Name: measuring retinal vessel diameter from mouse fluorescent angiography images
Uploaded: 2023-05-19T13:20:00
Description: Watch this Scientific Journal Video about Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images at JoVE.com

This work was supported by a Postdoctoral Fellowship grant from the Icelandic Research Fund (217796-052) (A.G.L.) and the Helga J&#243;nsd&#243;ttir and Sigurlidi Kristj&#225;nsson Memorial Fund (A.G.L and T.E.). The authors thank Prof. Eir&#237;kur Steingr&#237;msson for providing the mice.

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 Mitfmi-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...

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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...

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.

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			<td>1% Tropicamide (Mydriacyl)</td>
			<td>Alcon Inc Laboratories</td>
			<td></td>
			<td>Mydriatic agent</td>
		</tr>
		<tr>
			<td>2% Methocel</td>
			<td>OmniVision Eye Care</td>
			<td></td>
			<td>Hydroxypropryl methylcellulose gel</td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td>Wild type mice</td>
		</tr>
		<tr>
			<td>Chanazine 2% (xylazine)</td>
			<td>Chanelle Animal Health UK</td>
			<td>BN I21322/I</td>
			<td>Anesthesia IP</td>
		</tr>
		<tr>
			<td>Excel for Microsoft 365</td>
			<td>Microsoft Inc</td>
			<td></td>
			<td>Software package</td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>28803-100G</td>
			<td>Fluorescent angiography</td>
		</tr>
		<tr>
			<td>Matlab 8.0</td>
			<td>The MathWorks, Inc.</td>
			<td></td>
			<td>Software package</td>
		</tr>
		<tr>
			<td>Micron IV rodent fundus camera</td>
			<td>Phoenix-Micron</td>
			<td>40-2200</td>
			<td>Fundus photography</td>
		</tr>
		<tr>
			<td>Phenylephrine 10% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Mydriatic agent</td>
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		<tr>
			<td>Phosphate Buffered Saline - 100 tablets</td>
			<td>Gibco</td>
			<td>18912-014</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Sigmaplot 13</td>
			<td>Jandel Scientific Software</td>
			<td></td>
			<td>Software package</td>
		</tr>
		<tr>
			<td>S-Ketamine, 25 mg/mL</td>
			<td>Pfizer Inc.</td>
			<td>PAA104470</td>
			<td>Anesthesia IP</td>
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measuring retinal vessel diameter from mouse fluorescent angiography images

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.

Figure 1&#160;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&#39;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...

Watch this Scientific Journal Video about Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images at JoVE.com

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

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.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

This protocol provides an easy and reliable method to automatically measure the diameter of retinal vessels in anesthetized mice due to gene mutations or retinal disease. The main advantage of this technique is that the custom written MATLAB program here is practical, easy to use, convenient and reliable. Retinal vessel diameter changes commonly occur in degenerative diseases like RP and other retinal vascular conditions.Mouse models offer insights into treatment efficacy and their impact on retinal vessels. The method may be potentially useful as quantifiable clinical markers following the progression of vascular disorders in the eye, and for monitoring potential treatments. To begin, prepare a syringe with fluorescein working solution and administer it intra-peritoneally to the anesthetized animal and apply a drop of 2%methyl cellulose gel to the corneal surface.Then place the animal on the positioning stage of the imaging system. Position the retinal imaging fundus camera lens to directly and gently touch the mouse's cornea, and slightly adjust the alignment to place the optic nerve head in the middle of the visual field. Then switch to the green fluorescence channel on the fundus camera and focus on the retinal vessels to take the images.Capture images at the desired time points following the fluorescein injection to determine the ideal time point and visualize the fluorescein fundus image. First, open the MATLAB program. Then download and save the fundus diameter.m code. Next, open the fundus image analysis folder and drag and drop the fundus diameter. m code into the analysis folder.Drag and drop an FFA image into the image analysis folder. In the MATLAB program, click twice on the code file to activate it. Select the Run option.A popup menu to select the image will appear. Select the image, and a popup menu for analysis parameters will appear on the screen. Set the analysis parameters in the popup menu and press OK.Select the center of the optic disc and then select the edge of the optic disc.The analysis will be performed in the background and the analysis results will appear on the screen. The table contains the values of mean, standard deviation, and standard error for each vessel. Save the table.And then save the figure with the mean thickness set. Open the analysis folder with the saved files and then open the saved figure. Open the saved table, copy the values, and paste them into an Excel file.After the fluorescein injection, images from a control mouse and an MITFMI-VGA9/mutant mouse were obtained, for which the shortest distance was measured from the optic disc center to the end points on the vessels in a clockwise direction. The mean vessel diameter was obtained as a function of the vessel number for the wild type and mutant mice. The most important things to remember are the positioning of the fundus camera and the centering of the optic disc in the fundus image.In addition to this technique, molecular techniques can be performed in order to explore new drugs, diagnose different vascular conditions in the eye, and to better understand the functioning of the retinal vasculature.

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