Functional Deficits Precede Structural Lesions in Mice With High-Fat Diet-Induced Diabetic Retinopathy

Abstract

Obesity predisposes to human type 2 diabetes, the most common cause of diabetic retinopathy. To determine if high-fat diet-induced diabetes in mice can model retinal disease, we weaned mice to chow or a high-fat diet and tested the hypothesis that diet-induced metabolic disease promotes retinopathy. Compared with controls, mice fed a diet providing 42% of energy as fat developed obesity-related glucose intolerance by 6 months. There was no evidence of microvascular disease until 12 months, when trypsin digests and dye leakage assays showed high fat-fed mice had greater atrophic capillaries, pericyte ghosts, and permeability than controls. However, electroretinographic dysfunction began at 6 months in high fat-fed mice, manifested by increased latencies and reduced amplitudes of oscillatory potentials compared with controls. These electroretinographic abnormalities were correlated with glucose intolerance. Unexpectedly, retinas from high fat-fed mice manifested striking induction of stress kinase and neural inflammasome activation at 3 months, before the development of systemic glucose intolerance, electroretinographic defects, or microvascular disease. These results suggest that retinal disease in the diabetic milieu may progress through inflammatory and neuroretinal stages long before the development of vascular lesions representing the classic hallmark of diabetic retinopathy, establishing a model for assessing novel interventions to treat eye disease.

Figure 1

HFD-fed mice develop obesity and glucose intolerance. Male C57BL/6J mice were fed standard chow or an HFD providing 42% of energy from fat. A: HFD-fed mice develop significant increases in body mass compared with chow-fed controls beginning at 6 months of age. B: Whole-body adiposity was measured using MRI, showing that mice fed the HFD have significantly more fat mass compared with controls beginning at 6 months. C: Measurements from MRI demonstrate mild increases in lean mass in HFD-fed animals at 6 months but not at 12 months of age. Traces from intraperitoneal GTTs performed after 6 h of fasting at 3 (D), 6 (E), and 12 (F) months of age are shown. G: Calculated AUC for GTTs at each indicated age. H: Mean ± SEM were calculated for AUCs of intraperitoneal insulin tolerance tests performed on animals fed each diet at the indicated ages. I: Insulin levels from fasting serum samples were measured using an ELISA-based method at 3, 6, and 12 months of age. Values represent mean ± SEM from at least five independent experiments in each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Mos, months.

Figure 2

Animals fed the HFD or chow have no differences in gross retinal thickness. Total and laminar retinal thicknesses were measured using hematoxylin and eosin–stained paraffin-embedded retinal sections obtained from 12-month-old animals. Representative examples of stained retinas from whole-globe hemisections obtained at the optic nerve from aged chow-fed (A) or HFD-fed (B) animals. Measurements of total retinal thickness (photoreceptor outer segments − the internal limiting membrane distance) (C) or of the inner nuclear layer (D) in 12-month-old animals fed the HFD or chow. All thicknesses were measured at the indicated distances from the optic nerve head. Distances marked as negative (−) and positive (+) were taken toward the dorsal and ventral borders of the retina, respectively. Scale bars = 25 μm. Values represent mean distances measurements in μm ± SEM, from 5 animals (10 eyes) in each group. No significant differences were observed between groups for any thickness comparison by ANOVA.

Figure 3

At an advanced age, HFD-fed mice develop typical lesions of DR. Retinal vasculature networks were analyzed in 6- and 12-month-old animals by trypsin digest of isolated and fixed retinas, followed by periodic acid Schiff/hematoxylin staining. A: Representative example, under low-power magnification, of a trypsin-digested and stained retina. Grading of vascular lesions was performed under high-power magnification in the midperipheral retina at standardized areas across all samples, as indicated by the black boxes. B: Shown are examples of healthy-appearing pericytes (top three panels, arrowheads) or typical examples of vascular pathologies, including pericyte ghosts (middle three panels, arrows) and atrophic capillaries (bottom panels). Scale bar = 25 μm. HFD-fed animals develop significantly more atrophic capillaries (C) and pericyte ghosts (D) at 12 months, but not 6 months, of age, compared with chow-fed controls. Shown are quantifications of vascular lesions, as assessed by a masked grader. Values represent mean ± SEM from 10 independent experiments in each group. **P < 0.01, ***P < 0.001 by ANOVA.

Figure 4

HFD feeding promotes retinal vascular permeability. EB dye (25 mg/kg) was injected intravenously into anesthetized animals and allowed to circulate for 180 min. A: Enucleated globes are shown from chow-fed (left) or HFD-fed (right) animals. Leakage of the EB dye into the anterior chamber is visible in the eye from a 12-month-old animal fed the HFD compared with the relatively clear aqueous fluid in the control animal. After dye circulation, retinal flat mounts were imaged by fluorescence microscopy. Dye leakage from capillaries into the neural retina is represented by a less defined pattern of fluorescence within the vascular space and a more diffuse “ground-glass” appearance to the background fluorescence. Shown are illustrative examples from chow-fed animals (B and D) and HFD-fed animals (C and E) at 12 months of age. F: Quantification of dye fluorescence from aqueous paracentesis samples demonstrates significantly greater leakage in HFD-fed animals compared with controls at 12 months of age but not at 6 months of age. Dye leakage at 12 months coincides with elevated expression of VEGF-A (G), intercellular adhesion molecule-1 (ICAM-1) (H), and glial fibrillary acidic protein (GFAP) (I), as assessed by RT-PCR from retinal lysates. Values represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA.

Figure 5

HFD feeding does not alter major retinal responses to light stimuli. ERG responses were recorded from animals in scotopic conditions after overnight dark adaptation. A: A high-pass filter was applied to the raw ERG to isolate the OPs for separate analysis from the a-wave and b-wave measurements. ERG a-wave amplitudes recorded at 3, 6, and 12 months of age are plotted against stimulus luminance (B, D, and F), with corresponding b-wave amplitudes (C, E, and G). Values represent the mean ± SEM from at least five animals at each age. Decline in amplitudes of responses are seen with increasing age. At all tested ages, no significant differences were observed between animals on different diets with regard to either major ERG component (ANOVA).

Figure 6

Aged HFD-fed mice have decreased OP amplitudes compared with controls. Visual function in HFD- or chow-fed animals was assessed at 3, 6, and 12 months of age by ERG under scotopic, dark-adapted conditions. OPs were extracted from the raw ERG trace using a high-pass filter. The root mean square (RMS) of all OP peaks and troughs were normalized to the amplitude of the maximal b wave. RMS of OP amplitudes are plotted against stimulus luminance at 3 (A), 6 (B), and 12 (C) months of age. At 12 months of age, mice fed the HFD demonstrate significant declines in OP amplitudes relative to controls. Values represent the mean ± SEM from at least five animals at each age. *P < 0.05 by ANOVA. D–F: Reductions in OP amplitudes correlate with glucose intolerance beginning at 6 months of age. Correlation plots of normalized OP amplitude with the AUC for the intraperitoneal GTT are shown for animals at 3 (D), 6 (E), and 12 (F) months of age. Each data point represents a unique animal. Pearson correlation analyses were performed for animals fed each diet at each tested age, with R² and P values shown in the lower right corner of each graph. G: Representative ERG tracings from mice fed each of the two indicated diets, with the HFD-fed mice displaying reductions in OP amplitudes compared with chow-fed controls.

Figure 7

Delays in OP implicit times are seen in HFD animals before the development of vascular lesions. Implicit times of the first four OPs (OP1–4) were measured from ERGs generated by a 0.25 cd · s/m² white light stimulus. Recordings were performed at each indicated age. At 6 months of age, HFD-fed mice show significant delays in the implicit times of the first two OP peaks compared with chow-fed mice. This difference persists through 12 months of age. Shown are mean values ± SEM for each of the four measured peaks, with individual values for each animal tested superimposed (at least five animals were used in each diet group at each age). *P < 0.05 by ANOVA.

Figure 8

HFD promotes early inflammasome activation in the inner retina. A–E: Retinal lysates from HFD-fed or chow-fed animals at each indicated age were probed for NLRP3, IL-1β, immature caspase-1 (45 kD), active caspase-1 (10 kD), and phosphorylated (p)JNK and total JNK. NLRP3, IL-1β, and pJNK levels were normalized against total JNK, whereas caspase-1 p10 was quantitated relative to the corresponding levels of immature isoform (p45). Values represent mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001. Mo, months. F: Representative retinal sections from 3-month-old animals coimmunostained for the adaptor protein ASC and NLRP3. Staining for both molecules is strongest within the retinal ganglion cell and inner nuclear layers. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; RGCL, retinal ganglion cell layer. G: A high-power image from a HFD-fed animal shows specks (arrows) in retinal ganglion cell and inner nuclear layers. No costaining is observed at large-diameter retinal vessels (*). Scale bar = 20 μm.