Figure 4.

Morphological and Degenerative Features of the Dhdds^flx/flx iCre⁺ Retina

(A and B) Confocal micrographs of (A) Dhdds^flx/flx iCre⁻ and (B) Dhdds^flx/flx iCre⁺ mouse retinas at PN 5 weeks of age. Note the shortening of rod outer segments (detected using anti-opsin; red channel) in Dhdds^flx/flx iCre⁺ retina (B) and the accompanying gliosis (detected using anti-GFAP; green channel). GFAP labeling was limited to Műller glial endfeet and astrocytes (comprising the internal limiting membrane at the vitreoretinal interface) in controls (A). Dhdds^flx/flx iCre⁺ mice did not exhibit anti-opsin immunostaining in either the inner segments (IS) or the photoreceptor perinuclear space, suggesting unhindered opsin trafficking (scale bars, 20 μm).

(C and D) Electron micrographs of Dhdds^flx/flx iCre⁺ outer retina at PN 5 weeks, indicating grossly shortened and poorly aligned, but otherwise ultrastructurally normal, outer segments (OS) (blue arrows, D) and functional RPE phagocytosis (yellow arrow, D). Note pyknotic nuclei (blue arrowheads, C) and thinning of ONL (6 nuclei in a row, yellow bracket, C). Scale bars: 10 μm in (C) and 500 nm in (D).

(E) Dhdds^flx/flx iCre⁻ retina did not exhibit TUNEL labeling. Iba-1 immunoreactivity of microglial cell bodies and arbors was limited to few cell bodies in PN 4- to 5-week-old Dhdds^flx/flx iCre⁻ retina (see Figure S8D) (scale bar: 20 μm).

(F) Retinal dystrophy in Dhdds^flx/flx iCre⁺ mice was characterized by photoreceptor-specific autonomous cell death, as evidenced by TUNEL labeling (white arrows, 488-nm channel). Autonomous cell death in Dhdds^flx/flx iCre⁺ mice was accompanied by infiltration of Iba-1-positive microglia (red channel) into the ONL and subretinal space (whitearrows. Furthermore, we observed microglial phagocytosis of TUNEL-negative photoreceptors (“phagoptosis”) leading to non-autonomous cell death (denoted by asterisks) (scale bar: 20 μm).

(G) Western blot analysis and semi-quantitative densitometry indicates significant up-regulation of GFAP (marker of gliosis) and ICAM-1 (facilitates trans-endothelial migration of leukocytes and breakdown of the blood-retinal barrier). GLUL and ACTB served as loading controls for Müller glia and total protein, respectively. (H) Quantification of GFAP levels in Dhdds^flx/flx iCre⁻ (black) vs. Dhdds^flx/flx iCre⁺ (gray) mouse retinas on Western blots (see G), normalized to ACTB. (I) Quantification of ICAM1 levels in Dhdds^flx/flx iCre⁻ (black) vs. Dhdds^flx/flx iCre⁺ (gray) mouse retinas on Western blots (see G), normalized to ACTB. ∗p < 0.05, ∗∗p < 0.01; Welch's (unpaired) t test.