Fig 2: Effect of TRPA1 activation on the retinal expression of Ca(2+)/CaMKII/CREB signaling pathways under AOH. (A-C) The mRNA level of (A) Trpa1, (B) Camk2a and (C) Camk2b in retina at day 7 in AOH eyes without or with conditional knock out of Trpa1 in RGCs compared with the contralateral control eyes (n = 4 for each group, unpaired t-test, **P < 0.02, ***P < 0.01). (D) Western blot and the relative optical densities analysis of the level of TRPA1 in (F)Trpa1fl/fl;Thy1-Cre− group and (G)Trpa1fl/fl;Thy1-Cre+ group without or with AOH (n = 4 for Trpa1fl/fl;Thy1-Cre− group, n = 3 for Trpa1fl/fl;Thy1-Cre+ group, paired t-test, *P < 0.05). (H) The relative phosphorylation of CaMKII (calculated as p-CaMKII/total CaMKII) in Trpa1fl/fl;Thy1-Cre− group and Trpa1fl/fl;Thy1-Cre+ group (n = 4 for Trpa1fl/fl;Thy1-Cre− group, n = 3 for Trpa1fl/fl;Thy1-Cre+ group, unpaired t-test, *P < 0.05). (E) Western blot and (I) the relative level of CREB1 phosphorylation (n = 4 for Trpa1fl/fl;Thy1-Cre− group, n = 3 for Trpa1fl/fl;Thy1-Cre+ group, unpaired t-test, *P < 0.05). (J) Immunofluorescent staining of the inner retina (GCL + IPL + INL), showing co-localization and expression of CaMKII and p-CaMKII, and (K) CREB1 and p-CREB1. (L) Western blot and the optical densities analysis of the relative level of (M) CaMKII, (N) CaMKII phosphorylation and (O) CREB1 phosphorylation (n = 3 for each group, unpaired t-test, **P < 0.02, ***P < 0.01). GAPDH was used as the internal controls. All data were presented as mean ± SD. AOH, acute ocular hypertension

Fig 3: 2D and 3D observation of TRPA-mediated selective RGC damage pattern. (A) Schematic illustration of brain stereotaxic injection of CTB-488 and CTB-555 for grouping of RGCs. (B) Immunostaining of retina flatmount showing distribution of Plgn RGC and Mlgn RGC together with TRPA1 expression under control or AOH conditions. Scale bar: 50 μm. Plgn RGC and Mlgn RGC were retrogradely labeled by CTB. Note that in all other conditions where RGCs and their central projections were labeled with CTB, an intravitreal dose of CTB was applied for anterograde labeling. (C-D) Comparison of the relative TRPA1 intensity between Plgn RGC and Mlgn RGC in (C) control retina and (D) AOH retina (n = 10 for each group, *P < 0.05, N.S., non-significant). (E) 3D-reconstructed whole brain showing the changes of RGC central projection under AOH with or without conditional knock-out of Trpa1 in RGCs. Different brain regions were labeled, and the original data (left column) were surfaced (right column). AOH refers to the side of the brain where the RGCs of AOH model eye projected to. Scale bar: 1000 μm. (F) Heatmaps showing the relative value of the surface area, volume and sum of intensity of whole RGC central projection, OT, LGN, SC, and PrGPC, PrGMC in the AOH side compared with the control side in mice with or without conditional knock-out of Trpa1 in RGCs (n = 3 for each group, P values stand for differences between Trpa1fl/fl;Thy1-Cre− and Trpa1fl/fl;Thy1-Cre+ AOH side. *P < 0.05, **P < 0.02, ***P < 0.01). (G-H) Comparison of the extent of decrease in both the area, volume and sum of intensity between PrGPC and PrGMC (n = 3 for each experiment, paired t-test, **P < 0.02) in (G)Trpa1fl/fl;Thy1-Cre− and (H)Trpa1fl/fl;Thy1-Cre+ mice. (I) 3D-reconstructed whole brain. AOH refers to the side of the brain where the RGCs of AOH model eye projected to. Scale bar: 1000 μm. (J) Heatmaps showing the relative value of the surface area, volume and sum of intensity of different brain regions in the AOH side of brains compared with the control side with or without HC intravitreal injection (n = 3, *P < 0.05, ***P < 0.01 for comparison of AOH and AOH + HC group). Comparison of the extent of decrease in both the area, volume and sum of intensity between PrGPC and PrGMC (n = 3 for each experiment, paired t-test, *P < 0.05) in AOH mice without or with HC treatment were also shown (n = 3, *P < 0.05). (K) Direct comparison of PrGPC and PrGMC under AOH after HC treatment (n = 3 for each experiment, unpaired t-test, n.s., non-significant). Ar, surface area; CTB, cholera toxin subunit B; DCM, dichloromethane; LGN, lateral geniculate nucleus; OC, optic chiasm; ON, optic nerve; OT, optic tract; PrGMC, magnocellualr part; PrGPC, parvocellular part; SC, superior colliculi; SOI, sum of intensity; Vol, volume

Fig 4: 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