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 &#38; Tissue kit</td>
			<td>QIAGEN</td>
			<td>69506</td>
			<td>To isolate DNA from mouse ear</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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 ...

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12.4K 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

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

The Helsinki Rat Microsurgical Sidewall Aneurysm Model

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced into clinical practice. Furthermore, experimental models are needed to elucidate the complex aneurysm biology leading to rupture of saccular aneurysms.
Several different kinds of experimental models for saccular aneurysms have been established in different species. Many of them, however, require special skills, expensive equipment, or special environments, which limits their widespread use. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various institutions.
The microsurgical rat abdominal aortic sidewall aneurysm model combines the possibility to study both novel endovascular treatment strategies and the molecular basis of aneurysm biology in a standardized and inexpensive manner. Standardized grafts by means of shape, size, and geometry are harvested from a donor rat&#39;s descending thoracic aorta and then transplanted to a syngenic recipient rat. The aneurysms are sutured end-to-side with continuous or interrupted 9-0 nylon sutures to the infrarenal abdominal aorta.
We present step-by-step procedural instructions, information on necessary equipment, and discuss important anatomical and surgical details for successful microsurgical creation of an abdominal aortic sidewall aneurysm in the rat.

Microsurgical sidewall aneurysms in rats are created by end-to-side anastomosis of an aortic graft to the abdominal aorta. We present step-by-step instructions and discuss anatomical and surgical details for successful experimental saccular aneurysm creation.

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &amp;#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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 ...

Porcine Model of Infrarenal Abdominal Aortic Aneurysm

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal abdominal aortic aneurysms (AAA) in swine. To achieve this, we use a combination of balloon angioplasty, elastase and collagenase, and a lysyl oxidase inhibitor, called &#946;-aminopropionitrile (BAPN), to create clinically significant infrarenal aortic aneurysms, analogous to human disease.
Noncastrated male swine are fed BAPN for 7 days prior to surgery to achieve a steady state in the blood. A midline laparotomy is performed and the infrarenal aorta is circumferentially dissected. An initial measurement is recorded prior to aneurysm induction with a combination of balloon angioplasty, elastase (500 units)/collagenase (8000 units) perfusion, and topical elastase application. Swine are fed BAPN daily until terminal procedure on either postoperative day 7, 14, or 28, at which time the aneurysm is measured, and tissue procured. BAPN + surgery pigs are compared to pigs that underwent surgery alone.
Swine treated with BAPN and surgery had a mean aortic dilation of 89.9% &#177; 47.4% at day 7, 105.4% &#177; 58.1% at day 14, and 113.5% &#177; 30.2% at day 28. Pigs treated with surgery alone had significantly smaller aneurysms compared to BAPN + surgery animals at day 28 (p &#60; 0.0003). The BAPN + surgery group had macroscopic and immunohistochemical evidence of end stage aneurysmal disease.
Clinically significant infrarenal AAA can be induced using balloon angioplasty, elastase/collagenase perfusion and topical application, supplemented with oral BAPN. This model creates large, clinically significant AAA with hallmarks of human disease. This has important implications for the elucidation of AAA pathogenesis and testing of novel therapies and devices for the treatment of AAA. Limitations of the model include variation in BAPN ingested by swine, quality of elastase perfusion, and cost of BAPN.&#160;

This novel model creates robust infrarenal abdominal aortic aneurysms in swine using a combination of balloon angioplasty, elastase/collagenase perfusion, topical elastase application, and oral compound &#946;-aminopropionitrile administration, which interferes with collagen cross-linking.

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal ...