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 vasculature^3,4. 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 diseases^6,7. 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 Mitf^9,11,12,13. 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

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 mice (n = 7) were bred and raised at animal facilities in the Biomedical Center at the University of Iceland. In the present study, 3-month-old animals were used; however, the protocol applies even to 1 month and older animals.

1. Experimental preparation

1. Prepare the anesthesia mixture. Take one ampule of a 2 mL ketamine stock solution (25 mg/mL) and 250 µL xylazine stock solution (2%) to prepare a working solution of ketamine/xylazine mixture (25 mg/mL ketamine and 20 mg/mL xylazine) (see Table of Materials).
2. Anesthetize the mouse with an intraperitoneal injection of the ketamine/xylazine mixture, with a volume of fluid that is four times the mouse's body weight; for example, a mouse 20 g in weight needs 35 µL of xylazine/ketamine working solution to obtain a working dose of xylazine (4 mg/kg body weight) and ketamine (40 mg/kg body weight) mixture.
3. Dilate the pupils with an eye drop of 10% phenylephrine hydrochloride and 1% tropicamide immediately following anesthesia.
4. Wait until the animal is fully anesthetized and its pupils are widely dilated.
5. Prepare fluorescein salt solution. Add 9 mL of phosphate-buffered saline (PBS; 1x) to 1 mL of fluorescein solution (100 mg/mL stock concentration) (see Table of Materials). The final concentration working solution is 10 mg/mL.

2. In vivo imaging of retinal vasculature using a rodent retinal imaging system

1. Administer fluorescein working solution intraperitoneally (5 µL/g body weight) to the anesthetized animal.
2. Apply a drop of 2% methylcellulose gel to the corneal surface and then place the animal on the positioning stage of the imaging system (see Table of Materials).
3. Position the retinal imaging fundus camera lens to touch the mouse's cornea directly and gently. To place the optic nerve head in the middle of the visual field, slightly adjust the alignment.
4. Switch to the green fluorescent channel on the fundus camera.
5. Focus on the retinal vessels to take images.
6. To find the ideal time point, take a number of photos at 1, 3, 5, and 10 min (but no longer than 10 min) following the fluorescein injection.
   NOTE: The FA treatment must be completed within 10 min, as after that time the fluorescein may become too diffused, and the vessels become undetectable.
7. On completion of imaging, while the mouse is still anesthetized, euthanize it by cervical dislocation.

3. Analysis of the retinal vessel's diameter

1. Open the MATLAB program (see Table of Materials).
2. Download and save the "fundusDiameter.m" code (see Supplementary Coding File 1, Supplementary Coding File 2, and Supplementary Coding File 3).
3. Open the folder where the code was saved. Drag the code and drop it over the Current Folder in MATLAB.
4. Drag and drop the FFA image (fundus fluorescence angiography image) or images one wishes to analyze over the Current Folder in MATLAB.
5. Press the Run tool from the MATLAB toolbar.
6. A pop-up window will appear. Write the file name of the image of interest in the Enter filename box and press OK.
   NOTE: Do not change or modify the rest of the parameters.
7. Select the center of the optic disc, and then select the edge of the optic disc. The software now calculates the intensity of pixels in the mouse fundus images in a circle with a radius that is twice that of the optic disk, clockwise from the optic disk's center (Figure 1).
8. Next, ensure that the software plots the mean vessel diameter (in pixels) of each vessel in the fundus as a function of vessel number (Figure 2).
9. Following that, ensure that the software transfers the measurement data for each vessel into an Excel document, where the mean, median, and standard deviation of these values are calculated (Table 1 and Table 2).
10. Move the values from the results table to a spreadsheet program by selecting all the values in the table. Paste the values into the spreadsheet.
11. Plot the graphs and perform statistical analysis using the data pasted into a spreadsheet program of choice (see Table of Materials).

Results

Figure 1 shows the process used to analyze the retinal vasculature, which is applied to mouse FFA images from all the tested mice. A radius that is twice as large as the optic disc is used to measure the intensity of pixels in a circular, clockwise direction from the optic disc's center. It marks pixels with a start or end point when it comes across points above and below a user-specified threshold, respectively. This is repeated 30 times, each time going a little bit further away f...

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
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