Figure 4

Combined (not separate) cell death pathway inhibition reduces retinal cell death. (a) Scheme: EGF-treated retinal explants were cultured until DEV 3 with different cell death pathway inhibitors (Table S1) either separately or in combination, and were compared to solvent controls (CTRL). (b,e) Immunofluorescence images and (c,d) quantitative analysis of cultured retinal explant sections stained for cell death markers TUNEL and activated caspase 3 (aCASP3) indicated a reduction in cell death depending on the respective inhibitors added: Simultaneous application of necrostatin1 (Nec1) and the pan-caspase inhibitor Z-VAD-FMK (ZVAD) prevent cell death more efficiently than single inhibitors. Nec1 combined with a CASP8 inhibitor (Z-IETD-FMK, IETD) was less efficient than Nec1 with the pan-caspase inhibitor ZVAD (N = 4, each treatment). The ZVDL mixture (ZVAD, Z-DEVD-FMK, and Z-LEHD-FMK) (N = 4) strongly reduced cell death when compared to Nec1+ ZVAD (N = 3). Data are presented as mean ± SEM; N ≥ 3. (f,g) Gene expression analysis of cell death regulators, receptor-interacting serine/threonine-protein kinases 1 and 3 (Ripk1, Ripk3) and Casp3, Casp7 and Casp8, by QPCR of RNA derived from whole retinal explant samples: (f) Temporal analysis indicated gene expression related to apoptotic and necrotic cell death in retinal explants at DEV 2, 4, and 7 compared to uncultured control (DEV 0). (g) Gene expression upon cell death inhibitor treatments showed differential expression levels in death pathway genes (N = 4, each inhibitor treatment). Cq values from gene expression data were normalized to the housekeeping gene (Actb) and respective solvent control (ddCq method). All QPCR reactions were run in technical duplicate, N ≥ 3 biological replicates (see Table S4). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; DEV, days ex vivo. *P < 0.05; **P < 0.01; ***P < 0.001 with Student’s t-test (unpaired, two-tailed). Scale bars: 50 µm. See Figs S3 and S4.