This work was supported, in part, by a Joint Biology Consortium Microgrant provided under NIH grant P30AR070253 (P.-Y.C.), and HL135582 (M.S.). We are grateful to R. Webber and L. Coon for maintaining the mice used in this study.

mT/mG (stock no. 007676), and Apoe-/- (stock no. 002052) mice were purchased from the Jackson Laboratory. Myh11-CreERT2 mice were a gift from Stefan Offermanns (available from the Jackson Laboratory as stock no. 019079). Tgfbr2fl/fl mice were obtained from Harold L. Moses (Vanderbilt University). All animal procedures were performed using protocols approved by the Yale University Institutional Animal Care and Use Committee.
1. Mice
<ol>
	<li>Produce MYH11-CreERT2;mT/mGf/f;Apoe-/- and MYH11-CreERT2;Tgfbr2f/f;mT/mGf/f;Apoe-/- mice as previously described7. Breed mutant strains to the C57BL/6J background for more than ten&#160;generations. 
	NOTE: The Myh11-CreERT2 Cre mouse line provides a powerful tool for studying the role of smooth muscle cells in vascular homeostasis and vascular pathology. The Cre allele is inserted into the Y chromosome; thus, female mice do not express this construct.</li>
</ol>
2. Mouse genotyping, tamoxifen induction, and high cholesterol high fat diet feeding
<ol>
	<li>Perform mouse genotyping using mouse ear DNA and PCR analysis. Mouse ear DNA should be isolated using the blood and tissue DNA isolation kit (Table of Materials) according to the manufacturer&#8217;s instructions. PCR primers are listed in Table 1.</li>
	<li>Induce Cre-Lox recombination by tamoxifen injection at 1 mg/day i.p. for 5 days in 6 week-old MYH11-CreERT2;mT/mGf/f;Apoe-/- and MYH11-CreERT2;Tgfbr2f/f;mT/mGf/f;Apoe-/- male mice.</li>
	<li>Induce atherosclerosis by placing 8 week-old male mice (2 weeks after tamoxifen treatment) on a HCHF diet&#160;(40% kcal fat, 1.25% cholesterol, 0% cholic acid) for 16 weeks.</li>
</ol>
3. Reagents and dissection tool preparation
<ol>
	<li>Stock Oil Red O solution preparation: dissolve 1 g of Oil Red O in 100 mL of isopropyl alcohol.</li>
	<li>Working Oil Red O solution preparation: mix 24 mL of stock Oil Red O solution with 16 mL of dH2O. Filter the diluted Oil Red O with 0.45 &#956;m sterile syringe filters (the solution is only good for 1&#8211;2 h).</li>
	<li>60% isopropyl alcohol preparation: mix 60 mL of isopropyl alcohol with 40 mL of dH2O.</li>
	<li>4% formaldehyde in 1x DPBS preparation: dilute 10 mL of 16% formaldehyde in 30 mL of 1x DPBS.</li>
	<li>Clean all dissection tools with 70% ethanol (Figure 1).</li>
</ol>
4. Euthanasia (Figure 2A)
<ol>
	<li>Measure the mouse&#8217;s weight prior to euthanasia.</li>
	<li>Euthanize the mouse by intraperitoneal injection of ketamine and xylazine (each milliliter contains 10 mg/mL ketamine and 2 mg/mL xylazine).</li>
	<li>Place the mouse in supine position (belly side face-up).</li>
</ol>
5. Opening of chest and abdominal cavity and heart perfusion (Figure 2B)
<ol>
	<li>Prepare a 10 mL syringe with 10 mL of 1x DPBS. Cap with a 25 G needle. The syringe will be used to flush the heart.</li>
	<li>Hold up the skin with tweezers (Style 5) and cut with fine scissors from the base of the abdomen to the top of the neck.</li>
	<li>Open the abdominal wall below the ribcage.</li>
	<li>Lift the sternum with tweezers (Style 5) and cut the diaphragm, then cut away the ribcage to expose the thoracic cavity.</li>
	<li>Make a small incision in the right atrium of the heart.</li>
	<li>Perfuse through the apical left ventricular puncture by slowly injecting 10 mL of 1x DPBS. Once thoroughly perfused, the liver and kidney become light brown in color.</li>
	<li>Clean the chest cavity of extraneous blood and fluid by using a non-woven sponge to absorb the material.</li>
</ol>
6. Isolation of aorta and branches (Figure 2C)
<ol>
	<li>Remove organs (i.e., lung, liver, spleen, and gastrointestinal and reproductive organs) and cut the clavicle using tweezers (Style 5) and fine scissors while leaving the heart, kidney, and aorta intact in situ. 
	NOTE: Make sure not to lacerate the heart or any major blood vessels.</li>
	<li>Place the mouse under a stereomicroscope.</li>
	<li>Dissect aorta and aorta branches including brachiocephalic artery, carotid arteries, subclavian arteries, renal arteries, common iliac arteries, and femoral arteries using tweezers (Style 4) and spring scissors. 
	NOTE: Cover the aorta with a wet, non-woven sponge to avoid dehydration while dissecting the aorta branches.</li>
	<li>Carefully dissect and remove adventitial adipose and connective tissue around the aorta and aorta branches using tweezers (Style 4) and spring scissors. 
	NOTE: Since inflammation is prominent in the aneurysm hyperlipidemia mouse, it is difficult to remove adventitia. Be careful not to tear or nick the aorta and aorta branches. This step requires practice and patience.</li>
</ol>
7. Fixing of heart and aorta (Figure 2D,E)
<ol>
	<li>Prepare a 10 mL syringe with 10 mL of 4% formaldehyde in 1x of DPBS. Cap with a 25 G needle. 
	CAUTION: Formaldehyde is hazardous. Read the MSDS before working with this chemical. Wear gloves and safety glasses and produce the dilution solutions inside a fume hood. 
	NOTE: 4% formaldehyde solution degrades over time. It is important to use freshly made 4% formaldehyde for fixation.</li>
	<li>Fix the vascular tree through apical left ventricular puncture by slowly injecting 10 mL of 4% formaldehyde. 
	NOTE: Formaldehyde fixation interferes with several downstream applications, such as cell culture, FACS analysis, and single-cell RNA sequencing analysis. Skip this step if the aorta will be used for any of these applications.</li>
	<li>Clean the chest cavity of any extraneous fluid with a non-woven sponge to absorb the material.</li>
	<li>Separate the heart from the aorta by holding the heart with tweezers (Style 4) and using micro-dissecting spring scissors. 
	NOTE: To perform en face Oil Red O staining after this step, it is recommended to cut the aorta open in situ instead of ex vivo and proceed to section 8. This makes it easy for the en face aorta to lay flat.</li>
	<li>Isolate and excise the aorta and its major from 1 mm above the carotid artery to the end of femoral artery using tweezers (Style 4) and spring scissors.</li>
	<li>Transfer the vessel into a wax Petri dish or 1.5 mL microcentrifuge tube and fill with 1x DPBS until it covers the aorta. 
	NOTE: The protocol can be paused here.</li>
</ol>
8. Oil Red O staining and imaging of unopened whole aorta (Figure 3)
<ol>
	<li>Pin the vessel onto a wax Petri dish using minutien pins (Figure 3A).</li>
	<li>Rinse the vessel once with 1x DPBS.</li>
	<li>Pour 25 mL of fresh Oil Red O solution into the Petri dish (Figure 3B). 
	NOTE: (1) Isopropanol is hazardous and a flammable liquid. Use proper personal protective equipment. (2) Oil Red O solution can easily precipitate. The precipitated particles can interfere with subsequent staining. It is important to remove the precipitate by filtering the Oil Red O solution through a 0.45 &#956;m filter before use. (3) It is best to prepare fresh Oil Red O solution and discard any unused solution. (4) In addition to Oil Red O, Sudan IV is another chemical compound used for staining of lipids, triglycerides, and lipoproteins. However, Oil Red O has gradually replaced Sudan IV because the red color produced by Oil Red O is more intense and can thus make fat much easier to see.</li>
	<li>Stain the aorta for 60 min at room temperature (RT). Oil Red O will stain lipid-rich plaque red, leaving other non-plaque containing areas pale in color.</li>
	<li>Wash once for 20 min with 60% isopropanol at RT. 
	NOTE: Over-rinsing can destain the plaque.</li>
	<li>Rinse the aorta 3x with dH2O for 5 min to remove isopropanol.</li>
	<li>Under a stereomicroscope, gently clean all perivascular adipose tissue around the aorta using tweezers (Style 4) and spring scissors (Figure 3C,D). 
	NOTE: It is important to clean all perivascular adipose tissue around the aorta and its branches after staining, because Oil Red O-stained perivascular adipose tissue can yield false background and interfere with plaque morphometry and plaque area quantification. Make sure not to remove a portion of the aortic wall. Fill the wax dish with dH2O until it covers the stained aorta during cleaning. This step requires practice and patience.</li>
	<li>Transfer the vessel to a clean, glass microscope slide.</li>
	<li>Acquire digital micrographs using a camera connected to a light microscope. Save high resolution images, preferably in tagged image file format (TIFF) (Figure 3E). 
	NOTE: The protocol can be paused here. To prevent the aorta from drying, transfer the vessel into a 1.5 mL microcentrifuge tube and fill with 1x DPBS until it covers the aorta. Store at 4 &#176;C.</li>
</ol>
9. En face aorta mounting (Figure 4, Figure 5)
<ol>
	<li>Transfer the vessel to a wax Petri dish and fill with 1x DPBS until it covers the aorta.</li>
	<li>Sever the carotid, subclavian arteries of the aortic arch and iliac arteries in the abdominal aorta 1&#8211;2 mm after bifurcations. Sever the renal arteries. (Figure 4A)</li>
	<li>Longitudinally cut open the aorta preparation along the inner curvature (Figure 4B1) and alone iliac arteries (Figure 4B2) with micro-dissecting spring scissors.</li>
	<li>Cut open the three branches of the aortic arch (i.e., innominate, left common carotid, left subclavian artery) along the greater curvature until the base level of inner curvature (x-mark) (Figure 4B3&#8211;B8) with micro-dissecting spring scissors.</li>
	<li>Pin the aorta flat (lumen side face-up) in a wax dish with minutien pins and apply 1x DPBS until it covers the aorta to prevent it from drying (Figure 4C). 
	NOTE: (1) It is important to make the rolled-up aorta flat and pin it en face without stretching. This step will take a few days depending on the severity of atherosclerosis. (2) For aortas from Apoe-/- or Ldlr-/- animals, it is recommended to pin the aorta flat for 24 h. (3) The protocol can be paused here.</li>
	<li>Clean the glass microscope slides with 70% ethanol and delicate task wipers (Figure 5A).</li>
	<li>Transfer the aorta into a clean glass microscope slide and put 15 drops of optimal cutting temperature (OCT) compound onto another clean glass microscope slide (Figure 5B).</li>
	<li>Carefully place the glass microscope slide with OCT compound over the aorta and avoid trapping air bubbles on the slide (Figure 5C).</li>
	<li>Label the slides with sample names (Figure 5D). 
	NOTE: The mounted en face aorta slides can be stored in the moisture chamber at 4 &#176;C for several months.</li>
</ol>
10. Imaging and lesion quantification of en face aorta (Figure 6)
<ol>
	<li>Acquire digital micrographs using a camera connected to a light microscope. Save high resolution images, preferably in tagged image file format (TIFF) (Figure 6A).</li>
	<li>Transfer images of the en face stained whole aorta to a computer equipped with ImageJ software.</li>
	<li>In ImageJ, select the &#8220;Freehand selection&#8221; tool and circle all Oil Red O-stained plaque manually (intense red spots) while pressing the &#8220;Alt&#8221; key (for Windows PC) or &#8220;Shift&#8221; key (for Mac). Then, click &#8220;Measure&#8221; in the &#8220;Analyze&#8221; menu to display lesion areas in the result window (Figure 6B left). 
	NOTE: There are several pitfalls of the quantification of atherosclerotic lesions: (1) any small pieces of stained adventitial fat that remained attached to the aorta from step 8.7 can yield false background and interfere with plaque quantification; (2) removing a portion of the aortic wall or damaging the aorta from steps 6.4 and 8.7 can interfere with plaque quantification; (3) bubbles and folds formed in the aorta after mounting (step 9.8) can interfere with plaque quantification; and (4) atherosclerotic plaque is a 3D phenomenon, and measurements performed in a 2D plane may not reflect the true extent of the plaque. In addition to analysis of the en face aorta plaque area, it is recommended to analyze plaque size in the aortic root, brachiocephalic artery, ascending aorta, and abdominal aorta separately8.</li>
	<li>Circle the outer border line of the aorta and click &#8220;Measure&#8221; in the &#8220;Analyze&#8221; menu to display the aorta area in the result window (Figure 6B right).</li>
	<li>Export all measurements to an Excel file.</li>
	<li>Calculate the ratio of plaque area from the total aorta area and normalize the value as the percentage of total Oil Red O surface area.</li>
	<li>Calculate the ratio of plaque area in 8&#8211;10 Apoe-/- and 8&#8211;10 TGF&#946;R2iSMC-Apoe mice. Present the data as mean &#177; SEM (Figure 6C).</li>
	<li>Perform an unpaired Student&#8217;s t-test for statistical analysis of the ratio of plaque area data compared to another mouse group. Consider the differences in mean values as significant at p &#60; 0.05.</li>
</ol>

<ol>
	<li>Lusis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis.">Atherosclerosis.</a> Nature. 407, 233-241 (2000).</li><li>Emini Veseli, B., 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=Animal+models+of+atherosclerosis.">Animal models of atherosclerosis.</a> European Journal of Pharmacology. 816, 3-13 (2017).</li><li>Plump, A. S., 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=Severe+hypercholesterolemia+and+atherosclerosis+in+apolipoprotein+E-deficient+mice+created+by+homologous+recombination+in+ES+cells.">Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.</a> Cell. 71, 343-353 (1992).</li><li>Zhang, S. H., Reddick, R. L., Piedrahita, J. A., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+hypercholesterolemia+and+arterial+lesions+in+mice+lacking+apolipoprotein+E.">Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.</a> Science. 258, 468-471 (1992).</li><li>Ishibashi, S., 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=Hypercholesterolemia+in+low+density+lipoprotein+receptor+knockout+mice+and+its+reversal+by+adenovirus-mediated+gene+delivery.">Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.</a> Journal of Clinical Investigation. 92, 883-893 (1993).</li><li>Nakashima, Y., Plump, A. S., Raines, E. W., Breslow, J. L., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ApoE-deficient+mice+develop+lesions+of+all+phases+of+atherosclerosis+throughout+the+arterial+tree.">ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree.</a> Arteriosclerosis Thrombosis. 14, 133-140 (1994).</li><li>Chen, P. 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=Smooth+muscle+cell+reprogramming+in+aortic+aneurysms.">Smooth muscle cell reprogramming in aortic aneurysms.</a> Cell Stem Cell. 26, 542-557 (2020).</li><li>Andres-Manzano, M. J., Andres, V., Dorado, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil+Red+O+and+Hematoxylin+and+Eosin+Staining+for+Quantification+of+Atherosclerosis+Burden+in+Mouse+Aorta+and+Aortic+Root.">Oil Red O and Hematoxylin and Eosin Staining for Quantification of Atherosclerosis Burden in Mouse Aorta and Aortic Root.</a> Methods in Molecular Biology. 1339, 85-99 (2015).</li><li>Chen, P. 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=Endothelial+TGF-beta+signalling+drives+vascular+inflammation+and+atherosclerosis.">Endothelial TGF-beta signalling drives vascular inflammation and atherosclerosis.</a> Nature Metabolism. 1, 912-926 (2019).</li><li>Mehlem, A., Hagberg, C. E., Muhl, L., Eriksson, U., Falkevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+neutral+lipids+by+oil+red+O+for+analyzing+the+metabolic+status+in+health+and+disease.">Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease.</a> Nature Protocols. 8, 1149-1154 (2013).</li><li>Ferruzzi, J., Madziva, D., Caulk, A. W., Tellides, G., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compromised+mechanical+homeostasis+in+arterial+aging+and+associated+cardiovascular+consequences.">Compromised mechanical homeostasis in arterial aging and associated cardiovascular consequences.</a> Biomechanics and Modeling Mechanobiology. 17, 1281-1295 (2018).</li></ol>

The authors declare no competing financial interests.

Apolipoprotein E (Apoe) and low density lipoprotein receptor (Ldlr) deficient mice are useful for studying development and treatment of atherosclerosis. Investigators can evaluate the impact of genetics and therapeutic manipulations on atherosclerosis-related diseases initiation, progression, and regression using Oil Red O staining of the whole aorta9. Aorta Oil Red O staining and lesion quantification is the gold standard endpoint for atherosclerosis research. This technique is ...

Coronary artery disease, a leading cause of mortality in the US, is usually caused by atherosclerosis, a process that leads to the buildup of plaque inside arterial walls1. Hyperlipidemia-prone Apoe- and Ldlr-deficient mice are central to investigations of atherosclerosis and its complications and development of therapies2,3,4,5. Quantification of atherosclerotic lesions from an en face aorta is an important endpoint analysis for evaluating the impact of genetic manipulation in different cell types. It also helps to study novel therapies designed to affect atherosclerotic disease initiation, progression, and regression. Atherosclerotic lesions can form anywhere in the aorta and its branches (i.e., brachiocephalic, carotid and subclavian arteries in the chest, as well as renal, common iliac and femoral arteries below the diaphragm)6. A comprehensive evaluation of atherosclerosis burden and appropriate therapy requires assessment of disease burden in different locations, a challenge that is often overlooked.
This protocol describes how to perform a comprehensive analysis of atherosclerotic lesions, starting with an unopened whole aorta and proceeding to en face preparation, in a single mouse. Unopened whole aorta Oil Red O staining allows rapid, qualitative assessment of lipid-laden plaques in the entire aorta and its branches, while en face preparation provides a quantitative assessment of atherosclerotic lesion distribution in the mouse aorta.
The technique uses 8 week-old mice with a smooth muscle cell-specific TGF&#946;R2 deletion on the Apoe-/- hyperlipidemic background (MYH11-CreERT2;Tgfbr2f/f;mT/mGf/f;Apoe-/-; hereafter referred to as TGF&#946;R2iSMC-Apoe mice) and littermate Apoe-/- controls (MYH11-CreERT2;mT/mGf/f;Apoe-/-; hereafter referred to as Apoe-/- mice). The animals are kept for 16 weeks on a high cholesterol&#160;high fat diet (HCHFD) as study materials7. At study termination, the unopened whole aortas are stained and imaged (including all major branches) with Oil Red O for qualitative assessment of lipid-laden plaques. The aortas are cut open via en face preparation, and all atherosclerotic lesions are imaged and quantified. This protocol can be used to study atherosclerotic lesion development in Apoe-/- or Ldlr-/- hyperlipidemia mice models and extended to general aorta-related vascular biology applications.

<table><tbody><tr><td>1.5 mL Eppendorf tube</td><td>DENVILLE</td><td>C2170</td><td></td></tr><tr><td>10 mL syringe</td><td>BD</td><td>302995</td><td></td></tr><tr><td>16% Formaldehyde</td><td>Polysciences</td><td>18814-10</td><td></td></tr><tr><td>70% ethanol</td><td>VWR</td><td>RC2546.70-5</td><td>To clean the dissection tools</td></tr><tr><td>Black dissection wax</td><td>CR Scientific</td><td>C3541</td><td></td></tr><tr><td>Corn oil</td><td>Sigma</td><td>C8267</td><td>Solvent for Tamoxifen</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue kit</td><td>QIAGEN</td><td>69506</td><td>To isolate DNA from mouse ear</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-buffered saline (1X DPBS), pH 7.4</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14059-11</td><td>To cut the mouse skin and open the ribcage</td></tr><tr><td>Fisherbrand Economy Plain Glass Microscope Slides</td><td>Fisher Scientific</td><td>12-550-A3</td><td></td></tr><tr><td>High cholesterol high fat diet</td><td>Research Diets</td><td>D12108</td><td>To induce atherosclerosis</td></tr><tr><td>Imaging software</td><td>National Institutes of Health</td><td>Image J</td><td>Aortic lesion quantification</td></tr><tr><td>Isopropanol</td><td>VWR</td><td>JT9079-5</td><td></td></tr><tr><td>Kimwipes</td><td>Fisher Scientific</td><td>06-666A</td><td>To clean the glass microscope slides</td></tr><tr><td>McPherson-Vannas Micro Dissecting Spring Scissors</td><td>ROBOZ</td><td>RS-5602</td><td>To separate the heart and the aorta and to cut open the aorta and aorta branches</td></tr><tr><td>Microscope control software</td><td>Olympus</td><td>DP Controller</td><td>For aorta imaging</td></tr><tr><td>Minutien pins</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Needle-25G</td><td>BD</td><td>305124</td><td></td></tr><tr><td>NonWoven Sponge</td><td>McKesson</td><td>94442000</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O-0625</td><td>To stain the atherosclerosis lesions</td></tr><tr><td>Pall Acrodisc Sterile Syringe Filters with Super Membrane</td><td>VWR</td><td>28143-312</td><td>To filter working Oil Red O solution</td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools</td><td>15021-15</td><td>To dissect and clean the aorta</td></tr><tr><td>Statistical software</td><td>GraphPad</td><td>Prism 8</td><td>Statical analysis</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1000</td><td>For aorta dissection</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td>For aorta imaging</td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>T5648</td><td>To induce Cre-loxP recombination</td></tr><tr><td>Tissue-Tek O.C.T Compound, Sakura Finetek</td><td>VWR</td><td>25608-930</td><td></td></tr><tr><td>Tweezer Style 4</td><td>Electron Microscopy Sciences</td><td>0302-4-PO</td><td>To cut the mouse skin and open the ribcage</td></tr><tr><td>Tweezer Style 5</td><td>Electron Microscopy Sciences</td><td>0302-5-PO</td><td>To dissect and clean the aorta</td></tr></tbody></table>

imaging and analysis of oil red o-stained whole aorta lesions in an aneurysm hyperlipidemia mouse model

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for atherosclerosis research. They are used to study the impact of a various genetic factors and different cell types on atherosclerotic lesion formation and as well as test the development of new therapies. Isolation, excision of the whole aorta, and quantification of Oil Red O-stained atherosclerotic lesions are basic morphometric methods used to evaluate atherosclerotic burden. The goal of this protocol is to describe an optimized, step-by-step surgical method to dissect, perfuse-fix, isolate, stain, image and analyze atherosclerotic lesions in mouse aortas with Oil Red O. Because atherosclerotic lesions can form anywhere in the entire aortic tree, this whole aorta Oil Red O staining method has the advantage of evaluating lipid-laden plaques in the entire aorta and all branches in a single mouse. In addition to Oil Red O staining, fresh isolated whole aortas can be used for variety of in vitro and in vivo experiments and cell isolations.

In this protocol, atherosclerotic lesions in TGF&#946;R2iSMC-Apoe mice were analyzed after 4 months on a HCHF diet7. In addition to extensive atherosclerosis, these mice developed both thoracic and abdominal aortic aneurysms, as previously reported. Compared to Apoe-/- mice, TGF&#946;R2iSMC-Apoe mice aortic walls showed severe atherosclerosis, making it difficult to dissect the lesions (Figure 2C,D,E). In addition, the...

Watch this Scientific Journal Video about Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model at JoVE.com

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for ...

imaging-analysis-oil-red-o-stained-whole-aorta-lesions-an-aneurysm

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Medicine

Unit 1

Blood Vessels and Circulation

SCIENTISTS IN ACTION

12.9K Views.  Yale University School of Medicine. This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.

Video: Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Immunology and Infection

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Isolation and Analysis of Aortic Arch and Root Lesions in an Atherosclerotic Mouse Model

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study introduces a comprehensive protocol for the isolation and histological analysis of aortic arch and root lesions in a widely used atherosclerotic mouse model, low-density lipoprotein receptor knock-out (Ldlr-/-) mice. The aortic arch and root are key sites for atherosclerotic lesions, and their examination is critical for assessing the onset, progression, or regression of atherosclerosis, predicting cardiovascular event risks, and identifying potential therapeutic targets. This protocol outlines methods for quantifying atherosclerotic burden in the aortic arch and root, including tissue isolation, fixation, Oil Red O staining, aortic root sectioning, Hematoxylin and Eosin (HE) staining, Verhoeff-Van Gieson (VVG) staining, and image analysis. Oil Red O staining measures plaque area in the aortic arch, evaluating the severity of atherosclerosis, while HE staining of the aortic root reveals plaque components such as the lipid core and fibrous cap, facilitating the assessment of plaque stability and rupture risk. VVG staining can stain collagen fibers within tissues, providing further insights into plaque composition and related information. This thorough analysis offers valuable insights into the mechanisms of lesion development and may guide the creation of novel therapeutic strategies for preventing and treating atherosclerosis.

This protocol provides a comprehensive methodology for the quantitative analysis of atherosclerotic plaques to facilitate more in-depth research into the mechanisms and progression of atherosclerosis.

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study ...

Optimized Tissue-Clearing Protocols for 3D Imaging of the Murine Aorta 

Three-Dimensional Imaging of Aortic Tissues in Atherosclerosis

Recent research has advanced the understanding of atherosclerosis as a transmural chronic inflammatory disease involving all three layers of the arterial wall, including the intima plaque, the media, and the adventitia, which forms the outer connective tissue coat of arteries. Our recent studies have suggested that the adventitia is used by the peripheral nervous system as a conduit for reaching all tissue cells. We also found that the peripheral nervous system, that is, the sensory and sympathetic nervous system, undergoes major remodeling processes involving the neogenesis of axon networks adjacent to atherosclerotic plaques. In this context, understanding the structure of the neural network and its interactions with vascular components of diseased arteries holds major promises for a better understanding of cardiovascular disease pathogenesis. To achieve these objectives, methods to visualize the subcellular architecture of the intact healthy and diseased arteries together with their surrounding perivascular compartments are needed. Tissue clearing allows intact deep-tissue imaging of larger tissue compartments that are otherwise inaccessible. It allows volumetric imaging of intact arteries through the integration of labeling, clearing, advanced microscopic imaging, and image processing tools. Here, we describe two distinct but complementary passive tissue clearing approaches, that is, aqueous-based 2, 2-thiodiethanol (TDE) clearing and solvent-based immunolabeling-enabled three-dimensional imaging of solvent-cleared organ (iDISCO) clearing to image isolated aortic segments or whole aorta in-situ in the whole mouse.

Here, we present optimized tissue-clearing protocols to image the murine aorta in three dimensions (3D). We delineate state-of-the-art procedures for immunostaining, optical clearing, and imaging with the intent to define the anatomical proximity of the peripheral nervous system with atherosclerotic plaques and the adventitia in atherosclerosis.

Recent research has advanced the understanding of atherosclerosis as a transmural chronic inflammatory disease involving all three layers of the arterial ...