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, and consequently any dysfunction or atrophy, can have significant effects on both the bifurcations of the retinal vasculature and the diameter of arteries, demonstrating an interplay between the retinal cells and the vasculature3,4. It is known that numerous eye conditions, including retinopathy of prematurity (ROP), diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma, and corneal neovascularization, can result in abnormal ocular angiogenesis5. In the case of the retinal vasculature, mouse models of retinal degeneration often exhibit changes that are comparable to those seen in human vascular diseases6,7. The Myc supergene family of fundamental helix-loop-helix-zipper transcription factors includes the microphthalmia-associated transcription factor (Mitf) gene expressed in the retinal pigment epithelium (RPE)8,9,10.
Numerous organs, including the eye, ear, immune system, central nervous system, kidney, bone, and skin, have been demonstrated to be regulated by Mitf9,11,12,13. We have discovered that the structure and function of the RPE are affected in mice carrying various mutations in the Mitf gene, resulting in some cases of retinal degeneration and, ultimately, vision loss10. Recently, it has been shown that the number of vessels and vessel diameter differ significantly between Mitf mutant and wild-type mice14. Researchers and physicians can now precisely quantify the retinal vasculature in vivo due to retinal imaging developments. Since the 1800s, researchers and physicians have taken advantage of the benefit of visualizing the retinal vasculature, and fluorescein angiography (FA) has shown both retinal blood flow and degradation of the blood-retinal barrier15.
This article demonstrates how to analyze the retinal vessel diameter from mouse FA images with a custom-written code in MATLAB software.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by a Postdoctoral Fellowship grant from the Icelandic Research Fund (217796-052) (A.G.L.) and the Helga Jónsdóttir and Sigurlidi Kristjánsson Memorial Fund (A.G.L and T.E.). The authors thank Prof. Eiríkur Steingrímsson for providing the mice.
Materials
| 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 |
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