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>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+retina+to+study+dementia+and+stroke.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vasculature+development+in+health+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+retinal+vessel+attenuation+in+eyes+with+retinitis+pigmentosa.">Quantitative analysis of retinal vessel attenuation in eyes with retinitis pigmentosa.</a> Investigative Ophthalmology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vessel+oxygen+saturation+and+vessel+diameter+in+retinitis+pigmentosa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenic+therapy+for+ischemic+retinopathies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+optic+axon+dystrophy+and+vacuslar+changes+in+rd+mice.">Progressive optic axon dystrophy and vacuslar changes in rd mice.</a> Investigative Ophthalmology &amp; Visual Science. 41 (2), 537-545 (2000).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+cells+influence+retinal+vascular+degeneration+in+mouse+models+of+retinal+degeneration+and+diabetes.">Photoreceptor cells influence retinal vascular degeneration in mouse models of retinal degeneration and diabetes.</a> Investigative Ophthalmology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocytes+and+the+microphthalmia+transcription+factor+network.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+of+the+microphthalmia+locus+and+its+gene.">The discovery of the microphthalmia locus and its gene.</a> Mitf. Pigment Cell &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microphthalmia-associated+transcription+factor+(Mitf)+gene+and+its+role+in+regulating+eye+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+promoter+use+in+eye+development:+the+complex+role+and+regulation+of+the+transcription+factor+MITF.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitf+induction+by+RANKL+is+critical+for+osteoclastogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+development+of+signaling+pathways+inhibitors+of+melanogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+microphthalmia-associated+transcription+factor+(Mitf)+mutations+affect+the+structure+of+the+retinal+vasculature.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+retinal+vasculature.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+vessel+diameter+quantification+and+vessel+tracing+for+OCT+angiography.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+evaluation+of+neurovascular+coupling+in+the+murine+retina+by+dynamic+retinal+vessel+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+anesthetic+protocols+for+functional+blood+flow+imaging+in+the+rat+eye.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>
		</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/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/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/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/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/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

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470 Views.  University of Iceland. 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. 

Video: Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

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 ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...