Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Paula V. Subirada; María C. Paz; María V. Vaglienti; José D. Luna; Pablo F. Barcelona; María  C. Sánchez

doi:10.3791/63126

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Method Article

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

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DOI:

10.3791/63126

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March 12th, 2022

Paula V. Subirada¹^,² , María C. Paz¹^,²^,⁴ , María V. Vaglienti¹^,² , José D. Luna³ , Pablo F. Barcelona¹^,² , María C. Sánchez¹^,²

¹Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, ²Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), ³Vitreo-retinal Department, Centro Privado de Ojos Romagosa-Fundación VER, ⁴Unidad de Investigación y Desarrollo en Tecnología Farmacéutica (UNITEFA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)

Summary

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Abstract

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

Introduction

The eyes are nourished by two arterio-venous system: the choroidal vasculature, an external vascular network that irrigates retinal pigmented epithelium and photoreceptors; and the neuro-retinal vasculature that irrigate the ganglion cells layer and the inner nuclear layer of the retina¹. The retinal vasculature is an organized network of vessels that deliver nutrients and oxygen to the retinal cells and harvest waste products to ensure proper visual signaling transduction. This vasculature has some distinct features, including: the lack of autonomous innervation, the regulation of vascular tone by intrinsic retinal mechanisms and the possessio....