Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition

Abstract

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. This study shows that diabetic retinal vascular leakage and nonperfusion are temporally and spatially associated with retinal leukocyte stasis (leukostasis) in the rat model of streptozotocin-induced diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule-1 (ICAM-1). ICAM-1 blockade with a mAb prevents diabetic retinal leukostasis and vascular leakage by 48.5% and 85.6%, respectively. These data identify the causal role of leukocytes in the pathogenesis of diabetic retinopathy and establish the potential utility of ICAM-1 inhibition as a therapeutic strategy for the prevention of diabetic retinopathy.

Figures

Figure 1

Time course of diabetic retinal leukostasis and vascular leakage. (A) Leukostasis was quantified serially by using AOLF. Nondiabetic animals (day 0) and animals with streptozotocin-induced diabetes of varying duration were studied. (B) Radioactive albumin permeation into retinal tissue was quantitated at the same time points by using the isotope dilution technique. All data show the means ± SD.

Figure 2

Static leukocytes are in flux, block capillary flow, and transmigrate. Serial AOLF of static leukocytes in the same retinal area after 7 (A) and 8 (C) days of diabetes shows their complete replacement within a 24-h period. The arrows point to a static leukocyte (A and B) that seems to have transmigrated (B). After 1 day, AOLF and fluorescein angiography show that the leukocyte has disappeared (C and D). The arrowheads show a patent capillary (B) that subsequently becomes obstructed by a static leukocyte 24 h later (C and D). (Bars = 100 μm; 3.2 pixel = 1 μm.)

Figure 3

Leukocyte-induced nonperfusion and reperfusion. Serial studies were completed 1 (A and B), 2 (C and D), and 4 (E and F) weeks after diabetes induction by using both AOLF (A, C, and E) and fluorescein angiography (B, D, and F). The arrows show a patent capillary (B) that subsequently becomes occluded downstream from a static leukocyte (C and D) and then opens up when the leukocyte disappears (E and F). The arrowheads show a patent capillary (B) that becomes occluded downstream from a static leukocyte (C and D) and then remains closed after the leukocyte has disappeared (E and F). (Bars = 100 μm; 3.2 pixel = 1 μm.)

Figure 4

ICAM-1 gene expression in diabetic retina. (A) The ribonuclease protection assay shows that retinal ICAM-1 levels were increased significantly 7 days after diabetes induction. Each lane shows the signal from the two retinas of a single animal. The lane labeled “Probes” shows a 100-fold dilution of the full-length ICAM-1 and 18S riboprobes. The lanes labeled “RNase - (0.1)” and “RNase - (0.01)” show the 10-fold and 100-fold dilutions, respectively, of the full-length riboprobes without sample or RNase. (B) When normalized to 18S RNA, the retinal ICAM-1 levels after 7 days of diabetes were 2.2-fold higher (n = 4; P < 0.05) than in the nondiabetic controls. NS = not significant.

Figure 5

In vivo retinal leukostasis inhibition. Representative retinal leukostasis observed with AOLF in nondiabetic (A), diabetic (B), diabetic + 5 mg/kg mouse control IgG1 (C), and diabetic + 5 mg/kg anti-ICAM-1 mAb-treated animals (D). (Bars = 100 μm; 3.2 pixel = 1 μm.)

Figure 6

Effect of anti-ICAM-1 mAb on leukostasis and vascular leakage in diabetic retina. When ICAM-1 bioactivity was inhibited via systemic administration of the ICAM-1 neutralizing antibody, retinal leukostasis (A) and albumin permeation (B) were decreased 48.5% (5 mg/kg; n = 5; P < 0.001) and 85.6% (5 mg/kg; n = 4; P < 0.0001), respectively. NS = not significant.