Intravitreal triamcinolone acetonide inhibits breakdown of the blood-retinal barrier through differential regulation of VEGF-A and its receptors in early diabetic rat retinas

Abstract

Objective: To elucidate the mechanism of the unique beneficial effect of intravitreal steroid therapy on diabetic macular edema, we investigated the effect of locally administered triamcinolone acetonide (TA) on the expression of vascular endothelial growth factor (VEGF)-A and its receptors in retinas of rats with streptozotocin (STZ)-induced diabetes. We then correlated the expression of these proteins with breakdown of the blood-retinal barrier (BRB).

Research design and methods: Thirty-two eyes of 16 diabetic and nondiabetic rats were divided into four groups. TA was injected into the vitreous of the right eye, and saline was injected into the left eye (control) 3.5 weeks after induction of diabetes. Retinas were harvested 48 h following treatment. mRNA and protein expression of VEGF-A, VEGF-A receptor 1 (fms-like tyrosine kinase [FLT]-1), and VEGF-A receptor 2 (fetal liver kinase [FLK]-1) were determined by real-time RT-PCR and immunohistochemistry. BRB permeability was quantitated by measuring extravasated endogenous albumin and retinal thickness.

Results: Diabetes-induced retinal thickness and albumin extravasation were significantly reduced in TA-treated diabetic retinas to a level similar to that in sham-treated nondiabetic eyes. A close correlation between albumin leakage and increased expression of both Vegf-a and Flk-1 was noted in the diabetic retinas. TA downregulated the expression of Vegf-a and Flk-1 but upregulated the expression of Flt-1. TA did not alter the expression of these genes in nondiabetic retinas.

Conclusions: Intravitreal injection of TA stabilizes the BRB in association with regulation of Vegf-a, Flk-1, and Flt-1 expression in retinas in the early stages of diabetes.

FIG. 1.

mRNA expression ratios for Vegf-a, Flk-1 and Flt-1. A: Sham-treated diabetic retina/sham-treated nondiabetic retina. B: IVTA-treated diabetic retina/sham-treated diabetic retina. C: IVTA-treated nondiabetic retina/sham-treated nondiabetic retina.

FIG. 2.

Quantification analysis of VEGF-A, FLK-1, and FLT-1 distribution in the retina from different treatment groups. Number of positive cells for VEGF-A (A), FLK-1 (B), and FLT-1 (C) per millimeter (means ± SEM, n = 8). ***P < 0.001.

FIG. 3.

Retinal thickness was measured on H&E-stained sections at the similar location. The micrograph shows representative samples from IVTA-treated nondiabetic retina (A), sham-treated nondiabetic retina (B), IVTA-treated diabetic retina (C), and sham-treated diabetic retina (D). The yellow bar in A demonstrates one measurement of retinal thickness. E: Table showing mean (± SEM) retinal thickness (*** P < 0.001). Original magnifications ×200. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium layer; OS, inner and outer photoreceptor segment; CH, choroidal layer. To view a high-quality digital representation of this image, go to http://dx.doi.org/db07-0982.

FIG. 4.

Immunohistochemical analysis of retinal albumin distribution. Albumin was detected (in red) within the vessels in sham-treated nondiabetic retinas (A). A similar pattern of albumin distribution was observed in IVTA-treated diabetic retina, but with slightly higher expression in extra vascular space (B). Diffuse extravascular albumin was observed in the inner nuclear, outer plexiform, and outer nuclear layers in sham-treated diabetic retina (C). Extravasated albumin (arrows in C) was marked green in panel A1, B1, and C1 (corresponding to panels A, B, and C) by IPP4.5. Intravascular albumin was excluded by the analysis (arrow heads in C1). D: Quantification of albumin distribution/leakage in the four treatment groups. Values were presented as IAU. Data are means ± SEM, n = 8; *** P < 0.001. Original magnifications ×400. To view a high-quality digital representation of this image, go to http://dx.doi.org/db07-0982.