Fig. 3

CD4⁺ T cells are required for glaucomatous RGC loss. An elevated intraocular pressure (EIOP)-induced glaucoma model was established after a single microbead (MB) injection. PBS-injected mice served as control (Ctr). A Pooled data from three independent experiments conducted to determine the IOP curve (the total number of enrolled mice: MB, n = 30; Ctr, n = 30). B, C MB-injected mice were sacrificed and retinas were collected at day 30 (GL₃₀) and 45 (GL₄₅) after MB injection, respectively. Immunofluorescence staining for Brn3a (used as a RGC marker) was performed to quantify RGC number, presented as the loss in relative to Ctr retinas. B Illustration of the six areas (each area size: 0.1 mm²) that were examined as indicated in each quadrant of one retina flat-mount. Red boxes represent the central areas, green boxes represent the middle-peripheral areas, and blue boxes represent the peripheral areas. Brn3a-labeled RGCs per 0.1mm² area in one retina flat-mount from each mouse was quantified in 4 quadrants, and the average loss of six areas was also calculated. C–H Shown are results representing one of three independent experiments. C Representative Brn3a-stained retina flat-mounts and RGC number is shown (scale bar: left, 500 μm; right, 20 μm). Sample size: Ctr, n = 8; GL₃₀, n = 12; GL₄₅, n = 9. n refers to the number of retinas used for RGC density analysis and one retina was analyzed per mouse. *P < 0.05, ***P < 0.001, Kruskal–Walls test followed by Dunn’s multiple comparisons test. D–G, Retina tissues were collected from MB-injected mice on day 20 (GL₂₀) or Ctr (scale bar: 500 μm; inset, 20 μm). Retina flat-mounts were prepared and stained for D SMI32, E SMI34, F GFAP, and G Iba1. For determination of D SMI32 and E SMI34 expression, fluorescence intensity (FI) was quantified. For determination of F GFAP and G Iba1, the percentage of antibody-labeled areas per microscopic field (size: 319.45 μm²) was quantified. D-G n ≥ 4. n refers to the number of retinas used for SMI32, SMI34, GFAP, or Iba1 staining. Only one retina per mouse was used for each marker. H PBMC were collected from mice (Ctr, n = 8; GL₂₀, n = 6; GL₃₀, n = 8; GL₄₅, n = 6. n refers to the number of enrolled mice) and flow cytometry was performed to determine the difference of Freq. CD62L⁻ CD4⁺ T cells in the blood among indicated groups. The kinetics of RGC loss (the red-dashed line, derived from C, right Y axis) is also shown. I Pooled data from multiple independent experiments are shown (the total number of mice in each group: Ctr = 60; GL₂₀ = 40; GL₃₀ = 68; GL₄₅ = 36). Retinal cells were collected from Ctr or GL mice at indicated timepoints. Flow cytometry was performed to determine the number of retinal CD4⁺ T cells. (left) Representative flow cytometric plots were shown. (right) Quantification of retinal CD4⁺ T cells (cell number in every 10⁵ live retinal cells) in Ctr (n = 15), GL₂₀ (n = 10), GL₃₀ (n = 17), and GL₄₅ (n = 9). n refers to the number of flow cytometry tests. Four retinas from 4 individual mice (one retina from each mouse) were combined for each flow cytometry test. J Shown is representative immunofluorescence staining for DAPI and Isolectin in retina flat-mounts from GL mice with CD4-specific expression of Tomato (a red fluorescent protein, scale bar: 20 μm) (3 independent experiments were conducted, 4 mice per group in each experiment). K Representative immunofluorescence staining (from 8 mice) for DAPI, CD4, and Brn3a in retina flat-mounts from GL mice showing the location of CD4⁺ T cells with the Brn3a⁺ RGC layer (scale bar: upper and right, 20 μm; lower, 5 μm). L Experimental design: glaucoma was induced and mice were intraperitoneally injected with anti-CD4 (aCD4) or isotype antibody on day 15, 25, 40 after MB injection. M Shown are results representing one of three independent experiments and RGC number was evaluated (n = 6 each group. n refers to the number of retinas used for RGC density analysis and one retina was analyzed per mouse) as described in C. H, I ***P < 0.001 versus Ctr group, Kruskal–Walls test followed by Dunn’s multiple comparisons test. D–G, M **P < 0.01, two-tailed unpaired Student’s t test was performed