We thank all past members of G&#246;ran K Hansson&#8217;s experimental cardiovascular research unit that helped develop this protocol over the past quarter-century. We are particularly grateful for the contributions by Antonino Nicoletti, Xinghua Zhou, Anna-Karin Robertson, and Inger Bodin. This work was supported by project grant 06816 and Linnaeus support 349-2007-8703 from the Swedish Research Council, and by grants from the Swedish Heart-Lung Foundation, Stockholm County Council, Professor Nanna Svartz foundation, Loo and Hans Osterman Foundation for Medical Research, Karolinska Institutet&#8217;s Research Foundation and Foundation for Geriatric Diseases at Karolinska Institutet.

<ol>
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A., 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=Low+density+lipoprotein+receptor-negative+mice+expressing+human+apolipoprotein+B-100+develop+complex+atherosclerotic+lesions+on+a+chow+diet:+no+accentuation+by+apolipoprotein(a).">Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: no accentuation by apolipoprotein(a).</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (8), 4544-4549 (1998).</li><li>Gistera, A., 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=Vaccination+against+T-cell+epitopes+of+native+ApoB100+reduces+vascular+inflammation+and+disease+in+a+humanized+mouse+model+of+atherosclerosis.">Vaccination against T-cell epitopes of native ApoB100 reduces vascular inflammation and disease in a humanized mouse model of atherosclerosis.</a> Journal of Internal Medicine. , (2017).</li><li>Johnson, J. 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The authors have nothing to disclose.

Cardiovascular disease is the main killer in the world and new preventive measurements are needed2. Mouse models of the disease provide a comprehensive platform for investigation of pathophysiology and experimental treatments13. Reliable lesion size quantification is essential for this approach. However, quantification methods differ between laboratories. Standardization and optimization have been an ongoing process since the 1980&#8217;s13,27,33,34. Aortic roots have emerged as the most popular site to quantify experimental atherosclerosis. Cross-sections of plaques enable comparison of plaque volume between groups. En face preparations are favored for lesion quantification in larger segments of the aorta. The en face method visualizes plaque quantity and enables quantification of plaque area coverage, but do not take plaque thickness in account. The biological relevance for observed differences is substantiated by coherent results at different locations in the vascular tree. Evaluating atherosclerosis development at different locations addresses possible site specific effects. The effect of transplanted hematopoietic cells on atherosclerosis development can be assessed in hypercholesterolemic Ldlr-/- chimeras. However, whole-body irradiation affects the atherosclerosis process with site specific effects. More prominent atherosclerotic lesions are developed in the aortic root, while reduced lesion development is observed in aortic arches35.
Importantly, not only lesion size needs to be addressed in studies of experimental atherosclerosis. Lesion composition is also a key parameter. Several plaque features have been associated with manifestations of the disease in humans36. Serial sectioning of the aortic root leaves several sections available for careful analysis of plaque composition. Plaque rupture in humans is characterized by a thin fibrous cap with few smooth muscle cells, sparse collagen content and signs of inflammation in the plaques36. Although plaque rupture is a rare event in mouse models of atherosclerosis, markers for plaque stability are informative to evaluate. Translational approaches could confirm mechanistic findings from mouse models and uncover important features of human disease31. Inflammatory status of atherosclerotic plaques could be determined by immunohistochemistry staining of VCAM-1, MHC class II, macrophages, and lymphocytes30. Some protocols use longitudinal sections in the coronal plane of the aortic arch or the brachiocephalic artery for measuring atherosclerotic lesion size and composition37. However, this alternative method leaves only few sections to be analyzed, which limits its applications.
An initial critical step in this protocol is the ability to harvest aortas efficiently. Hand-eye coordination under the microscope requires practice and is crucial both for the microdissection and the subsequent pinning of the aortic arch. The next critical step in this protocol is the collection of serial sections from the aortic root. Eighty consecutive sections should be collected for each mouse, which requires both focus and patience. Methodological proficiency could speed up the described processes considerably. Nevertheless, atherosclerotic lesion quantification is still a time-consuming task. New technology, automated handling, and small animal imaging might facilitate quantification of experimental atherosclerosis in the future. The progression of atherosclerosis is slow and most experimental protocols in mouse models take more than four months to complete13. Therefore, aortas need to be collected in an optimized way at study endpoints. This protocol provides a comprehensive guide to harvest aortas efficiently and the proposed processing prepares aortas for multi-purpose use including lesion quantification in aortic root, aortic arch, and brachiocephalic artery. Hopefully the protocol can reduce experimental variability, enhance reliability of results, and lead to findings that will pave the way for new treatments against atherosclerosis.

Cardiovascular disease is the main cause of death in the world with ischemic heart disease and stroke accounting for one in every four deaths1. Most cases are caused by atherosclerosis, a disease characterized by a slow build-up of lipid-laden plaques with signs of chronic inflammation in large- and medium-sized arteries2. The disease usually remains unnoticed over several decades until a rupture or erosion of the plaque elicits an arterial thrombosis that leads to ischemic tissue damage.
A normal artery consists of an intima layer with endothelial cells and sparsely distributed smooth muscle cells, a media layer with smooth muscle cells and elastic lamellae, and a surrounding adventitial layer with loose connective tissue3. An intimal retention of LDL offsets atherosclerosis development4. Accumulation and modification of lipoproteins lead to aggregation and entrapment within the arterial intima5. An inflammatory response is evoked by the trapped and modified lipoproteins6. Endothelial cells start to express adhesion molecules, such as VCAM-1 at sites in the arterial tree with turbulent blood flow, leading to recruitment of circulating monocytes and other leukocytes7. The infiltrating monocytes differentiate into macrophages that engulf lipid with ensuing transformation to macrophage foam cells8.
Atherosclerosis has been studied in mouse models with increasing frequency since the mid-1980s. C57BL/6 is the most commonly used inbred mouse strain for these studies, and it is used as the genetic background for the majority of genetically modified strains9. This strain was established in the 1920&#8217;s10, and its genome was published in 200211. Experiments in mouse models have several benefits: the colonies reproduce fast, housing is space-efficient, and inbreeding reduces experimental variability. The model also allows for genetic manipulations, such as targeted gene deletions and insertion of transgenes. This has led to new pathophysiological understanding of the disease and new therapy targets12.
Wild-type C57BL/6 mice are naturally resistant to atherosclerosis. They have most of the circulating cholesterol in HDL, and complex atherosclerotic lesions are not formed even when fed a high-fat and high-cholesterol diet13. Hypercholesterolemic mice, such as Apoe-/- on the C57BL/6-background, are therefore used as experimental models of atherosclerosis14,15. The lack of ApoE impairs hepatic uptake of remnant lipoproteins and severely perturbs lipid metabolism. In Apoe-/- mice, circulating cholesterol is predominantly in VLDL particles, and the mice develop complex atherosclerotic plaques on a regular chow diet.
Ldlr-/- mice mimic the development of atherosclerosis seen in humans with familial hypercholesterolemia16. The Ldlr-/- mice need a Western type diet to develop atherosclerosis17. Western diet mimics human food intake and usually contains 0.15% cholesterol. The LDL receptor recognizes ApoB100 and ApoE and mediates uptake of LDL particles through endocytosis. LDL receptors are fundamental for liver clearance of LDL from circulation, while LDL receptor expression in hematopoietic cells does not influence this process. This opens the possibility for bone marrow transplantation of Ldlr+/+ cells into hypercholesterolemic Ldlr-/- recipients and assessment of atherosclerosis development. Bone marrow chimeras have commonly been used to study the participation of hematopoietic cells in experimental atherosclerosis. However, bone marrow transplantation could influence the size and composition of atherosclerotic plaques, making interpretation of results ambiguous.
Different variants of Apoe-/- and Ldlr-/- mice with additional genetic alterations have been developed to study specific processes of the disease18. One example is human APOB100-transgenic Ldlr-/- (HuBL) mice that carry the full-length human APOB100 gene19,20. These mice develop hypercholesterolemia and atherosclerosis on a regular chow diet. However, the development of complex atherosclerotic plaques takes at least six months and shorter experimental protocols usually use Western diet21. A large fraction of plasma cholesterol is circulating in LDL particles, which gives HuBL mice a more human-like dyslipidemic lipoprotein profile compared to Apoe-/- and Ldlr-/- mice. HuBL mice also allow studies of human apoB as an autoantigen22.
The mouse models of atherosclerosis develop complex atherosclerotic plaques with shared features of human disease. However, the plaques are fairly resistant to rupture with ensuing myocardial infarction. Atherothrombosis is only sporadically detected and experimentally challenging to assess23,24,25. Special models of plaque rupture have been developed, but the experimental field lacks a reliable and reproducible model for assessment of plaque stabilizing agents.
Quantification of atherosclerosis has been reported in numerous ways in the literature. Recent efforts have tried to standardize experimental design, execution, and reporting of animal studies26. Investigators have different preferences and techniques adapted to their laboratories. Most research projects are also unique in a way that they require some protocol modifications. Due to the multifactorial nature of the disease, optimal controls vary between projects. Local conditions and lack of standardization may cause observed differences in disease development, which hampers advances of the research field. Differences in experimental variability also means that statistical power calculations need to be based on pilot studies under local conditions.
Quantification of atherosclerosis is recommended at several locations in the vascular tree. This protocol describes how to obtain results from the aortic root, the aortic arch, and the brachiocephalic artery in a single mouse, in addition to leaving the rest of the thoracoabdominal aorta for other analyses. En face preparations allow rapid quantification of lipid-laden plaques in the aortic arch. Disease burden in the brachiocephalic artery can also be quantified if the specimens are carefully displayed. The more time consuming cross-sectioning of the aortic root leaves several sections available for detailed evaluation of plaque composition.

<table>
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>VWR Chemicals</td>
			<td>20066.296</td>
			<td>For fixation of sections for immunohistochemistry.</td>
		</tr>
		<tr>
			<td>Black electrical insulation tape (50 mm wide)</td>
			<td>Any specialized retailer</td>
			<td>-</td>
			<td>To create pinning beds for aortic arches.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5417C</td>
			<td>Benchtop microcentrifuge.</td>
		</tr>
		<tr>
			<td>Cork board</td>
			<td>Any specialized retailer</td>
			<td>-</td>
			<td>For cutting hearts in the preparation to cryomount aortic roots.</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific</td>
			<td>Microm HM 560</td>
			<td>For serial cryosectioning of aortic roots.</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>-</td>
			<td>-</td>
			<td>For rinsing and preparation of solutions.</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Leica Microsystems</td>
			<td>DC480</td>
			<td>5.1 megapixel CCD for high-resolution images of aortic arches and aortic root sections.</td>
		</tr>
		<tr>
			<td>Dissecting scissors (10 cm, straight)</td>
			<td>World Precision Instruments</td>
			<td>14393</td>
			<td>For general dissection of organs.</td>
		</tr>
		<tr>
			<td>Dumont forceps #5 (11 cm, straight)</td>
			<td>World Precision Instruments</td>
			<td>500341</td>
			<td>For microdissection of aorta.</td>
		</tr>
		<tr>
			<td>Ethanol 70% (v/v)</td>
			<td>VWR Chemicals</td>
			<td>83801.290</td>
			<td>Highly flammable liquid and vapour, store in a well-ventilated place, and keep cool.</td>
		</tr>
		<tr>
			<td>Ethanol absolute &#8805;99.8%</td>
			<td>VWR Chemicals</td>
			<td>20821.310</td>
			<td>Highly flammable liquid and vapour, store in a well-ventilated place, and keep cool.</td>
		</tr>
		<tr>
			<td>Formaldehyde 4% stabilised, buffered (pH 7.0)</td>
			<td>VWR Chemicals</td>
			<td>9713.1000</td>
			<td>Harmful by inhalation, in contact with skin and if swallowed.</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>-</td>
			<td>Image analysis software.</td>
		</tr>
		<tr>
			<td>Iris forceps (10 cm, curved, serrated)</td>
			<td>World Precision Instruments</td>
			<td>15915-G</td>
			<td>Used as anatomical forceps.</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck</td>
			<td>1096341011</td>
			<td>Flammable liquid, causes serious eye irritation, and may cause drowsiness or dizziness.</td>
		</tr>
		<tr>
			<td>Kaiser&#39;s glycerol gelatine</td>
			<td>Merck</td>
			<td>1092420100</td>
			<td>Aqueous mounting medium containing phenol. Suspected of causing genetic defects.</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica Microsystems</td>
			<td>DM LB2</td>
			<td>For analysis during sectioning and documentation of Oil Red O stained micrographs.</td>
		</tr>
		<tr>
			<td>Mayer&#39;s hematoxylin</td>
			<td>Histolab</td>
			<td>1820</td>
			<td>Non-toxic staining solution without chloral hydrate, but causes serious eye irritation.</td>
		</tr>
		<tr>
			<td>Mayo scissors (17 cm, straight)</td>
			<td>World Precision Instruments</td>
			<td>501751-G</td>
			<td>For general dissection.</td>
		</tr>
		<tr>
			<td>Micro Castroviejo needle holder (9 cm, straight)</td>
			<td>World Precision Instruments</td>
			<td>503376</td>
			<td>For pinning of aortic arches.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Corning</td>
			<td>MCT-175-C</td>
			<td>Polypropylene microtubes with snaplock cap.</td>
		</tr>
		<tr>
			<td>Microlance 3 needles, 23 gauge</td>
			<td>BD</td>
			<td>300800</td>
			<td>For blood collection.</td>
		</tr>
		<tr>
			<td>Microlance 3 needles, 27 gauge</td>
			<td>BD</td>
			<td>302200</td>
			<td>For perfusion of mice.</td>
		</tr>
		<tr>
			<td>Microvette 500 &#181;L, K3 EDTA</td>
			<td>Sarstedt</td>
			<td>20.1341.100</td>
			<td>For blood collection.</td>
		</tr>
		<tr>
			<td>Microvette 500 &#181;L, Lithium Heparin</td>
			<td>Sarstedt</td>
			<td>20.1345.100</td>
			<td>For blood collection.</td>
		</tr>
		<tr>
			<td>Minutien insect pins, 0.10 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>For pinning of aortic arches.</td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>O0625</td>
			<td>Not classified as a hazardous substance or mixture.</td>
		</tr>
		<tr>
			<td>Optimum cutting temperature (OCT) cryomount</td>
			<td>Histolab</td>
			<td>45830</td>
			<td>For embedding tissue.</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM992</td>
			<td>Paraffin wax film used to create pinning beds for aortic arches.</td>
		</tr>
		<tr>
			<td>Petri dishes (100x20 mm)</td>
			<td>Any cell culture supplier</td>
			<td>-</td>
			<td>Proposed as a storage container for pinned aortas.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>-</td>
			<td>-</td>
			<td>Sterile and RNase-free solution is required for perfusion of mice.</td>
		</tr>
		<tr>
			<td>Qualitative filter paper (grade 1001)</td>
			<td>Munktell</td>
			<td>120006</td>
			<td>For filtering Oil Red O working solution (typical retention 2-3 &#181;m).</td>
		</tr>
		<tr>
			<td>RNAlater RNA stabilization reagent</td>
			<td>Qiagen</td>
			<td>76106</td>
			<td>For stabilization of RNA in tissue samples</td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td>A surface decontamination solution that destroys RNases on contact.</td>
		</tr>
		<tr>
			<td>Scalpel handle #3 (13 cm)</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td>For cutting hearts in the preparation to cryomount aortic roots.</td>
		</tr>
		<tr>
			<td>Standard scalpel blade #10</td>
			<td>World Precision Instruments</td>
			<td>500239</td>
			<td>For cutting hearts in the preparation to cryomount aortic roots.</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>MZ6</td>
			<td>For dissection and en face documentation</td>
		</tr>
		<tr>
			<td>Sudan IV</td>
			<td>Sigma-Aldrich</td>
			<td>S4261</td>
			<td>Not classified as a hazardous substance or mixture.</td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td>To collect aortic root sections.</td>
		</tr>
		<tr>
			<td>Tissue forceps (15 cm)</td>
			<td>World Precision Instruments</td>
			<td>501741-G</td>
			<td>For general dissection.</td>
		</tr>
		<tr>
			<td>Tissue-Tek cryomolds (10x10x5 mm)</td>
			<td>Sakura</td>
			<td>4565</td>
			<td>For embedding aortic roots in OCT.</td>
		</tr>
		<tr>
			<td>Vannas scissors (8 cm, straight)</td>
			<td>World Precision Instruments</td>
			<td>503378</td>
			<td>For microdissection of aorta.</td>
		</tr>
	</tbody>
</table>

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.

In mouse models of atherosclerosis the most prominent lesions tend to develop in the aortic root and aortic arch. This protocol describes quantification of atherosclerosis in the aortic root, the aortic arch, and the brachiocephalic artery in a single mouse. Measurable lesions in thoracic descending aorta and abdominal aorta are only present in animals with advance disease. In this protocol, these parts are not analyzed for atherosclerotic burden, but saved for subsequent analysis of mRNA levels or other analyses. Serial sections of atherosclerotic lesions in the aortic root are usually displayed in a graph with lesion size on the y-axis and distance to the aortic sinus on the x-axis28. True cross-sections are crucial for lesion size quantification. Oblique sections can overestimate lesion sizes and a tilting of only 20&#176; could overestimate the absolute lesion surface by 15%29. However, calculating the lesion fraction of total vessel area makes the result less sensitive to possible angle differences during sectioning (Figure 4A). An appropriate statistical method to detect differences between groups is usually a regular 2-way analysis of variance (ANOVA). Bonferroni post-tests are then carried out to detect differences at certain levels. Fisher&#39;s least significant difference could also be used as a follow-up test to ANOVA. It reduces the likelihood of type II statistical errors, but do not account for multiple comparisons. In addition, it could be illustrative to calculate area under the curve or the average lesion size per mouse and present the data in a dot plot to further visualize individual variation within the groups (Figure 4B).
Oil Red O is a fat-soluble bright red diazo dye, which stains neutral lipids. Polar lipids in cell membranes are not stained. Oil Red O staining can be performed on fresh, frozen, or formalin-fixed samples, but not on paraffin-embedded samples due to the removal of lipids in the required deparaffinization process. A quantification of lesional lipid accumulation could be performed by color thresholding the Oil Red O positive area of total lesion area (Figure 4C). Hematoxylin produces a blue staining of cell nuclei, which is helpful to visualize plaque morphology. The right and left coronary arteries usually diverge from the aorta around 250 &#181;m from the aortic sinus27, which often coincide with the most prominent lesion sizes. Cross-sections from this region is often displayed as representative results (Figure 4D).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59828/59828fig4.jpg" /> 
Figure 4: Atherosclerotic lesions in the aortic root. (A) Twenty-eight weeks old male bone marrow chimeras fed Western diet for eight weeks were evaluated to determine the effect of Smad7-deficient T cells on atherosclerosis development. Experimental Ldlr-/- chimeras received Cd4-Cre+Smad7fl/fl bone marrow and controls received Cd4-Cre+Smad7fl/+ bone marrow. The graph shows quantification of atherosclerotic lesion area from eight consecutive sections, 100 - 800 &#181;m from the aortic sinus displayed as lesion fraction of total vessel surface (Cd4-Cre+Smad7fl/+/Ldlr-/- n=6, Cd4-Cre+Smad7fl/fl/Ldlr-/- n = 9, 2-way ANOVA with Bonferroni&#8217;s post test, graph shows mean &#177;SEM, braces indicate significance level for strain comparison). (B) The combined dot plot and bar graph shows the mean atherosclerotic lesion area from the aortic root sections (Cd4-Cre+Smad7fl/+/Ldlr-/- n=6, Cd4-Cre+Smad7fl/fl/Ldlr-/- n = 9, Student&#8217;s t-test) (C) Fraction of Oil Red O-stained area in the lesions (Cd4-Cre+Smad7fl/+/Ldlr-/- n = 4, Cd4-Cre+Smad7fl/fl/Ldlr-/- n = 6, Student&#8217;s t-test, ns=non-significant) (B-C) Dots represent individual mice and bars show mean &#177;SEM. (D) Representative micrographs showing Oil Red O staining (in red color) of neutral lipids in the aortic root 300 &#181;m from aortic sinus (50x magnification), Scale bar =&#160;500 &#181;m. *p&#160;&#8804; 0.05, ***p&#160;&#8804; 0.001. This figure has been modified from Gister&#229; et al.31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59828/59828fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Oil Red O could be used for staining of en face prepared aortas, but this protocol uses Sudan IV, another convenient fat-soluble diazo dye. Sudan IV clearly visualizes atherosclerotic plaques in an orange-red color by staining lipids, triglycerides, and lipoproteins. Removing the dark background in representative images of the en face aortic arches could enhance the visual display (Figure 5A). Usually lesion size is normally distributed within groups, allowing statistical testing with Student&#8217;s t-test between groups. A dot plot that shows both individual mice and the mean, which is compared between groups, is an informative way to display the results (Figure 5B-C). Since the variation within groups typically is different between locations in the vascular tree, separate power calculations are usually needed. Unnecessary variation can be avoided by method proficiency and protocol standardization. Obtaining statistically significant results is important, but the biological relevance for an observed difference always needs to be considered as well.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59828/59828fig5.jpg" /> 
Figure 5: Atherosclerotic lesions in aortic arch and brachiocephalic artery. (A) Representative en face micrographs of aortic arches with lipid-laden plaques stained with Sudan IV (in orange color) from 20 weeks old mice fed Western diet for ten weeks, visualized together. Scale bar = 2 mm. Human APOB100-transgenic Ldlr-/- (HuBL) mice were used as controls and the experimental group consisted of TCR-transgenic mice with LDL-reactive T cells (BT1) crossbred to HuBL mice. (B) Atherosclerotic lesions in the aortic arch (HuBL n = 10, BT1xHuBL n = 12; Student&#8217;s t-test). (C) Atherosclerotic lesions in the brachiocephalic artery (HuBL n = 8, BT1xHuBL n = 9, Student&#8217;s t-test). (B-C) Dots represent individual mice, bars show mean &#177;SEM. *p&#160;&#8804; 0.05. This figure has been modified from Gister&#229; et al.32. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59828/59828fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Alternative organization of slides for serial sections of the aortic root. A simplified systematic slide organization for collection of sections from the aortic root. The collection enables Oil Red O staining for lipids and immunohistochemistry or immunofluorescence staining. Dedicated slides for Picrosirius red staining of collagen are omitted. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59828/2019-JoVE-Figure-S1.eps">Please click here to download this file.</a>

Watch this Scientific Journal Video about Quantification of Atherosclerosis in Mice at JoVE.com

Quantification of Atherosclerosis in Mice

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 ...

quantification-of-atherosclerosis-in-mice

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

36.7K Views.  Karolinska Institutet, Karolinska University Hospital. 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.

Video: Quantification of Atherosclerosis in Mice

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...