Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

Summary

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

Abstract

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Introduction

Possibly the most researched vascular bed in the body is the retinal vasculature. With ever-improving technical sophistication, retinal vasculature is easily photographed in living patients and used in many research fields¹. Additionally, the mouse retinal vasculature during development has proven to be a very effective model system for research into the fundamental biology of vascular growth. The primary purpose of the retinal vasculature is to provide the inner portion of the retina with metabolic support through a laminar capillary meshwork that permeates the neural tissue². Nevertheless, the condition of the retina, ....

Protocol

All experiments were approved by the Icelandic Food and Veterinary Authority (MAST license No. 2108002). All animal studies were conducted according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Male and female C57BL/6J and Mitf^mi-vga9/+ mice were used in this study. C57BL/6J mice (n = 7) were used as a control. The wild types were commercially obtained (see Table of Materials), but all mutant mic.......

Discussion

The present article is the first to present a method to analyze retinal vessel diameter and retinal vasculature from mouse FA images. Since only fundus imaging was utilized to capture images of the retinal vasculature, the method has several drawbacks, one of which is that one can only infer alterations in the superficial layers of that the retinal vasculature in the mice examined in this study; any differences in the deeper layers are yet unknown.

A unique optical coherence tomography angiog.......

Materials

Name	Company	Catalog Number	Comments
1% Tropicamide (Mydriacyl)	Alcon Inc Laboratories		Mydriatic agent
2% Methocel	OmniVision Eye Care		Hydroxypropryl methylcellulose gel
C57BL/6J	Jackson Laboratory	000664	Wild type mice
Chanazine 2% (xylazine)	Chanelle Animal Health UK	BN I21322/I	Anesthesia IP
Excel for Microsoft 365	Microsoft Inc		Software package
Fluorescein sodium salt	Sigma-Aldrich	28803-100G	Fluorescent angiography
Matlab 8.0	The MathWorks, Inc.		Software package
Micron IV rodent fundus camera	Phoenix-Micron	40-2200	Fundus photography
Phenylephrine 10% w/v	Bausch & Lomb		Mydriatic agent
Phosphate Buffered Saline - 100 tablets	Gibco	18912-014	Dilution
Sigmaplot 13	Jandel Scientific Software		Software package
S-Ketamine, 25 mg/mL	Pfizer Inc.	PAA104470	Anesthesia IP