Name: quantification of vascular parameters in whole mount retinas of mice with non-proliferative and proliferative retinopathies
Uploaded: 2022-03-12T15:00:00
Description: 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

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....

<ol>
	<li>Kur, J., Newman, E. A., Chan-Ling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+physiological+mechanisms+underlying+blood+flow+regulation+in+the+retina+and+choroid+in+health+and+disease.">Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease.</a> Progress in Retinal and Eye Research. 31 (5), 377-406 (2012).</li><li>McDougal, D. H., Gamlin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+control+of+the+eye.">Autonomic control of the eye.</a> Comprehensive Physiology. 5 (1), 439-473 (2015).</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>Wei, 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=Age-related+alterations+in+the+retinal+microvasculature,+microcirculation,+and+microstructure.">Age-related alterations in the retinal microvasculature, microcirculation, and microstructure.</a> Investigative Ophthalmology &amp; Visual Science. 58 (9), 3804-3817 (2017).</li><li>Lavia, C., 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=Reduced+vessel+density+in+the+superficial+and+deep+plexuses+in+diabetic+retinopathy+is+associated+with+structural+changes+in+corresponding+retinal+layers.">Reduced vessel density in the superficial and deep plexuses in diabetic retinopathy is associated with structural changes in corresponding retinal layers.</a> PLoS One. 14 (7), 0219164 (2019).</li><li>Rosenblatt, T. 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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>Han, N., Xu, H., Yu, N., Wu, Y., Yu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-203a-3p+inhibits+retinal+angiogenesis+and+alleviates+proliferative+diabetic+retinopathy+in+oxygen-induced+retinopathy+(OIR)+rat+model+via+targeting+VEGFA+and+HIF-1&#945;.">MiR-203a-3p inhibits retinal angiogenesis and alleviates proliferative diabetic retinopathy in oxygen-induced retinopathy (OIR) rat model via targeting VEGFA and HIF-1&#945;.</a> Clinical and Experimental Pharmacology &amp; Physiology. 47 (1), 85-94 (2020).</li><li>Paz, M. C., 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=Metabolic+syndrome+triggered+by+fructose+diet+impairs+neuronal+function+and+vascular+integrity+in+ApoE-KO+mouse+retinas:+Implications+of+autophagy+deficient+activation.">Metabolic syndrome triggered by fructose diet impairs neuronal function and vascular integrity in ApoE-KO mouse retinas: Implications of autophagy deficient activation.</a> Frontiers in Cell and Developmental Biology. 8, 573987 (2020).</li><li>Connor, K. 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=Quantification+of+oxygen-induced+retinopathy+in+the+mouse:+a+model+of+vessel+loss,+vessel+regrowth+and+pathological+angiogenesis.">Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.</a> Nature Protocols. 4 (11), 1565-1573 (2009).</li><li>Zarb, 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=Ossified+blood+vessels+in+primary+familial+brain+calcification+elicit+a+neurotoxic+astrocyte+response.">Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response.</a> Brain. 142 (4), 885-902 (2019).</li><li>Smith, L. 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=Oxygen-induced+retinopathy+in+the+mouse.">Oxygen-induced retinopathy in the mouse.</a> Investigative Ophthalmology &amp; Visual Science. 35 (1), 101-111 (1994).</li><li>Subirada, P. V., 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=Effect+of+autophagy+modulators+on+vascular,+glial,+and+neuronal+alterations+in+the+oxygen-induced+retinopathy+mouse+model.">Effect of autophagy modulators on vascular, glial, and neuronal alterations in the oxygen-induced retinopathy mouse model.</a> Frontiers in Cellular Neuroscience. 13, 279 (2019).</li><li>Lutty, G. A., McLeod, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+hyaloid,+choroidal+and+retinal+vasculatures+in+the+fetal+human+eye.">Development of the hyaloid, choroidal and retinal vasculatures in the fetal human eye.</a> Progress in Retinal and Eye Research. 62, 58-76 (2018).</li><li>Kim, C. B., D'Amore, P. A., Connor, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+mouse+model+of+oxygen-induced+retinopathy.">Revisiting the mouse model of oxygen-induced retinopathy.</a> Eye and Brain. 8, 67-79 (2016).</li><li>Guaiquil, V. 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=A+murine+model+for+retinopathy+of+prematurity+identifies+endothelial+cell+proliferation+as+a+potential+mechanism+for+plus+disease.">A murine model for retinopathy of prematurity identifies endothelial cell proliferation as a potential mechanism for plus disease.</a> Investigative Ophthalmology &amp; Visual Science. 54 (8), 5294-5302 (2013).</li><li>Mezu-Ndubuisi, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+angiography+quantifies+oxygen-induced+retinopathy+vascular+recovery.">In vivo angiography quantifies oxygen-induced retinopathy vascular recovery.</a> Optometry and Vision Science: Official Publication of the American Academy of Optometry. 93 (10), 1268-1279 (2016).</li><li>Scott, A., Powner, M. 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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. 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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. 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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 r...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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 &#181;L&#160; 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...

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, ...

In retinopathies, vascular alterations are the first sign to be detected by clinicians. They follow the progression of the disease, and they choose a treatment according to these changes. Several animal models have been developed in order to study different stages of the pathology.In this work, we are going to show how to measure different vascular alterations. For that, we are going to employ two animal models. One of these is the Oxygen-Induced Retinopathy mouse model, which reproduces the retinopathy of prematurity and some aspects of the proliferative diabetic retinopathy.Here, we are going to measure avascular areas, neovascular areas, and the dilatation and tortuosity of the arterioles. In our laboratory, a metabolic syndrome mouse model was developed, which induces a nonproliferative retinopathy. Vascular density and branching were evaluated in this work.Upon mice sacrifice, enucleatize with scissors. Fix enucleated eyes with freshly-prepared paraformaldehyde for one hour at room temperature. 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. Place the retinas in 200 microliter tubes. Then block and permeabilize the retinas in 100 microliters of TBS containing 5%bovine serum albumin Triton during six hours at four degrees with gentle agitation in shaker.Then invert the tube to place the retina in the cup and remove the blocking solution with pipette. Add 100 microliter solution containing isolectin. Wrap the tubes with aluminum foil to protect samples from light.Incubate the retinas with lectin solution overnight at four degrees with gentle agitation in shaker. Wash the retinas with 100 microliters of TBS-Triton for 20 minutes with gentle agitation in shaker. Repeat this step three times.Place the retina in a slide and add a drop of PBS. Under dissecting microscope, perform four equally-distant cuts from the edges of the retina towards the optic nerve. To unfold the petals of the retina, use forceps or small pieces of filter paper.Ensure that photoreceptor side is facing down. Remove the remaining PBS off the slide with filter paper. Then add a mounting median and coverslip.Let it dry for one hour at room temperature. Store slides at four degrees protected from light. At the confocal microscope, place the slide in the plate face down.Focus the superior plexus of the retinal vasculature and select an area to start the image acquisition. Set general laser parameters in the software. Set the coordinates in X, Y, and Z axes.Initialize the scanning. Once scanning process finishes, open the image and save it with a preferred extension. In the ImageJ FIJI software, go to the Menu Bar and click File, Open.Select the image to process. In the Bio-Formats Import Option window, select Display OME-XML Metadata and click OK.In the OME Metadata window, search for pixel size information. Copy this data.For image calibration, go to the Menu Bar and select Image Properties. Copy the information and click OK.Assign a preferred lut to the image. To visualize all stack in a single image, go to the Menu Bar and select Image, Stacks, Z Project.In the projection window displayed, select all the stacks where vessels are visualized. In Projection Type, choose Average Intensity and click OK.Go to the Menu Bar, Image, Adjust, Brightness/Contrast. Click Apply and save changes.Copy the parameters selected for brightness and contrast. These must be identical for all images. In the Menu Bar, choose the Wand tool.Select the retinal area that lacks formed vessels. Press T on the keyboard to record the select areas in the ROI Manager. Click Measure in the ROI Manager to obtain the avascular area information.Repeat these steps to measure the total area of the retina. In the Menu Bar, choose the Wand tool. Zoom in the image to better visualize neovessels.Select the neovessels one by one with the Wand tool. Press the T letter on the keyboard to record the selected area in the ROI Manager. Click Measure in the ROI Manager to obtain the area data.In the Menu Bar, select the Straight Line tool. Zoom in the image to better visualize the vessel wall. Perform three transversal lines to the main vessel before the first branch.Press T on the keyboard to record the selected areas in the ROI Manager. Click Measure in the ROI Manager to obtain the distance data. In the Menu Bar, click the Straight Line tool icon with the right button of the mouse and select Segmented Line.Draw a line inside the vessel from the optic nerve until the first vessel ramification, following the vascular shape. Press the T letter on the keyboard to record the selected area in the ROI Manager. In the Menu Bar, select the Straight Line tool.Draw a line from the optic nerve until the first vessel ramification. Press the T letter on the keyboard to record the selected area in the ROI Manager. Click Measure on the ROI Manager to obtain the distances data.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. Press the T letter on the keyboard to record the selected area in the ROI Manager.Manually, count the number of primary branches arising from main vessels inside the selection area. Transform the image to 8-bit mode. Go to Plugins in the Menu Bar, select Vessel Analysis, Vascular Density.Define an area with the Square tool of the Menu Bar where you want to measure vessel density. Click OK.The program will display a new window with the information required. In the first experimental example, the Oxygen-Induced Retinopathy mouse model was employed.Intravitreal injections were performed at postnatal day 12 to determine the effectiveness of a drug as an antiangiogenic. Mice injected with vehicle were employed as control. The avascular areas are defined as the zones that lacks retinal blood vessels.From the images acquired, avascular areas can be quantified as the sum of the regions without vessels divided 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.Neovessels are small caliber vessels that originate from pre-existing vessels of the superior vascular plexus. We calculated the area occupied by neovessels'tufts by quantifying the neovascular area of the retina occupied by the total area of the retina. As neovessels'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. For the analysis of the dilatation, we measured the main vessel diameter at three heights before the first branch. 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 micrometers. We suggest to perform the analysis, and later, average of at least six animals per condition. Regarding to tortuosity, we measure the distance traveled by the main vessel following its shape, respect to the straight-line distance between the optic nerve and the first branching point.As we can see in the image, there is heterogeneity in the sinuosity of 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 a metabolic syndrome model exhibiting non-proliferative retinopathy.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. From the images acquired, the vascular density was quantified as the area occupied by vessels divided by the area of the ROI, which was positioned in different places in each microphotograph. Avoid quantifying areas with mechanical damage.If there are more than two areas with punctures, the retina must be discarded. In summary, in this article, we have shown classical, well-established and reproducible techniques to quantify some of the most relevant parameters taking account in the clinical practice.

Research

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Neuroscience

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