Retinal microangiopathy in a mouse model of inducible mural cell loss

Abstract

Diabetes can lead to vision loss because of progressive degeneration of the neurovascular unit in the retina, a condition known as diabetic retinopathy. In its early stages, the pathology is characterized by microangiopathies, including microaneurysms, microhemorrhages, and nerve layer infarcts known as cotton-wool spots. Analyses of postmortem human retinal tissue and retinas from animal models indicate that degeneration of the pericytes, which constitute the outer layer of capillaries, is an early event in diabetic retinopathy; however, the relative contribution of specific cellular components to the pathobiology of diabetic retinopathy remains to be defined. We investigated the phenotypic consequences of pericyte death on retinal microvascular integrity by using nondiabetic mice conditionally expressing a diphtheria toxin receptor in mural cells. Five days after administering diphtheria toxin in these adult mice, changes were observed in the retinal vasculature that were similar to those observed in diabetes, including microaneurysms and increased vascular permeability, suggesting that pericyte cell loss is sufficient to trigger retinal microvascular degeneration. Therapies aimed at preventing or delaying pericyte dropout may avoid or attenuate the retinal microangiopathy associated with diabetes.

Figure 1

Characterization of inducible mouse model of mural cell dropout. Levels of plasma insulin (A) and blood glucose (B) in M-Cre and iDTR;M-Cre mice are similar to those observed in C57BL/6J mice (0.637 ng/mL and 159 mg/dL, respectively). C: Retinal flat mounts in M-Cre (left panel) and iDTR;M-Cre (middle and right panels) mice stained for α-actin (green) to detect mural vascular cells and with TUNEL (using an in situ cell death detection kit, red). As a positive control for TUNEL staining, retinal flat mounts from M-Cre mice were treated with DNase. TUNEL-positive pericytes (arrow) as well as missing mural vascular cells (arrowhead) are observed in iDTR;M-Cre mice (middle and right panels) 5 days after DT injection. D: A TUNEL-positive cell, localized to capillary bifurcation, is observed in iDTR;M-Cre mice (arrow), demonstrating specificity of cell death in this model. E: Quantification of TUNEL-positive nuclei accomplished by counting positive cells in M-Cre and iDTR;M-Cre mice. Data are expressed as means ± SEM (A, B, and E). n = 3 M-Cre mice (E); n = 5 iDTR;M-Cre mice (E). ^∗∗P < 0.01. Scale bars: 100 μm (C and D).

Figure 2

Pericyte loss in the retinal microvasculature is associated with microaneurysms and microvascular permeability. Microaneurysms (A) and their quantification (B) in retinal flat mounts labeled with isolectin-B4 to detect ECs (green) and TUNEL (red). Microaneurysms were quantified by assessing fluorescence microscopy images of M-Cre mice given tamoxifen and DT and iDTR;M-Cre mice given tamoxifen and DT. C: Microaneurysm (arrow) in retinal elastase digests stained for PAS and counterstained with hematoxylin from an iDTR;M-Cre mouse given tamoxifen and DT. D: DT-treated M-Cre and iDTR;M-Cre mice were perfused with fluorescein dextran before retina imaging by using fluorescence microscopy. E: Areas of vascular leakage (arrows) are observed in DT-treated iDTR;M-Cre mice. E: Hyperpermeable areas per retina quantified in M-Cre mice given tamoxifen and DT and iDTR;M-Cre mice given tamoxifen and DT. Data are expressed as means ± SEM (B and E). n = 3 M-Cre mice (B and E); n = 5 iDTR;M-Cre mice (B); n = 4 iDTR;M-Cre mice (E). ^∗P < 0.05, ^∗∗P < 0.01. Scale bars: 50 μm (A and C); 250 μm (D). PAS, periodic acid-Schiff.

Figure 3

Loss of pericytes in retinal microvasculature of an inducible model of mural cell loss. Vessels were digested from retinas by using elastase and stained with PAS and hematoxylin. A: Elastase-digested vasculature from iDTR;M-Cre retina showed pericytes (white arrowheads) and ECs (black arrows). Automated image analysis was used to quantify ECs (green) and pericytes (blue, numbered) in isolated vasculatures from iDTR;M-Cre and DT-treated iDTR;M-Cre mice. Red represents cells with overlapping nuclei that were not included in analysis. Only cells from capillaries were counted. The number of pericytes per millimeter capillary length (B) and the number of ECs per millimeter capillary length (C) were analyzed as in A by using 15 selected images per retina in iDTR;M-Cre mice given tamoxifen and no DT, M-Cre mice given tamoxifen and DT, and iDTR;M-Cre mice given tamoxifen and DT. Data are expressed as means ± SEM (B and C). n = 8 iDTR;M-Cre (−DT) mice (B and C); n = 7 M-Cre mice (B and C); n = 8 iDTR;M-Cre (+DT) mice (B and C). ^∗P = 0.0251. Scale bar = 50 μm. PAS, periodic acid-Schiff.

Figure 4

Microvascular abnormalities of retinal vessels in the retinal vasculature of DT-inducible mural cell loss. A: The vasculatures were digested from iDTR;M-Cre, DT-treated M-Cre, and DT-induced iDTR;M-Cre retinas and stained with PAS and hematoxylin. Arrows indicate acellular capillaries in elastase digests of retinal vessels of DT-treated iDTR;M Cre⁺ mice. B: The number of acellular capillaries per millimeter capillary length evaluated in elastase digests by using quantitative image analysis automation in 10 selected images per retina in iDTR;M-Cre mice given tamoxifen and no DT, M-Cre mice given tamoxifen and DT, and iDTR;M-Cre mice given tamoxifen and DT. Data are expressed as means ± SEM (B). n = 8 iDTR;M-Cre (−DT) mice (B); n = 7 M-Cre mice (B); n = 8 iDTR;M-Cre (+DT) mice (B). ^∗P < 0.05. Scale bar = 50 μm. PAS, periodic acid-Schiff.