Figure 5

Retinal cell death inhibition prevents Müller glia proliferation. (a–d) Combined (not separate) death pathway block prevented Müller glia proliferation in EGF-treated retinal explants (data related to Fig. 4). (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 normalized to solvent controls (CTRL). (b) Immunofluorescence images and (c,d) quantitative analysis of retinal sections immunostained for proliferation marker KI67 and MG marker SOX2+ showed that (c) total (KI67+) and (d) MG (SOX2+ KI67+ double-positive cells) cell proliferation could be inhibited by cell death pathway signaling inhibitors compared to solvent controls. Cell proliferation was almost entirely prevented by combined, but not separate, application of necrostatin1 (Nec1) and pan-caspase inhibitor Z-VAD-FMK (ZVAD). ZVAD reduced cell death (Fig. 4) but not cell proliferation, suggesting compensatory mechanisms. Nec1 combined with a CASP8 inhibitor (Z-IETD-FMK, IETD) also prevented MG proliferation. Compared to Nec1+ ZVAD, the ZVDL mixture (ZVAD, Z-DEVD-FMK, and Z-LEHD-FMK) similarly reduced cell death (Fig. 4) and prevented MG proliferation. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. DEV, days ex vivo. Data are presented as mean ± SEM; N = 4 for each inhibitor. *P < 0.05; **P < 0.01; ***P < 0.001 with Student’s t-test (unpaired, two-tailed). Scale bars: 50 µm. See Fig. S5.