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.
