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 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
<ol>
	<li>Prepare the anesthesia mixture. Take one ampule of a 2 mL ketamine stock solution (25 mg/mL) and 250 &#181;L 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).</li>
	<li>Anesthetize the mouse with an intraperitoneal injection of the ketamine/xylazine mixture, with a volume of fluid that is four times the mouse&#39;s body weight; for example, a mouse 20 g in weight needs 35 &#181;L 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.</li>
	<li>Dilate the pupils with an eye drop of 10% phenylephrine hydrochloride and 1% tropicamide immediately following anesthesia.</li>
	<li>Wait until the animal is fully anesthetized and its pupils are widely dilated.</li>
	<li>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.</li>
</ol>
2. In vivo imaging of retinal vasculature using a rodent retinal imaging system
<ol>
	<li>Administer fluorescein working solution intraperitoneally (5 &#181;L/g body weight) to the anesthetized animal.</li>
	<li>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).</li>
	<li>Position the retinal imaging fundus camera lens to touch the mouse&#39;s cornea directly and gently. To place the optic nerve head in the middle of the visual field, slightly adjust the alignment.</li>
	<li>Switch to the green fluorescent channel on the fundus camera.</li>
	<li>Focus on the retinal vessels to take images.</li>
	<li>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.</li>
	<li>On completion of imaging, while the mouse is still anesthetized, euthanize it by cervical dislocation.</li>
</ol>
3. Analysis of the retinal vessel&#39;s diameter
<ol>
	<li>Open the MATLAB program (see Table of Materials).</li>
	<li>Download and save the &#34;fundusDiameter.m&#34; code (see Supplementary Coding File 1, Supplementary Coding File 2, and Supplementary Coding File 3).</li>
	<li>Open the folder where the code was saved. Drag the code and drop it over the Current Folder in MATLAB.</li>
	<li>Drag and drop the FFA image (fundus fluorescence angiography image) or images one wishes to analyze over the Current Folder in MATLAB.</li>
	<li>Press the Run tool from the MATLAB toolbar.</li>
	<li>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.</li>
	<li>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&#39;s center (Figure 1).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Plot the graphs and perform statistical analysis using the data pasted into a spreadsheet program of choice (see Table of Materials).</li>
</ol>

<ol><li>Cheung, C. Y., Ikram, M. K., Chen, C., Wong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+retina+to+study+dementia+and+stroke%5BTitle%5D)+AND+%22Progress+in+Retinal+and+Eye+Research%22%5BJournal%5D)">Imaging retina to study dementia and stroke.</a> Progress in Retinal and Eye Research. 57, 89-107 (2017).</li>
<li>Selvam, S., Kumar, T., Fruttiger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+vasculature+development+in+health+and+disease%5BTitle%5D)+AND+%22Progress+in+Retinal+and+Eye+Research%22%5BJournal%5D)">Retinal vasculature development in health and disease.</a> Progress in Retinal and Eye Research. 63, 1-19 (2018).</li>
<li>Ma, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+retinal+vessel+attenuation+in+eyes+with+retinitis+pigmentosa%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Quantitative analysis of retinal vessel attenuation in eyes with retinitis pigmentosa.</a> Investigative Ophthalmology & Visual Science. 53 (7), 4306-4314 (2012).</li>
<li>Eysteinsson, T., Hardarson, S. H., Bragason, D., Stefansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+vessel+oxygen+saturation+and+vessel+diameter+in+retinitis+pigmentosa%5BTitle%5D)+AND+%22Acta+Ophthalmologica%22%5BJournal%5D)">Retinal vessel oxygen saturation and vessel diameter in retinitis pigmentosa.</a> Acta Ophthalmologica. 92 (5), 449-453 (2014).</li>
<li>Al-Latayfeh, M., Silva, P. S., Sun, J. K., Aiello, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antiangiogenic+therapy+for+ischemic+retinopathies%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Perspectives+in+Medicine%22%5BJournal%5D)">Antiangiogenic therapy for ischemic retinopathies.</a> Cold Spring Harbor Perspectives in Medicine. 2 (6), 006411(2012).</li>
<li>Wang, S., Villegas-Perez, M. P., Vidal-Sanz, M., Lund, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+optic+axon+dystrophy+and+vacuslar+changes+in+rd+mice%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Progressive optic axon dystrophy and vacuslar changes in rd mice.</a> Investigative Ophthalmology & Visual Science. 41 (2), 537-545 (2000).</li>
<li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoreceptor+cells+influence+retinal+vascular+degeneration+in+mouse+models+of+retinal+degeneration+and+diabetes%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Photoreceptor cells influence retinal vascular degeneration in mouse models of retinal degeneration and diabetes.</a> Investigative Ophthalmology & Visual Science. 57 (10), 4272-4281 (2016).</li>
<li>Steingrimsson, E., Copeland, N. G., Jenkins, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Melanocytes+and+the+microphthalmia+transcription+factor+network%5BTitle%5D)+AND+%22Annual+Review+of+Genetics%22%5BJournal%5D)">Melanocytes and the microphthalmia transcription factor network.</a> Annual Review of Genetics. 38, 365-411 (2004).</li>
<li>Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+discovery+of+the+microphthalmia+locus+and+its+gene%5BTitle%5D)+AND+%22Mitf.+Pigment+Cell+%26+Melanoma+Research%22%5BJournal%5D)">The discovery of the microphthalmia locus and its gene.</a> Mitf. Pigment Cell & Melanoma Research. 23 (6), 729-735 (2010).</li>
<li>Garcia-Llorca, A., Aspelund, S. G., Ogmundsdottir, M. H., Steingrimsson, E., Eysteinsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+microphthalmia-associated+transcription+factor+%28Mitf%29+gene+and+its+role+in+regulating+eye+function%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">The microphthalmia-associated transcription factor (Mitf) gene and its role in regulating eye function.</a> Scientific Reports. 9 (1), 15386(2019).</li>
<li>Bharti, K., Liu, W., Csermely, T., Bertuzzi, S., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+promoter+use+in+eye+development%3A+the+complex+role+and+regulation+of+the+transcription+factor+MITF%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Alternative promoter use in eye development: the complex role and regulation of the transcription factor MITF.</a> Development. 135 (6), 1169-1178 (2008).</li>
<li>Lu, S. Y., Li, M., Lin, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitf+induction+by+RANKL+is+critical+for+osteoclastogenesis%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">Mitf induction by RANKL is critical for osteoclastogenesis.</a> Molecular Biology of the Cell. 21 (10), 1763-1771 (2010).</li>
<li>Pillaiyar, T., Manickam, M., Jung, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+development+of+signaling+pathways+inhibitors+of+melanogenesis%5BTitle%5D)+AND+%22Cellular+Signalling%22%5BJournal%5D)">Recent development of signaling pathways inhibitors of melanogenesis.</a> Cellular Signalling. 40, 99-115 (2017).</li>
<li>Danielsson, S. B., Garcia-Llorca, A., Reynisson, H., Eysteinsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+microphthalmia-associated+transcription+factor+%28Mitf%29+mutations+affect+the+structure+of+the+retinal+vasculature%5BTitle%5D)+AND+%22Acta+Ophthalmologica%22%5BJournal%5D)">Mouse microphthalmia-associated transcription factor (Mitf) mutations affect the structure of the retinal vasculature.</a> Acta Ophthalmologica. 100 (8), 911-918 (2022).</li>
<li>Burns, S. A., Elsner, A. E., Gast, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+the+retinal+vasculature%5BTitle%5D)+AND+%22Annual+Review+of+Vision+Science%22%5BJournal%5D)">Imaging the retinal vasculature.</a> Annual Review of Vision Science. 7, 129-153 (2021).</li>
<li>Wei, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+vessel+diameter+quantification+and+vessel+tracing+for+OCT+angiography%5BTitle%5D)+AND+%22Journal+of+Biophotonics%22%5BJournal%5D)">Automated vessel diameter quantification and vessel tracing for OCT angiography.</a> Journal of Biophotonics. 13 (12), e202000248(2020).</li>
<li>Salas, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compact+akinetic+swept+source+optical+coherence+tomography+angiography+at+1060+nm+supporting+a+wide+field+of+view+and+adaptive+optics+imaging+modes+of+the+posterior+eye%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">Compact akinetic swept source optical coherence tomography angiography at 1060 nm supporting a wide field of view and adaptive optics imaging modes of the posterior eye.</a> Biomedical Optics Express. 9 (4), 1871-1892 (2018).</li>
<li>Albanna, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-invasive+evaluation+of+neurovascular+coupling+in+the+murine+retina+by+dynamic+retinal+vessel+analysis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Non-invasive evaluation of neurovascular coupling in the murine retina by dynamic retinal vessel analysis.</a> PLoS One. 13 (10), e0204689(2018).</li>
<li>Moult, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+anesthetic+protocols+for+functional+blood+flow+imaging+in+the+rat+eye%5BTitle%5D)+AND+%22Journal+of+Biomedical+Optics%22%5BJournal%5D)">Evaluating anesthetic protocols for functional blood flow imaging in the rat eye.</a> Journal of Biomedical Optics. 22 (1), 16005(2017).</li>
</ol>

The authors declare that no competing interests exist.

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 angiography (OCTA) image analysis method using automatic vessel tracing and vessel diameter has been presented16. However, this method requires a gradient-guided minimum radial distance (MRD) measurement, which is the basis of an automated framework for the quantitative characterization of the diameter of blood arteries, including individual capillaries. Furthermore, the sizes of tiny capillaries are probably overstated to some extent, since the OCT system has limited lateral resolution16. This method requires FA, which can capture a much wider area of the retinal and choroidal vasculature17. The Dynamic Vessel Analyzer (DVA) prototype can be used to analyze retinal vessels in mice18. Nevertheless, this device uses flicker light impulses applied at predetermined frequencies to enable dynamic vessel examination as a function of time. As shown by postmortem analysis, when comparing flicker-exposed with flicker-na&#239;ve retinae, visual stimulation during the recording method may put stress on the investigated retina18. This method allows the analysis of the retinal vasculature and retinal vessel diameter in both control and Mitf mutant animals under anesthesia without previous visual stimulation through the use of fluorescein salt to enhance contrast. However, other mouse models with different mutations, as well as albino mice, have not been investigated with the current method. Further investigation is needed to explore and analyze vessel diameter in other strains of mice. 
Since the fundus camera is equipped with a rat objective lens, we believe that this method could be applied to rats. As different anesthetics have been demonstrated to impact functional retinal blood flow in the rat eye, they are likely to also impact retinal vascular diameter19; as a result, it is crucial to emphasize that choosing an anesthetic requires careful thought. A combination of ketamine/xylazine was used in this study, and it is possible that this had some impact on how the retinal vascular sizes were measured. An alternative anesthesia method could be isofluorane inhalation via a mask, frequently used for similar procedures in mice.
It is crucial to time the acquisition and analysis of retinal vascular images so that the diameter measurements can be made 5 to 10 min following intraperitoneal fluorescein injection. In fact, defining the ideal period of picture acquisition as carried out in this study in a single example may be advised for each laboratory and study setup individually. The program intensity threshold was set to 80% on every image under analysis. The vessels were put through the MATLAB program after being carefully chosen. The reproducibility of the results was evaluated as a part of the MATLAB program's validation. It is also possible that the fundi of some of the Mitf mutant mice had background levels that are greater than the 80% cutoff, but the software would have recognized this, and is therefore equally likely to be a small error. Branching is a variable that affects the analysis of the retinal vasculature and must be properly considered while using the program to carry out the measurements. The process of determining vascular diameter must be entirely automatic in the future, but this has proven to be a challenging task thus far due to the variations in fluorescence intensities, potential vascular leakage, and other variations in the retinal vasculature between our Mitf mutant mouse model.

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.

<table><tbody><tr><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>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 &amp;amp; Lomb</td><td></td><td>Mydriatic agent</td></tr><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></tr></tbody></table>

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 from the center of the optic disk. FFA images are taken 5 min after fluorescein injection (Figure 1A and Figure 1B). The analysis software then calculates the shortest route between each of the 30 relevant end points and each of the start points. The outcome of this processing is a figure with the points and the measurements represented by a white line between them; examples of such images from a control and Mitf mutant mouse are shown in Figure 1A and Figure 1B,&#160;respectively.&#160;However,the black arrows shown in Figure 1B are minor errors that may occur, but the software does not read these errors as retinal vessels. Moreover, the program calculates the mean vessel diameter of each retinal vessel in pixels based on the 30 end points determined on the vessel, starting from the superior central vessel above the optic disk (as vessel 1) and then clockwise around the disk in numerical order. The software then presents the mean vessel diameter in a graph as a function of vessel number; examples of these graphs from a wild-type and mutant mouse are displayed in Figure 2A and Figure 2B, respectively. Along with the measurement values for each vessel, it also offers an Excel document with the mean, median, and standard deviation computations of vessel diameter in pixels. These tables are obtained for all the mice investigated in this study (Table 1 and Table 2) from the software; therefore, the values are not copied into another program. The value &#34;N&#34; in Table 1 and Table 2 corresponds to the number of points taken from each vessel and calculated by the program. When this value is less than or equal to 5, the software reads it as not applicable (N/A), and therefore it does not correspond to a retinal vessel.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64964/64964fig01.jpg" /> 
Figure 1: Fluorescein angiography images. The images from a control (A) and Mitfmi-vga9/+ mutant (B) mouse used in this study. The red dots correspond to the center and the edge of the optic disk. The white lines on the vessels show where measurements of the shortest distance between points are taken. The black arrows indicate possible errors in the code. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64964/64964fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64964/64964fig02.jpg" /> 
Figure 2: The mean vessel diameter as a function of vessel number. Mean vessel diameter (in pixels &#177; standard deviation [SD]) of the main retinal vessels in a fundus image from a wild-type (A) and Mitfmi-vga9/+ mutant (B) mouse. The abscissa in both panels indicates the vessel number of each vessel, counting from the superior central vessel above the optic disk as vessel number one, and then clockwise from that vessel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64964/64964fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Mean retinal vessel diameter (in pixels) in the fundus of a wild-type mouse. The values in the table are obtained from the analysis software. Each line represents data from one vessel, and the first line is the vessel in the superior central part of the fundus above the optic disk. The vessels are then numbered in a clockwise direction from that vessel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64964/64964_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Mean retinal vessel diameter (in pixels) in the fundus of a Mitfmi-vga9/+ mutant mouse. The same procedure is used as in Table 1. The values in the table are obtained from the analysis software. Each line represents data from one vessel, and the first line is the vessel in the superior central part of the fundus above the optic disk. The vessels are then numbered in a clockwise direction from that vessel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64964/64964_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File: &#34;fundusDiameter.m&#34; code for determining the retinal vessel&#39;s diameter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64964/fundusDiameter2023.zip" target="_blank">Please click here to download this File.</a>

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. ...

measuring-retinal-vessel-diameter-from-mouse-fluorescent-angiography

Nanyang Technological University

Research

JoVE Journal

Medicine

549 Views.  University of Iceland. Dit protocol biedt een eenvoudige en betrouwbare methode om automatisch de diameter van netvliesvaten te meten bij verdoofde muizen als gevolg van genmutaties of netvliesaandoeningen. Het belangrijkste voordeel van deze techniek is dat het op maat geschreven MATLAB-programma hier praktisch, gebruiksvriendelijk, handig en betrouwbaar is. Veranderingen in de diameter van het netvlies komen vaak voor bij degeneratieve ziekten zoals RP en andere vasculaire aandoeningen van het netvlies.Mui...

Video: Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Neuroscience

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

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