Figure 3

Retinal damage enables EGF-stimulated Müller glia proliferation. (a) Retinal stab wound damage was performed by stabbing with a common 200 μl sterile pipette tip through two of four wings of a retinal flatmount causing a circular lesion (see Fig. S2a). Retinas were prepared to achieve a low level of baseline cell death upon explantation and were EGF-treated (cC conditions, see Fig. 2). Top: Drawing of pipette tip dimensions. Middle: Retinal flatmount overview image with dashed lines indicating the stab wound areas that are shown at higher magnification in the lower image. For analysis, each lesion region of interest could be easily identified by a complete interruption and displacement of retinal layers (see Fig. 3b and S2). Two lesion areas of interest (440 µm width) were averaged and compared with internal unharmed control areas (areas >600 µm from the lesion area). (b) Cell death was increased upon stab wound injury: Representative image of a retinal lesion area immunostained for cell death (TUNEL), amacrine neurons (TFAP2A), and cell nuclei (DAPI). Quantitative analysis showed a higher number of TUNEL+ cell nuclei in stab wound lesion areas compared to control areas (CTRL). (c) Müller glia proliferation was increased in lesion areas compared to control. Image of a retinal lesion area immunostained for the MG marker SOX2 and cell proliferation marker KI67. Quantitative analysis of KI67 as well as SOX2+ KI67+ or SOX2+ BrdU+ double-positive cells indicated increased MG proliferation after stab wound compared to control areas (CTRL). S-phase cell proliferation marker BrdU was given cumulatively throughout explant culture. GCL, ganglion cell layer; INL, inner and ONL, outer nuclear layer. (b,c) Dashed white lines indicate retinal lesion. DEV, days ex vivo. Data are shown as mean ± SEM; N ≥ 3 (see Table S4). *P < 0.05 with Student’s t-test (unpaired, one-tailed). Scale bars: (a) scale 1000 µm, (b,c) 50 µm. Also see Fig. S2.