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The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Luhmann UF, Carvalho LS, Holthaus SM, Cowing JA, Greenaway S, Chu CJ, Herrmann P, Smith AJ, Munro PM, Potter P, Bainbridge JW, Ali RR - Hum. Mol. Genet. (2014)

Bottom Line: Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines.By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified.This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and u.luhmann@ucl.ac.uk.

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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 Crb1rd8/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, Ai, Bi, Ci, Di) and the photoreceptor nuclei of the ONL by DAPI (blue, Aii, Bii, Cii, Dii). Microglia and Müller glia activation in the inner retina was assessed by Iba1 (green, Aiii, Biii, Ciii, Diii) and GFAP (white, Aiv, Biv, Civ, Div) 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.
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DDU424F5: 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 Crb1rd8/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, Ai, Bi, Ci, Di) and the photoreceptor nuclei of the ONL by DAPI (blue, Aii, Bii, Cii, Dii). Microglia and Müller glia activation in the inner retina was assessed by Iba1 (green, Aiii, Biii, Ciii, Diii) and GFAP (white, Aiv, Biv, Civ, Div) 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.

Mentions: The prominent degenerative changes in the inferior retina of some Crb1rd8/rd8 mice lead us to further investigate an association between photoreceptor death and Müller and microglia activation (Fig. 5). Prominent loss of DAPI positive signal on retinal flat mounts (Fig. 5A, B, Aii and Bii) and TUNEL+ photoreceptor nuclei in sections (Fig. 5E) are consistent with the darker labelling of photoreceptor nuclei (denser chromatin, pyknotic cell nuclei) and inner segments observed at the base of the whorls in semithin histology (e.g. Fig. 2A and C) and all together indicated loss of photoreceptors at the ONL in inbred Crb1rd8/rd8/J and C57BL/6 Crb1rd8/rd8 (2) mice. Without exception, these areas were closely associated with localized activation of microglia (Fig. 5Aiii and Biii) and Müller glia cell (Fig. 5Aiv, BiV, E and F). However, no significant differences in expression of chemokine (Ccl2, Ccr2, Cx3cl1 and Cx3cr1) or microglia activation marker (iNos, Arg1 or TGFb) were detected in retinas of different Crb1rd8/rd8 lines relative to wild type supporting a localized response of microglia cells to these degenerative events (Supplementary Material, Fig. S2). In all these assessments, C57BL/6 Crb1rd8/rd8 (1) animals appeared similar to wild-type mice with the exception of the rare appearance of subtle aneurysm-like changes at the OPL (Fig. 5C, Ci–iV and G). Three-dimensional (3D) reconstruction of large inferior lesions revealed how activated Müller and microglia cells surround several photoreceptor columns at the OPL (Fig. 5I) and contribute to the formation of gliotic scars at the OLM (Fig. 5J). These processes separate photoreceptor nuclei that have lost their orderly arrangement within the ONL from still normally packed and healthy photoreceptors in their close vicinity (Fig. 5K and L).Figure 5.


The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Luhmann UF, Carvalho LS, Holthaus SM, Cowing JA, Greenaway S, Chu CJ, Herrmann P, Smith AJ, Munro PM, Potter P, Bainbridge JW, Ali RR - Hum. Mol. Genet. (2014)

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 Crb1rd8/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, Ai, Bi, Ci, Di) and the photoreceptor nuclei of the ONL by DAPI (blue, Aii, Bii, Cii, Dii). Microglia and Müller glia activation in the inner retina was assessed by Iba1 (green, Aiii, Biii, Ciii, Diii) and GFAP (white, Aiv, Biv, Civ, Div) 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.
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DDU424F5: 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 Crb1rd8/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, Ai, Bi, Ci, Di) and the photoreceptor nuclei of the ONL by DAPI (blue, Aii, Bii, Cii, Dii). Microglia and Müller glia activation in the inner retina was assessed by Iba1 (green, Aiii, Biii, Ciii, Diii) and GFAP (white, Aiv, Biv, Civ, Div) 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.
Mentions: The prominent degenerative changes in the inferior retina of some Crb1rd8/rd8 mice lead us to further investigate an association between photoreceptor death and Müller and microglia activation (Fig. 5). Prominent loss of DAPI positive signal on retinal flat mounts (Fig. 5A, B, Aii and Bii) and TUNEL+ photoreceptor nuclei in sections (Fig. 5E) are consistent with the darker labelling of photoreceptor nuclei (denser chromatin, pyknotic cell nuclei) and inner segments observed at the base of the whorls in semithin histology (e.g. Fig. 2A and C) and all together indicated loss of photoreceptors at the ONL in inbred Crb1rd8/rd8/J and C57BL/6 Crb1rd8/rd8 (2) mice. Without exception, these areas were closely associated with localized activation of microglia (Fig. 5Aiii and Biii) and Müller glia cell (Fig. 5Aiv, BiV, E and F). However, no significant differences in expression of chemokine (Ccl2, Ccr2, Cx3cl1 and Cx3cr1) or microglia activation marker (iNos, Arg1 or TGFb) were detected in retinas of different Crb1rd8/rd8 lines relative to wild type supporting a localized response of microglia cells to these degenerative events (Supplementary Material, Fig. S2). In all these assessments, C57BL/6 Crb1rd8/rd8 (1) animals appeared similar to wild-type mice with the exception of the rare appearance of subtle aneurysm-like changes at the OPL (Fig. 5C, Ci–iV and G). Three-dimensional (3D) reconstruction of large inferior lesions revealed how activated Müller and microglia cells surround several photoreceptor columns at the OPL (Fig. 5I) and contribute to the formation of gliotic scars at the OLM (Fig. 5J). These processes separate photoreceptor nuclei that have lost their orderly arrangement within the ONL from still normally packed and healthy photoreceptors in their close vicinity (Fig. 5K and L).Figure 5.

Bottom Line: Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines.By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified.This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and u.luhmann@ucl.ac.uk.

Show MeSH
Related in: MedlinePlus