sectioned, and nuclei were visualized by DAPI staining. (B) Thickness and (C) the number of cells of the ONL were measured (n ≥ 5 per group). (D) Apoptotic cells in frozen sections of retinas of control and minocycline (50 mg/kg)-treated Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice were examined by the TUNEL assay. (E) Proportion of TUNEL-positive cells in the control, Mino50, and Mino100 groups are shown. (B), (C), and (E): * indicates P < 0.05. (B): ** indicates P < 0.01. Gray and black asterisks indicate P values of control vs. Mino50 and control vs. Mino100, respectively. (A) and (D): Scale bar = 50 μm. DAPI, 4’,6-diamidino-2-phenylindole; ONL, outer nuclear layer; TUNEL, TdT-mediated dUTP nick end labeling.
Fig 6.
Long-term minocycline administration ameliorates photoreceptor cell death in Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice.
Minocycline (50 mg/kg; Mino50), minocycline (100 mg/kg; Mino100), or phosphate-buffered saline (PBS; control) was administered once daily to Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice from 8 to 16 weeks of age. Retinas were frozen and sectioned, and nuclei were visualized by DAPI staining (A) (scale bar = 50 μm). The number of CCR2-RFP positive cells in the subretinal space was counted. Values are the average of five independent samples with standard deviations. Thickness (B) and the number of cells (C) of the ONL were measured (n ≥ 5 per group). * indicates P < 0.05. (C): the gray and black asterisks indicate P values of control vs. Mino50 and control vs. Mino100, respectively. DAPI, 4’,6-diamidino-2-phenylindole; ONL, outer nuclear layer; RFP, red fluorescent protein.