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M ü ller Glia Are a Major Cellular Source of Survival Signals for Retinal Neurons in Diabetes

View Article: PubMed Central - PubMed

ABSTRACT

To dissect the role of vascular endothelial growth factor receptor-2 (VEGFR2) in Müller cells and its effect on neuroprotection in diabetic retinopathy (DR), we disrupted VEGFR2 in mouse Müller glia and determined its effect on Müller cell survival, neuronal integrity, and trophic factor production in diabetic retinas. Diabetes was induced with streptozotocin. Retinal function was measured with electroretinography. Müller cell and neuronal densities were assessed with morphometric and immunohistochemical analyses. Loss of VEGFR2 caused a gradual reduction in Müller glial density, which reached to a significant level 10 months after the onset of diabetes. This observation was accompanied by an age-dependent decrease of scotopic and photopic electroretinography amplitudes and accelerated loss of rod and cone photoreceptors, ganglion cell layer cells, and inner nuclear layer neurons and by a significant reduction of retinal glial cell line–derived neurotrophic factor and brain-derived neurotrophic factor. Our results suggest that VEGFR2-mediated Müller cell survival is required for the viability of retinal neurons in diabetes. The genetically altered mice established in this study can be used as a diabetic animal model of nontoxin-induced Müller cell ablation, which will be useful for exploring the cellular mechanisms of neuronal alteration in DR.

No MeSH data available.


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Analysis of Müller cell density with IHC for GS in conditional Vegfr2 KO mice at 4, 7, and 10 months after the onset of diabetes. A: Representative images showing gradual loss of GS-positive cells and alteration of cell shapes (*) in Müller glia of diabetic conditional Vegfr2 KO mice. Arrows: Müller cell–specific GS staining for quantification. B: Statistical analysis with t test. Scale bar: 50 µm. n = 6 for 10 months in diabetes group, n ≥ 4 for other groups. Error bar: SD; Ctrl, control; mo, months; ns, not significant. **P < 0.01; ***P < 0.001.
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Figure 2: Analysis of Müller cell density with IHC for GS in conditional Vegfr2 KO mice at 4, 7, and 10 months after the onset of diabetes. A: Representative images showing gradual loss of GS-positive cells and alteration of cell shapes (*) in Müller glia of diabetic conditional Vegfr2 KO mice. Arrows: Müller cell–specific GS staining for quantification. B: Statistical analysis with t test. Scale bar: 50 µm. n = 6 for 10 months in diabetes group, n ≥ 4 for other groups. Error bar: SD; Ctrl, control; mo, months; ns, not significant. **P < 0.01; ***P < 0.001.

Mentions: For retinal morphology, eyeballs were fixed at room temperature overnight in PerFix containing 4% paraformaldehyde, 20% isopropanol, 2% trichloroacetic acid, and 2% zinc chloride. The samples were kept in the 70% ethanol until paraffin embedding. Sections (5 μm thickness) were stained with hematoxylin and eosin (H&E) and examined under a light microscope. Outer nuclear layer (ONL) thickness was measured every 0.2 mm, as described previously (16). Inner nuclear layer (INL) thickness was determined by the average of eight distance points starting at 0.4 mm from the optic nerve head (ONH), as performed previously (9). For measurement of Müller cell, cone, ganglion cell layer (GCL), and TUNEL-positive cell density, the total number of positive cells in the retina was counted. The relative number of cells was determined by the ratio of total numbers versus the length of the retina. For IHC-stained Müller cells, those that demonstrated Müller glial-specific staining (line-like, white arrows in Fig. 1E and arrows in Fig. 2A) were considered positive.


M ü ller Glia Are a Major Cellular Source of Survival Signals for Retinal Neurons in Diabetes
Analysis of Müller cell density with IHC for GS in conditional Vegfr2 KO mice at 4, 7, and 10 months after the onset of diabetes. A: Representative images showing gradual loss of GS-positive cells and alteration of cell shapes (*) in Müller glia of diabetic conditional Vegfr2 KO mice. Arrows: Müller cell–specific GS staining for quantification. B: Statistical analysis with t test. Scale bar: 50 µm. n = 6 for 10 months in diabetes group, n ≥ 4 for other groups. Error bar: SD; Ctrl, control; mo, months; ns, not significant. **P < 0.01; ***P < 0.001.
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Figure 2: Analysis of Müller cell density with IHC for GS in conditional Vegfr2 KO mice at 4, 7, and 10 months after the onset of diabetes. A: Representative images showing gradual loss of GS-positive cells and alteration of cell shapes (*) in Müller glia of diabetic conditional Vegfr2 KO mice. Arrows: Müller cell–specific GS staining for quantification. B: Statistical analysis with t test. Scale bar: 50 µm. n = 6 for 10 months in diabetes group, n ≥ 4 for other groups. Error bar: SD; Ctrl, control; mo, months; ns, not significant. **P < 0.01; ***P < 0.001.
Mentions: For retinal morphology, eyeballs were fixed at room temperature overnight in PerFix containing 4% paraformaldehyde, 20% isopropanol, 2% trichloroacetic acid, and 2% zinc chloride. The samples were kept in the 70% ethanol until paraffin embedding. Sections (5 μm thickness) were stained with hematoxylin and eosin (H&E) and examined under a light microscope. Outer nuclear layer (ONL) thickness was measured every 0.2 mm, as described previously (16). Inner nuclear layer (INL) thickness was determined by the average of eight distance points starting at 0.4 mm from the optic nerve head (ONH), as performed previously (9). For measurement of Müller cell, cone, ganglion cell layer (GCL), and TUNEL-positive cell density, the total number of positive cells in the retina was counted. The relative number of cells was determined by the ratio of total numbers versus the length of the retina. For IHC-stained Müller cells, those that demonstrated Müller glial-specific staining (line-like, white arrows in Fig. 1E and arrows in Fig. 2A) were considered positive.

View Article: PubMed Central - PubMed

ABSTRACT

To dissect the role of vascular endothelial growth factor receptor-2 (VEGFR2) in M&uuml;ller cells and its effect on neuroprotection in diabetic retinopathy (DR), we disrupted VEGFR2 in mouse M&uuml;ller glia and determined its effect on M&uuml;ller cell survival, neuronal integrity, and trophic factor production in diabetic retinas. Diabetes was induced with streptozotocin. Retinal function was measured with electroretinography. M&uuml;ller cell and neuronal densities were assessed with morphometric and immunohistochemical analyses. Loss of VEGFR2 caused a gradual reduction in M&uuml;ller glial density, which reached to a significant level 10 months after the onset of diabetes. This observation was accompanied by an age-dependent decrease of scotopic and photopic electroretinography amplitudes and accelerated loss of rod and cone photoreceptors, ganglion cell layer cells, and inner nuclear layer neurons and by a significant reduction of retinal glial cell line&ndash;derived neurotrophic factor and brain-derived neurotrophic factor. Our results suggest that VEGFR2-mediated M&uuml;ller cell survival is required for the viability of retinal neurons in diabetes. The genetically altered mice established in this study can be used as a diabetic animal model of nontoxin-induced M&uuml;ller cell ablation, which will be useful for exploring the cellular mechanisms of neuronal alteration in DR.

No MeSH data available.


Related in: MedlinePlus