We thank Carlos Mas, Mar&#237;a Pilar Crespo, and Cecilia Sampedro of CEMINCO (Centro de Micro y Nanoscop&#237;a C&#243;rdoba, CONICET-UNC, C&#243;rdoba, Argentina) for assistance in confocal microscopy, to Soledad Mir&#243; and Victoria Blanco for dedicated animal care and Laura Gatica for histological assistance. We also thank to Victor Diaz (Pro-Secretary of Institutional Communication of FCQ) for the video production and edition and Paul Hobson for his critical reading and language revision of the manuscript.
This article was funded by grants from Secretar&#237;a de Ciencia y Tecnolog&#237;a, Universidad Nacional de C&#243;rdoba (SECyT-UNC) Consolidar 2018-2021, Fondo para la Investigaci&#243;n Cient&#237;fica y Tecnol&#243;gica (FONCyT), Proyecto de Investigaci&#243;n en Ciencia y Tecnolog&#237;a (PICT) 2015 N&#176; 1314 (all to M.C.S.).

C57BL/6J mice were handled according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Experimental procedures were designed and approved by the Institutional Animal Care and Use Committee (CICUAL) of the Faculty of Chemical Sciences, National University of C&#243;rdoba (Res. HCD 1216/18).
1. Preparation of buffer solutions and reagents
<ol>
	<li>Preparation of 1x phosphate buffer saline (PBS): Add 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 14.4 g of disodium-hydrogen-phosphate dihydrate (Na2HPO4 2H2O) and 0.24 g of potassium-dihydrogen phosphate (KH2PO4) to 800 mL of distilled water. Stir the solution until complete dissolution of the salts. Adjust the pH to 7.4 with chloride acid (HCl) and adjust the volume to 1 L with additional distilled water. Filter the solution and store it at 4 &#176;C.</li>
	<li>Preparation of 4% paraformaldehyde (PFA): Add 40 g of solid PFA to 800 mL of freshly prepared PBS. Stir the solution in a heating plate at a constant temperature of 60 &#176;C. Add 1 M sodium hydroxide (NaOH) until PFA is completely dissolved, checking that the pH is maintained at a range of 7.2-7.4. Top up the volume to 1,000 mL with PBS and mix. Turn off the heating plate and filter the solution when its temperature is under 35 &#176;C. Store the solution at 4 &#176;C for up to 3 days. 
	CAUTION: PFA is harmful if swallowed or if inhaled and is probably a mutagenic compound. Use gloves and face mask during the whole process and work in a safety cabin.</li>
	<li>Preparation of 1x Tris-buffered saline (TBS): Add 6.1 g of Tris and 9 g of NaCl to 800 mL of distilled water. Stir the solution until complete dissolution of the salts. Adjust pH to 7.4 with HCl and adjust the volume to 1 L with additional distilled water. Filter the solution and store it at 4 &#176;C.</li>
	<li>Preparation of TBS- 0.1% Triton X-100: Add 0.1 mL of TritonX-100 to 100 mL of TBS. Mix gently and store it at 4 &#176;C for up to 1 week. 
	NOTE: As Triton is a very dense detergent, slightly cut the terminal of the tip for better pipetting.</li>
	<li>Preparation of TBS-5%-Bovine Serum Albumin (BSA) -0.1% Triton-X-100: Weigh 5 g of BSA and add TBS- 0.1% triton X-100 solution up to a final volume of 100 mL. Stir gently. Aliquot and store at -20 &#176;C for up to 2 months.</li>
	<li>Isolectin GSA-IB4 Alexa fluor-488 conjugate (Isolectin GSA-IB4): Weight 36.75 mg of calcium chloride dihydrate (CaCl2&#183;2H2O) and add it to 500 mL of freshly prepared PBS. Stir the solution with a stir bar. Pipette 500 &#181;L of the solution of CaCl2/PBS and add it to the vial containing 500 &#181;g of lyophilized Isolectin GSA-IB4 powder. Mix gently and aliquot the solution in 0.5 mL tubes. Store them at -20 &#176;C protected from light.</li>
	<li>Poly(vinyl alcohol) in glycerol/Tris: Weigh 2.4 g of Poly(vinyl alcohol), add 6 g of glycerol and stir gently. Then, add 6 mL of water and continue stirring for several hours at room temperature. Add 12 mL of pre-warmed 0.2 M Tris solution (pH: 8.5) and heat at a constant temperature of 50 &#176;C for 10 min and mix occasionally. Finally, centrifuge the solution at 5,000 x g for 15 min for clarification. 
	&#8203;NOTE: Let the solution cool at room temperature, aliquot it and store it at -20 &#176;C.</li>
</ol>
2. Fluorescent lectin staining
<ol>
	<li>Upon mice sacrifice by carbon dioxide (CO2) inhalation, enucleate the eyes with scissors16. Fix the enucleated eyes with freshly prepared 4% PFA for 1 h at room temperature (RT). 
	NOTE: Fixation can also be performed by incubating the eyes in 4% PFA overnight (ON) at 4 &#176;C. Mice were sacrificed according to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Institutional Animal Care and Use Committee (CICUAL) of the Faculty of Chemical Sciences, National University of C&#243;rdoba (Res. HCD 1216/18).</li>
	<li>Under the dissecting microscope, remove the corneas with scissors by cutting along the limbus and dissect the whole retinas. Discard the anterior segment of the eye, and then separate the retina from the RPE-Choroid. 
	NOTE: Make sure to remove the remaining debris and hyaloid vessels out of the retina with forceps.</li>
	<li>Place the retinas in 200 &#181;L tubes. Then, block and permeabilize the retinas in 100 &#181;L of Tris-buffered saline (TBS) solution containing 5% Bovine Serum Albumin (BSA) and 0.1% Triton-X-100 during 6 h at 4 &#176;C with gentle agitation in the shaker. 
	NOTE: This step can be performed for 2 h at RT.</li>
	<li>Then, invert the tube to place the retina in the cap and remove the blocking solution with a pipette.</li>
	<li>Add 100 &#181;L of the solution containing 0.01 &#181;g/&#181;L of Isolectin GSA-IB4 (GSA-IB4). Wrap the tubes with aluminum foil to protect the samples from light. Incubate the retinas with the lectin solution ON at 4 &#176;C or for 2 h at RT with gentle agitation in the shaker.</li>
	<li>Wash the retina with 100 &#181;L of TBS containing 0.1% Triton-X-100 for 20 min with gentle agitation in the shaker. For proper washing, place the retina in the cap of the tube by inverting it. Return the tube to its standing position and remove the medium remaining in the container. Repeat this step three times.</li>
	<li>Place the retina in a slide and add a drop of PBS. Under the dissecting microscope, perform four equally distant cuts from the edges of the retina toward the optic nerve.</li>
	<li>To unfold the petals of the retina, use forceps or small pieces of filter paper. Ensure that the photoreceptor side is facing down.</li>
	<li>Remove the remaining PBS of the slide with filter paper. Then, add a mounting medium (Poly(vinyl alcohol) in glycerol/Tris) and coverslip. Let it dry for 1 h at RT.</li>
	<li>Store slides at 4 &#176;C protected from light. 
	&#8203;NOTE: Retinas can also be stored in PBS at 4 &#176;C until confocal microscopy visualization.</li>
</ol>
3. Confocal microscopy visualization and microphotograph acquisition
<ol>
	<li>Before visualization, incubate the slides for 10 min at RT covered from light.</li>
	<li>At the confocal microscope, place the slide in the plate face down.</li>
	<li>Focus the superior plexus of the retinal vasculature and select an area to start the image acquisition.</li>
	<li>Set general laser parameters in the software: Wavelength: 488 nm; Laser intensity; HV; Offset; Gain. 
	NOTE: Laser intensity, PMT, Offset, and gain may vary depending on the conditions and the usage of the lamp. The optimal settings provide a clear image of the vessels avoiding the saturation in the sensitivity of the photomultiplier tube to have a linear relationship with the fluorescence signal.</li>
	<li>Set other acquisition parameters: Mode; Size; Objective, without zoom; No Kalman; Step Size. 
	NOTE: Identical acquisition conditions must be used for imaging the control and experimental samples.</li>
	<li>Set the coordinates in x and y axis: Scan the retina in focus 2x. If the area shows vessels, select it by clicking twice the area in order to mark it for later image acquisition. 
	NOTE: It is highly recommendable to select retinal area by following the superior vascular plexus as the fluorescence of bigger vessels is higher.</li>
	<li>Move in the grid to a neighbor square, focus the retina, and select the area if it corresponds. Repeat this step until the selected area comprises the whole retina.</li>
	<li>Select one area and set the coordinates in z axis to define the thickness extent to scan. To do this, visualize the retina in focus 2x; with the micrometer, go to the top of the retina and click on Set at the Start position. Then, move to the bottom of the retina and click on Set at the End position.</li>
	<li>Repeat step 3.8 at every area selected in the grid. 
	NOTE: To verify the scanned area in depth, press Go at the Start position. If vessels are still visualized, readjust the position by moving the micrometer, and then click on Set. Repeat the process at the End position.</li>
	<li>Initialize automated scanning.</li>
	<li>Once the scanning process finishes, open the image.</li>
	<li>Select the Series format to record the image as a Mosaic and save it with the preferred extension. Finally, the stitched image will be shown on the screen.</li>
</ol>
4. Image processing
<ol>
	<li>In the ImageJ FIJI software, go to the Menu Bar and click on File &#62; Open. Select the image to process. In the Bio-Formats Import Options window, select the Display OME-XML metadata and click on OK. 
	NOTE: Images with similar name should be saved in separate folders.</li>
	<li>In the OME Metadata window, search for the pixel size information, named as PhysicalSizeX, PhysicalSizeY, and PhysicalSizeZ. Copy this data.</li>
	<li>For image calibration, go to the Menu Bar and select Image &#62; Properties. Copy the information of the image in step 4.2 and click on OK. The image opens as a window with several photos, where each one represents the microphotographs acquired at a z axis.</li>
	<li>Assign a preferred lut to the image in order to consign a color to the fluorescent label. To do that, go to the Menu Bar and click on the LUT icon.</li>
	<li>To visualize all stacks in a single image, go to the Menu Bar and select Image &#62; Stacks &#62; Z Project.</li>
	<li>In the Z Projection window displayed, select all the stacks where vessels are visualized. In Projection Type, choose Average Intensity and click on OK. 
	NOTE: The result of the sum of the stacks will be shown in a new image named AVG (image name).</li>
	<li>Adjust the brightness and contrast in the resulting image to reduce the background and highlight vessels. Go to the Menu Bar and click on Image&#62; Adjust&#62; Brightness/Contrast. In the B and C windows move the bars until a better intensity is observed. Click on Apply and save the changes.</li>
	<li>Copy the parameters selected for Brightness/Contrast; these must be identical for all images.</li>
	<li>Prior to image quantification, define the parameters that are going to be measured. In the Menu Bar, go to Analyze &#62; Set Measurements. In the procedures described here, it will be necessary to obtain Area and Perimeter (this is ultimately also needed to measure distances). Click on OK.</li>
	<li>Download the plugin required for vessel density quantification17&#160;and follow the installations instructions provided by the plugin.</li>
	<li>Continue with the quantification of a specific vascular parameter.</li>
</ol>
5. Quantification of avascular areas
<ol>
	<li>In the Menu Bar, choose the Wand Tool. Select the retinal area that lacks vessels. 
	NOTE: Bigger or smaller areas can be selected depending on the tolerance. To adjust the tolerance, click on the wand tool icon twice. Mode employed: Legacy.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 5.1 and 5.2 with all the avascular areas of the retina.</li>
	<li>Click on Measure in the ROI manager to obtain the avascular area information. The program will display a new window with the information required. Copy the data in another program or save the file. 
	NOTE: In some images, one may prefer to select the avascular areas manually. In this case, choose the Polygon Selection tool, draw short distances around the avascular area, and click on the image when a new distance is about to start. Close the selected area by clicking on the first point.</li>
	<li>Repeat steps 5.1, 5.2, and 5.3 to measure the total area of the retina.</li>
	<li>Calculate the avascular area of the retina as the sum of all the avascular areas measured divided by the total area of the retina.</li>
</ol>
6. Quantification of neovascular areas
<ol>
	<li>In the Menu Bar, choose the Wand tool.</li>
	<li>Zoom in the image to better visualize neovessels. Select the neovessels one by one with the wand tool. 
	NOTE: Adjust the tolerance no higher than 20 to ensure the selection of a single neovessel. This tolerance level must remain constant during all image quantifications.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 6.1 and 6.2 with all neovascular areas of the retina.</li>
	<li>Click on Measure in the ROI manager to obtain the area data. The program will display a new window with the information required. Copy the data in another program or save the file.</li>
	<li>Repeat steps 6.1, 6.2, and 6.3 to quantify the total area of the retina.</li>
	<li>Calculate the neovascular area of the retina as the sum of all the neovessels&#39; areas measured divided by the total area of the retina.</li>
</ol>
7. Quantification of vessel diameter
<ol>
	<li>In the Menu Bar, select the Straight-Line Tool.</li>
	<li>Zoom in the image to better visualize the vessel wall. Perform three transversal lines to the main vessel before the first branch. This must be done by drawing a line from the boundary of one wall of the vessel to the opposite. 
	NOTE: The line must be perfectly perpendicular to the wall vessel to avoid quantification errors.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. Repeat steps 7.1 and 7.2 in all the vessels that need to be quantified.</li>
	<li>Click on Measure in the ROI manager to obtain the distance data. The program will display a new window with the information required. Copy the data in another program or save the file.</li>
</ol>
8. Quantification of vessel tortuosity
<ol>
	<li>In the Menu Bar, click on the Straight-Line Tool icon with the right button of the mouse and select Segmented Line or Freehand Line. Draw a line inside the vessel from the optic nerve until the first vessel ramification following&#160;the vascular shape. 
	NOTE: The line must follow the vascular shape to avoid quantification errors.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager.</li>
	<li>In the Menu Bar, select the Straight-Line Tool. Draw a line from the optic nerve until the first vessel ramification.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager.</li>
	<li>Click on Measure in the ROI Manager to obtain the distances data. The program will display a new window with the information required. Copy the data in another program or save the file.</li>
	<li>Calculate the tortuosity index as follows: distance obtained with Segmented line divided by the distance obtained with Straight line.</li>
</ol>
9. Quantification of vascular branching
<ol>
	<li>With the Oval Tool of the Menu Bar, define an area applied equally to all experimental conditions. The selected area must be a concentric circle drawn around the optic nerve.</li>
	<li>Press the T letter on the keyboard to record the selected area in the ROI Manager. 
	NOTE: In this case, it is recommendable to save the ROI as the quantification is slow and the identical ROI must be used in all images. To do this, in the ROI Manager click on MORE &#62; Save.</li>
	<li>Manually, count the number of primary branches arising from main vessels inside the selection area.</li>
	<li>Repeat steps 9.2 and 9.3 in neighbor areas, maintaining the optic nerve-periphery criteria.</li>
</ol>
10. Quantification of vascular density
<ol>
	<li>Transform the image to 8-bit mode.</li>
	<li>Go to Plugins in the Menu Bar, select Vessel Analysis &#62; Vascular Density.</li>
	<li>Define an area with the Square tool of the Menu Bar, where the vessel density needs to be measured. Click on OK.</li>
	<li>The program will display a new window with the information required. Copy the data in another program or save the file.</li>
	<li>Repeat steps 10.2, 10.3, and 10.4 in the neighboring areas, maintaining the ROI but placing it nine times encompassing the periphery and the center of the whole retina.</li>
</ol>

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H., Wang, Z., Sun, Y., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ocular+angiogenesis:+from+development+to+pathologies.">Animal models of ocular angiogenesis: from development to pathologies.</a> FASEB Journal. 31 (11), 4665-4681 (2017).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+choroidal+and+retinal+neovascularization.">Animal models of choroidal and retinal neovascularization.</a> Progress in Retinal and Eye Research. 29 (6), 500-519 (2010).</li><li>Kern, T. S., Antonetti, D. A., Smith, L. E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+diabetic+retinopathy:+Contribution+and+limitations+of+laboratory+research.">Pathophysiology of diabetic retinopathy: Contribution and limitations of laboratory research.</a> Ophthalmic Research. 62 (4), 196-202 (2019).</li><li>Lorenc, V. E., 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=IGF-1R+regulates+the+extracellular+level+of+active+MMP-2,+pathological+neovascularization,+and+functionality+in+retinas+of+OIR+mouse+model.">IGF-1R regulates the extracellular level of active MMP-2, pathological neovascularization, and functionality in retinas of OIR mouse model.</a> Molecular Neurobiology. 55 (2), 1123-1135 (2018).</li><li>Ma, N., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+microglia+as+passenger+leukocytes+to+the+fate+of+intraocular+neuronal+retinal+grafts.">Contribution of microglia as passenger leukocytes to the fate of intraocular neuronal retinal grafts.</a> Investigative Ophthalmology &amp; Visual Science. 39 (12), 2384-2393 (1998).</li><li>Mazzaferri, J., Larriv&#233;e, B., Cakir, B., Sapieha, P., Costantino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+machine+learning+approach+for+automated+assessment+of+retinal+vasculature+in+the+oxygen+induced+retinopathy+model.">A machine learning approach for automated assessment of retinal vasculature in the oxygen induced retinopathy model.</a> Scientific Reports. 8 (1), 3916 (2018).</li><li>Milde, F., Lauw, S., Koumoutsakos, P., Iruela-Arispe, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+retina+in+3D:+quantification+of+vascular+growth+and+remodeling.">The mouse retina in 3D: quantification of vascular growth and remodeling.</a> Integrative Biology: Quantitative Biosciences from Nano to Macro (Camb). 5 (12), 1426-1438 (2013).</li><li>Yang, T., 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=Pericytes+of+indirect+contact+coculture+decrease+integrity+of+inner+blood-retina+barrier+model+in+vitro+by+upgrading+MMP-2/9+activity.">Pericytes of indirect contact coculture decrease integrity of inner blood-retina barrier model in vitro by upgrading MMP-2/9 activity.</a> Disease Markers. 2021, 7124835 (2021).</li><li>Huang, Q., Wang, S., Sorenson, C. M., Sheibani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDF-deficient+mice+exhibit+an+enhanced+rate+of+retinal+vascular+expansion+and+are+more+sensitive+to+hyperoxia-mediated+vessel+obliteration.">PEDF-deficient mice exhibit an enhanced rate of retinal vascular expansion and are more sensitive to hyperoxia-mediated vessel obliteration.</a> Experimental Eye Research. 87 (3), 226-241 (2008).</li><li>Jiang, H., Zhang, H., Jiang, X., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+D-amino+acid+oxidase+prevents+retinal+neurovascular+pathologies+in+diabetic+rats.">Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.</a> Diabetologia. 64 (3), 693-706 (2021).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Animal models of retinopathies are powerful tools for studying vascular development, remodeling, or pathological angiogenesis. The success of these studies in the field relies on the easy access to the tissue that allows to perform a wide variety of techniques, providing data from in vivo and postmortem mice26,27. Moreover, great correlation has been found between in vivo studies and clinical analysis, providing solid traceability and reliability to these models28. In this article, we present a simple and robust description of the steps to characterize the vascular network in mouse models of retinal vascular diseases. In the literature, readers will find other possible parameters to measure and parallel approaches to quantify those selected here. The compiled protocols have many benefits over others because they are reproducible, and they just require a free software to perform the analysis (Image J Fiji).
Moreover, these protocols are easy to be performed by students that do not have extensive knowledge about the program and most of the measurements do not need additional plugins (except from vascular density).
Sample extraction and fluorescence staining technique are simple and brief29. It is very important to work with fresh retinas, although they can be stored at 4 &#176;C in PBS with inhibitors of bacterial growth. As&#160;the tissue is thin and soft, then old samples frequently break down during the incubation or when unfolding the sample before mounting. Besides, excessive fixation with 4% PFA turns the retina excessively rigid and brittle, but this does not affect the lectin staining. Isolectin GSA-IB4 staining allows to visualize the complete vascular network at every layer, including neovessels and proliferating endothelial cells. Other markers, as CD 31, requires the incubation of the sample with concentrated antibodies and they have more background. On the other hand, in angiography, the fluorescent dye tends to diffuse out of the vessels, which increases the variability in the vessel caliber and tortuosity quantification. One disadvantage of Isolectin GSA-IB4 is that it can also label microglial cells30. Care should be taken when carrying out intravitreal injections or other experimental procedures that induce excessive infiltration because the microglial cells can form agglomerates.
Image acquisition could be performed with any confocal microscope. The use of a motorized plate couple to a software that includes the Stitching module will provide complete visualization of the whole mounted retina in a single image. When selecting the retinal area, make sure all extremes of the sample are included, otherwise the photo will be incomplete (Figure 1, yellow arrows). This is not very relevant in the mouse model of OIR, as an example, because vascular alterations are near the optic nerve. While in the rat model of OIR, this mistake can lead to inaccurate quantification, as the avascular and neovascular areas are next to the limbus. Another possibility is to take individual retinal microphotographs. The user can later organize them as a puzzle with a proper software. It is not recommendable to use epifluorescence microscope, especially when analyzing branching, because vessels sprout throughout the whole thickness of the retina and some vessels will not be detected in a solely z stack.
During image processing with ImageJ FIJI, it is recommended not to update the software until all the images are quantified, in order to keep identical conditions in the software. The calibration of the images (assignment of pixel size in microns) is a critical step as these protocols imply the quantification of distances and areas. When familiarized with the program, the user can explore other tools to select the neovascular and the avascular area in addition to the wand tool. The polygon selection tool is particularly useful to measure avascular areas that have been cut in two parts at the mounting step. Other researchers have created specific plugins to automate the selection of neovessels based on the fluorescence intensity of these structures. These methods are faster and less laborious, although it is recommended to check that small less-shiny neovessels have been selected prior to quantification31. The areas of neovessels will fluoresce more intensely than the surrounding normal vessels. The Magic Wand tool selects areas that are similarly colored and the tolerance determines how similar in color are the pixels selected. If a neovessel is of similar intensity to normal vessels, the tolerance may be adjusted downward to increase the sensitivity to subtle intensity differences. Although neovessel selection in every sample will be performed with an identical threshold, some neovessels are characterized by a minor fluorescence intensity. In this case, a lower tolerance will be required for proper selection.
Tortuosity and dilatation are measurements of distance. A comprehensive study of the vascular tree is helpful to select vessels of identical type (arteries or veins) and caliber. We measure the dilatation in vessels at three heights as shown in Figure 4. The angle of the straight selection respect to the vessel wall is another relevant point to take into account. This should be as close as possible to 90&#176; to avoid possible errors. When drawing the straight line, users can use the zoom tool to approach the vessel of interest.
Vascular branching can also be measured in two or more quantification areas of different heights, if desired. In this case, researchers should conserve the region of the retina analyzed (central, medium, or peripheral side of the optic nerve). Although branching is altered in several animal models of retinopathy, early stages of the retinopathy associated to MetS presented here still do not show such changes15. For extensive studies in retinal capillaries of the intermediate and deep plexus and vessel sprouting, 3D computational methods provide valuable data of subtle variations in vascular morphology and network32. Finally, the vascular density is determined by the area occupied by vessels with respect to the total area of a ROI selected. The measurement of this parameter requires the use of a plugin and an actualized version of ImageJ FIJI, where Auto Threshold and Geometry to Distance Map tools must be available. Despite these additional steps, the plugin is easy to use, reproducible and reliable. For the measurement of vascular density, a ROI of 0.015 mm2 was chosen, in order to make ten measurements of the fluorescence intensity in different regions of the retina. This size allowed us to cover the entire photo, thus obtaining a more representative average value and its deviation in each retina.
Overall, despite the fact that these collections of methods are not automated and require manual selection of the parameters, they are able to provide quantitative and rigorous data of the retinas. The main limitation regarding the above presented protocols for vascular measurements relies on the inter-examiner variability when analyzing the same image. To minimize this bias, it is important to pre-establish the general setting as vessel wall boundaries, identification of avascular areas and neovessels, and also exclusion criteria for damaged tissues. Regarding vessel tortuosity, an agreed starting point next to the optic nerve is required. For beginner users, it is highly recommended to average the data collected by two or more examiners. In trained users, no significant differences have been found in the measurement of various parameters, as long as agreement in the quantification criteria remain constant. Similarly, additional parameters can be added to the listed protocols, depending on the vascular alterations detected in the retinopathy. In non-proliferative retinopathies, the measurement of acellular capillary number, migrating pericytes, ratio of pericytes / endothelial cells number and blood retinal barrier permeability are the earliest parameters altered in the retinal vasculature33,34,35. For chronic models and mild retinopathies, it is advisable to include these additional measurements. A major number of parameters measured will provide a more faithful landscape of the events taking place in the vasculature. In summary, in this article, we have shown classical, well-established and reproducible techniques to quantify some of the most relevant parameters taken into account in the clinical practice.

The eyes are nourished by two arterio-venous system: the choroidal vasculature, an external vascular network that irrigates retinal pigmented epithelium and photoreceptors; and the neuro-retinal vasculature that irrigate the ganglion cells layer and the inner nuclear layer of the retina1. The retinal vasculature is an organized network of vessels that deliver nutrients and oxygen to the retinal cells and harvest waste products to ensure proper visual signaling transduction. This vasculature has some distinct features, including: the lack of autonomous innervation, the regulation of vascular tone by intrinsic retinal mechanisms and the possession of a complex retinal-blood barrier2. Therefore, retinal vasculature has been the focus of many researchers who have extensively studied not only vasculogenesis during the development, but also the alterations and the pathological angiogenesis that these vessels undergo in diseases3. The most common vascular changes observed in retinopathies are vessel dilatation, neovascularization, loss of vascular arborization and deformation of the retinal main vessels, which makes them more ziggaggy4,5,6. One or more of the described alterations are the earliest signs to be detected by clinicians. Vascular visualization provides a rapid, non-invasive, and inexpensive screening method7. The extensive study of the alterations observed in the vascular tree will determine whether the retinopathy is non-proliferative or proliferative and the further treatment. The non-proliferative retinopathies can manifest themselves with aberrant vascular morphology, decreased vascular density, acellular capillaries, pericytes death, macular edema, among others. In addition, proliferative retinopathies also develop increased vascular permeability, extracellular remodeling, and the formation of vascular tufts toward the vitreous cavity that easily breakdown or induce retinal detachment8.
Once detected, the retinopathy can be monitored through its vascular changes9,10. The progression of the pathology can be followed through the structural changes of the vessels, which clearly define stages of the disease11. The quantification of vascular alterations in these models allowed to correlate vessel changes and neuronal death and to test pharmacological therapies for patients in different phases of the disease.
In light of the above statements, we consider that the recognition and quantification of vascular alterations are fundamental in retinopathies studies. In this work, we will show how to measure different vascular parameters. To do that, we will employ two animal models. One of them is the Oxygen-induced retinopathy mouse model12, which mimics Retinopathy of Prematurity and some aspects of proliferative Diabetic Retinopathy13,14. In this model, we will measure avascular areas, neovascular areas and the dilatation and tortuosity of main vessels. In our laboratory, a Metabolic Syndrome (MetS) mouse model has been developed, which induces a non-proliferative retinopathy15. Here, we will evaluate vascular density and branching.

<table><tbody><tr><td>Aluminuim foil</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Merck</td><td>A4503</td><td>quality</td></tr><tr><td>Calcium chloride dihydrate</td><td>Merck</td><td>C3306</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Biopack</td><td>9632.08</td><td></td></tr><tr><td>Confocal Microscope FV1200</td><td>Olympus</td><td>FV1200</td><td>with motorized plate</td></tr><tr><td>Covers</td><td>Paul Marienfeld GmnH &amp;amp; Co.</td><td>111520</td><td></td></tr><tr><td>Dissecting Microscope</td><td>NIKON</td><td>SMZ645</td><td></td></tr><tr><td>Disodium-hydrogen-phosphate dihydrate</td><td>Merck</td><td>119753</td><td></td></tr><tr><td>200 &amp;micro;L&amp;nbsp; tube</td><td>Merck</td><td>Z316121</td><td></td></tr><tr><td>Filter paper</td><td>Merck</td><td>WHA5201090</td><td></td></tr><tr><td>Incubator shaker GyroMini</td><td>LabNet International</td><td>S0500</td><td></td></tr><tr><td>Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 488 Conjugate</td><td>Invitrogen</td><td>I21411</td><td></td></tr><tr><td>Poly(vinyl alcohol) (Mowiol 4-88)</td><td>Merck</td><td>475904</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Merck</td><td>158127</td><td></td></tr><tr><td>pHmeter</td><td>SANXIN</td><td>PHS-3D-03</td><td></td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>P9541</td><td></td></tr><tr><td>Potassium-dihydrogen phosphate</td><td>Merck</td><td>1,04,873</td><td></td></tr><tr><td>Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>S3014</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>S5881</td><td></td></tr><tr><td>Tris</td><td>Merck</td><td>GE17-1321-01</td><td></td></tr><tr><td>Triton X-100</td><td>Merck</td><td>X100-1GA</td><td></td></tr><tr><td>Vessel Analysis Fiji software</td><td>Mai Elfarnawany</td><td></td><td>https://imagej.net/Vessel_Analysis</td></tr></tbody></table>

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.

As described in the protocol section, from a single fluorescent staining assay you can obtain the vascular morphology and evaluate several parameters of interest quantitatively. The search of a specific alteration will depend on the type of retinopathy studied. In this article, avascular and neovascular areas, tortuosity, and dilatation were evaluated in a mouse model of proliferative retinopathy, whereas the vascular branching and density were analyzed in a MetS mouse model, which induces a non-proliferative retinopathy.
In the first experimental example, the Oxygen-Induced Retinopathy (OIR) mouse model, was employed, which resembles the Retinopathy of Prematurity and some features of the proliferative stage of Diabetic Retinopathy. In this model, litters are maintained with their nursing mother in a hyperoxic chamber from postnatal day (P)7 to P1218. Intravitreal injections were performed at P12 to determine the effectiveness of a drug as an antiangiogenic (condition named as Treatment). Mice were sacrificed at P17, the time point of the maximum neovessels formation19. Mice injected with vehicle were employed as control. Both samples were fixed and stained with Isolectin GSA-IB4 together. After confocal acquisition, images were analyzed with ImageJ FIJI software as indicated above. With the Stitching module integrated to Confocal Microscope, the complete whole mount was observed as a unique image (Figure 1). At P17, it is possible to observe the hyaloid vasculature, a transient vascular system essential during the intrauterine life (Figure 1, white arrows)20.
As a consequence of excessive oxygen provision in the hyperoxic chamber, OIR mice arrest the physiological vascular development and generate avascular areas in the retina. Then, the avascular areas are defined as the zones that lack retinal blood vessels (Figure 2). From the images acquired, avascular areas can be quantified as the sum of the regions without vessels divided by the total area of the retina. Areas with mechanical damage also show absence of vessels. To identify damaged areas, analyze the integrity of neuronal layers with Hoechst stain.
The avascular areas of the retina avoid oxygen delivery and, therefore, a strong pro-angiogenic response is set, inducing neovascularization16. Neovessels are small caliber vessels that originate from a pre-existing vessel of the superior vascular plexus. Their structure is disorganized, therefore, neovessels grow as tufts toward the vitreous cavity21. We calculated the area occupied by neovessels&#39; tufts by quantifying the neovascular area of the retina divided by the total area of the retina (Figure 3). As neovessels&#39; shape and size is variable, occasionally they can look similar to debris and artifacts. To distinguish neovessels, verify that the tuft grows from a mature vessel.
Vascular dilatation and tortuosity are other two frequent alterations, which have negative effects on vascular biology, as they produce turbulent blood circulation. For the analysis of the dilatation, we measured the main vessels diameter at three heights before the first branch22,23 (Figure 4). Researchers must define a predetermined distance to measure vessel diameter in order to reduce variability among results. Ideally, the furthest point from the optic nerve should be around 300 &#181;m. We suggest performing the analysis and later take an average of at least six mice per condition.
Regarding to tortuosity, we measure the distance covered by the main vessels following its shape with respect to the straight distance between the optic nerve and the first branching point24 (Figure 5). As we can see in the image, there is heterogeneity in the sinuosity of the main vessels. To obtain a representative result, not less than six mice per condition must be quantified.
The latest parameters, vascular branching and density, were analyzed in the MetS model exhibiting non-proliferative retinopathy. In MetS, both lipids and carbohydrates derangements are associated to retinal pericytes and endothelial cells dysfunction and death, leading to the formation of acellular capillaries or a decreased vascular branching25. In our model, ApoE-KO mice were fed with fructose added to the drinking water, at a concentration of 10% w/v. Control animals just received tap water. After 4 months of diet, mice were sacrificed, and the retinas processed as indicated before. For the measurement of vascular branching, we defined the quantification area by drawing a concentric circle, and then we counted, one by one, the primary branches arising from a main central vessel (Figure 6).
From the images acquired, the vascular density was quantified as the area occupied by vessels divided by the area of the ROI (a square of 0.015 mm2, see flatten in Figure 7), which was positioned in different places in each microphotograph, as it was explained in the protocol section. Avoid quantifying areas with mechanical damage (Figure 7, white arrows). If there are more than two areas with punctures, the retina must be discarded.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63126/63126fig01.jpg" /> 
Figure 1: Vascular staining of retinas whole mount labeled with Isolectin GSA-IB4: Representative confocal images of P17 OIR mice retinas injected with vehicle or treatment at P12. Fluorescent staining was performed in whole mount retinas with Isolectin GSA-IB4 Alexa 488 according to the protocol. Avascular areas (AV) and neovascular areas (NV) are indicated. Scale bar: 500 &#181;m. White arrows show the remnant hyaloid vasculature. Yellow arrows show incomplete scanning areas during confocal image acquisition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63126/63126fig01large.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/63126/63126fig02.jpg" /> 
Figure 2: Quantification of avascular areas: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Areas with vaso-obliteration are indicated in yellow. Scale bar: 500 &#181;m. (B) The AV area (%) was quantified as the ratio of central avascular area to whole retinal area. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean &#177; SEM. *** p &#60; 0.001. At least six animals were employed for each condition in the survival time examined. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63126/63126fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63126/63126fig03.jpg" /> 
Figure 3: Quantification of neovascular areas: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Areas with neovascularization are indicated in white. Scale bar: 500 &#181;m. (B) The NV area (%) was quantified as the ratio of the area occupied by neovessels to whole retinal area. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean &#177; SEM. *** p &#60; 0.001. At least six animals were employed for each condition in the survival time examined. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63126/63126fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63126/63126fig04.jpg" /> 
Figure 4: Quantification of vascular dilatation: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Left panels: ROIs selected for quantification. Right panels: zoom of the ROIs, showing straight lines performed in a main vessel to measure the vessel diameter. Three lines were traced transversally to the vessel and averaged. Scale bar: 100 &#181;m. (B) The vessel diameter (%) was quantified as the average diameter measured in major vessels of the retina. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean &#177; SEM. *p &#60; 0.05. At least six animals were employed for each condition in the survival time examined. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63126/63126fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63126/63126fig05.jpg" /> 
Figure 5: Determination of tortuosity index: (A) Representative images of whole mount retinas at P17 showing GSA-IB4 vascular staining in OIR-vehicle and OIR-treatment mice. Left panels: ROIs selected for quantification. Center panels: zoom of the ROIs, showing segmented lines traced in a main vessel from the optic nerve to the first branching point. Right panels: zoom of the ROIs, showing straight lines traced in a main vessel from the optic nerve to the first branching point. Scale bar: 100 &#181;m. (B) The tortuosity index was obtained by dividing the distance of the segmented line to the distance of the straight line. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean &#177; SEM. ns, non-significant. At least six animals were employed for each condition in the survival time examined. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63126/63126fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63126/63126fig06.jpg" /> 
Figure 6: Quantification of vascular branching: (A) Representative images of whole mount retinas showing Isolectin GSA-IB4 vascular staining in ApoE-KO and ApoE-KO + fructose fed mice. Circular quantification areas were defined in yellow. Scale bar: 100 &#181;m, 100x magnification. (B) The number of branches was quantified as the number of primary branches from a main vessel, since the optic nerve to the periphery. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean &#177; SEM. ns, non-significant. At least six animals were employed for each condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63126/63126fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63126/63126fig07.jpg" /> 
Figure 7: Quantification of vascular density: (A) Representative images of whole mount retinas showing Isolectin GSA-IB4 vascular staining in ApoE-KO and ApoE-KO + fructose fed mice. Square of 0.015 mm2 quantification area was defined in yellow. Scale bar: 100 &#181;m, 100x magnification. (B) Vascular density was quantified as the ratio of vascular area to total ROI area, which was positioned nine times in different places in each microphotograph. White arrows show areas with mechanical damage. Two-tailed unpaired t-test was used for statistical comparison. Data presented as mean &#177; SEM. ns, non-significant. At least six animals were employed for each condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63126/63126fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies at JoVE.com

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with 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, ...

quantification-vascular-parameters-whole-mount-retinas-mice-with-non

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.5K Views.  Universidad Nacional de Córdoba. Bij retinopathieën zijn vasculaire veranderingen het eerste teken dat door clinici wordt gedetecteerd. Ze volgen de progressie van de ziekte en ze kiezen een behandeling op basis van deze veranderingen. Verschillende diermodellen zijn ontwikkeld om verschillende stadia van de pathologie te bestuderen.In dit werk gaan we laten zien hoe we verschillende vasculaire veranderingen kunnen meten. Daarvoor gaan we twee diermodellen inzetten. Een daarvan is het zuurstof-geïnduceerde retinopat...

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

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 ...

Medicine

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

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

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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 ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

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

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.

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. ...

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

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