Figure 5.

Localized Müller and microglia activation occurs predominantly in the inferior retina and is associated with dying photoreceptors and vascular changes. (A–D) Overlay of projection images of confocal z-stacks of 30–40 µm height located around the outer plexiform layer (OPL) of the central inferior retina of respective Crb1^rd8/rd8 mouse lines and wild-type controls at 2 months of age. For orientation, the deep retinal vascular plexus was labelled by Isolectin B4 (red, A_i, B_i, C_i, D_i) and the photoreceptor nuclei of the ONL by DAPI (blue, A_ii, B_ii, C_ii, D_ii). Microglia and Müller glia activation in the inner retina was assessed by Iba1 (green, A_iii, B_iii, C_iii, D_iii) and GFAP (white, A_iv, B_iv, C_iv, D_iv) labelling. Scale bar: 75 µm. (E–H) Photoreceptor death in the retina was assessed qualitatively by TUNEL⁺ labelling (red) on superior to inferior oriented sagittal retinal sections and co-labelled with GFAP (white) for Müller cell activation. Images taken from the inferior central retina are shown. Localized GFAP activation was closely associated with TUNEL⁺ (red) photoreceptor nuclei (blue). Scale bar: 50 µm. (I–L) 3D reconstruction using Imaris software showing lectinB₄⁺ endothelial cells (red), Iba1⁺ microglia (green), DAPI-labelled photoreceptor nuclei in the ONL photoreceptor (blue) and GFAP⁺ Müller cells (white). (I) View at the OPL towards the ONL. (J) Pronounced gliotic scar located at the outer surface of the ONL facing the subretinal space. (J–I) Scale bar: 20 µm. 3D rotated side views of a single inferior lesion with (K) and without (L) representation of the ONL. Columns of photoreceptor nuclei that are elevated from the ONL (K) are surrounded by activated GFAP⁺ Müller cell processes and Iba1⁺ microglia (K, L) suggesting a localized glial response around these abnormally arranged photoreceptors. At the retinal ganglion cell layer, GFAP also labels astrocytes next to Müller cell processes. (K, L) Scale bar: 200 µm.