Non-invasive measurement of retinal permeability in a diabetic rat model.

Claire L Allen,Naseeb K Malhi,David O Bates,Jacqueline L Whatmore,Kenton P Arkill

doi:10.1111/micc.12623

Abstract

The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P<.01, P<.001, P<.0001). This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.

Highlights

Selective permeable barriers are vital for organ function and homeostasis
To determine whether permeability could be linked the data for all three diabetic and six controls over 28 days is shown (Figure 4B). These results show that the increase in permeability is progressive and that paired analysis can be used to determine a progressive increase in permeability with diabetic duration, allowing for a substantial reduction in number of animals per group as well as a reduction in overall numbers. These data show that the STZ model of diabetes in rats induces increased vascular permeability in the retina
Of note is that the permeability increases early including a trend at day 7, reaching significance by day 14, i.e. in early stages prior to gross vascular remodelling, as observed by measuring between the plexuses in the retina where there was no significant thickening of this layer

Summary

Introduction

Selective permeable barriers are vital for organ function and homeostasis. Such barriers include the EC barrier of blood vessels, the BBB[1], the BRB[2], the blood-spinal cord barrier[3] and the blood-placental barrier[4]. EB dye has a high affinity for albumin[6] and has a poor ability to cross selective permeable barriers under normal circumstances and remains predominantly within the blood circulation[5] When diseases such as diabetic retinopathy[7], sepsis[8], capillary leak syndrome[9] or cancer[10] result in disruption of these barriers there is an increase in vascular permeability and dyes such as EB may extravasate from the circulation into surrounding tissues. Leak of dyes such as this across blood vessels or the BBB/BRB can signify damage and breakdown of the barrier via trauma or cytokine release. EB has been used to measure breakdown in the blood-spinal cord barrier[16] [17], and the BRB [18] [19]

Methods

Results

Conclusion