Fig. 2

Retinal damage and TRPA1 change after AOH. (A) Schematic illustration of workflow for screening of relative expression of all the genes encoding TRP and PIEZO channel families in AOH retina. (B) Results for first-round screening and (C) results for second-round screening. The relative mRNA level was calculated as the fold change of respective genes in AOH retina compared with control retina (n = 6 for each experiment, paired t-test, *P < 0.05, **P < 0.02, ***P < 0.01). (D) Relative expression of TRPA1 in time sequence by RT-qPCR. At least three independent experiments were performed in duplicates (***P < 0.01). (E) Gel blot and densitometry analysis of TRPA1 showed TRPA1 upregulation in AOH (n = 6, paired t-test, **P < 0.02). (F-G) Expression pattern of TRPA1 in (F) retina slice and in (G) central and peripheral retinal flatmounts under physiological or AOH conditions. Statistical analysis of relative intensity of TRPA1 in GCL were shown in (H) and (I), respectively. Scale bar: 25 μm. (J-N) Flow cytometry analysis showing (L) decreased RGC proportion under AOH (n = 6, paired t-test, **P < 0.02) with (M) elevated TRPA1⁺ RGC percentage (n = 6, paired t-test, *P < 0.05) and (N) average TRPA1 expression indicated by FITC intensity (n = 6, paired t-test, **P < 0.02). Live Retinal cells and single cells were sorted, and cross gates divided all retinal cells in to TRPA1⁺ RGCs (Q2), TRPA1⁻ RGCs (Q3), TRPA1⁺ retinal cells other than RGCs (Q1) and TRPA1⁻ retinal cells other than RGCs (Q4). Results are presented as mean ± standard deviation (SD). AOH, acute ocular hypertension; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; ONL, outer nuclear layer