Impaired glucose tolerance plus hyperlipidaemia induced by diet promotes retina microaneurysms in New Zealand rabbits

Abstract

With the increasing prevalence of diabetes mellitus and metabolic syndrome worldwide, experimental models are required to better understand the pathophysiology and therapeutic approaches to preserve pancreatic beta cells, attenuate atherosclerosis and protect target organs. The aims of this study were to develop an experimental model of impaired glucose tolerance combined with hypercholesterolaemia induced by diet and assess metabolic alterations and target organ lesions. New Zealand male rabbits were fed high-fat/high-sucrose (10/40%) and cholesterol-enriched diet for 24 weeks, when they were sacrificed. Biochemistry, fundus photographs with fluorescein angiography and pathological analyses were performed. Cholesterol-fed and normal animals of same age were compared.

Results: The animals with diet-induced impaired glucose tolerance combined with hypercholesterolaemia gained weight, increased blood glucose, total cholesterol, LDL-C and triglycerides and decreased HDL-C (P < 0.05 vs. baseline). Fructosamine levels and the homeostasis model assessment of insulin resistance (HOMA-IR) index were increased, while there was a reduction in the HOMA-β (P < 0.05 for all vs. baseline). Histomorphologic findings of this model were aortic atherosclerosis, hepatic steatofibrosis and glomerular macrophage infiltration. Early clinical features of diabetic retinopathy with hyperfluorescent dots consistent with presence of retina microaneurysms were seen since week 12, progressing up to the end of the experiment (P < 0.0005 vs. baseline and 12 weeks). Our model reproduced several metabolic characteristics of human diabetes mellitus and promoted early signs of retinopathy. This non-expensive model is suitable for studying mechanistic pathways and allowing novel strategic approaches.

© 2011 The Authors. International Journal of Experimental Pathology © 2011 International Journal of Experimental Pathology.

Figures

Figure 1

Plots of glucose (mg/dl) and insulin (mU/l) after intraperitoneal infusion of a bolus of hypertonic glucose (50%, 1 g/kg of body weight) in diabetic rabbits at sacrifice. Mean values for glucose (mg/dl) are in the left axis, whereas insulin values (mU/l) are in the right axis. Areas under the curves (AUC) are presented for glucose (light grey) and insulin (dark grey). Mean values (SE) for glucose are 203 (64), 264 (10), 258 (11), 391 (131) and 419 (107) mg/dl for fasting, and 2, 5, 10 and 15 min, respectively. GlucoseAUC = 105,930 (38,297) mg/dl/min. Mean values for insulin (SE) are 1.4 (0.3), 1.1 (0.8), 1.1 (0.4), 1.2 (0.3) and 1.1 (0.2) mU/l for fasting, and 2, 5, 10 and 15 min, respectively. InsulinAUC = 3.1 (0.4) U/l/min.

Figure 2

Fundus photography (a and b) and fluorescein angiographies (c–f) representative of retinas from rabbits with diet-induced diabetes at 12 (c and d) or 24 weeks (e and f), showing hyperfluorescent dots consistent with microaneurysms. Magnification of microaneurysms is shown in (d) and (f) (arrows).

Figure 3

Means plots showing number of microaneurysms (a) and the level of retinopathy (b) in diabetic animals at baseline, 12 and 24 weeks. P<0.0001, anova repeated measures; 24 weeks > 12 weeks and baseline; 12 weeks > baseline.

Figure 4

Fluorescein angiographies of a 12-week cholesterol-fed rabbit (a) and a 24-week control rabbit (b) exhibiting few microaneurysms.

Figure 5

Photomicrographs of aortas from rabbits with diet-induced hyperglycaemia and hypercholesterolaemia. HE (a) and Verhoeff stain (b), Immunohistochemistry for macrophage (c) and for smooth muscle actin (d) (×100).

Figure 6

Photomicrographs of the liver (a–c), kidney (d–f) and pancreas (g–i) from rabbits with diet-induced hyperglycaemia and hypercholesterolaemia (a, b, d, e, g, h) or in cholesterol-enriched diet (c, e, f). Ballonization and steatosis in liver stained with HE (a, e) (×400) and fibrosis in picrosirius stain under polarized light (b) (×100). Kidney stained with picrosirius showing focal fibrosis (c) (×100), macrophages in glomerulus (d) (×400) and stained with HE (f) (×400). Pancreas showing preserved islet (g, i) (HE ×400) and immunohistochemistry with insulin antibody (h) (×400).

Figure 7

Representative photomicrographs of retina of normal (a) and hyperglycaemia/hypercholesterolaemia (b) rabbits. Immunohistochemistry with perlecan antibody (20×). S = sclera; R = retina.