Assessing Atherosclerosis in the Aorta from AAV8-PCSK9-Injected Mice by Oil Red O Staining

Summary

This protocol describes a step-by-step method for dissecting, perfusing, staining, imaging, and quantifying en face atherosclerotic lesions in mouse aortas using Oil Red O staining.

Abstract

Mouse models have long been fundamental tools in the study of atherosclerosis, providing critical insights into the molecular and cellular mechanisms underlying plaque formation, lipid metabolism, and cardiovascular inflammation. Beyond Apoe^-/- or Ldlr^-/- mice, AAV8-PCSK9^D377Y injection in normal C57BL/6J mice with concurrent western-type diet feeding emerges as a commonly used model for translational research in atherosclerosis. Oil Red O staining is a cornerstone technique for visualizing neutral lipid accumulation in vascular tissues. Its simplicity, cost-effectiveness, and high sensitivity for detecting lipid-laden lesions make it the gold standard for assessing atherosclerotic plaque burden in preclinical studies. In this study, we provide a detailed, reproducible protocol for the dissection, perfusion, longitudinal opening, Oil Red O staining, imaging, and semi-automated quantification of atherosclerotic burden in the en face aorta, without the need for pins or complex mounting steps. Our results demonstrate that the combination of AAV8-PCSK9^D377Y injection and western-type diet feeding effectively induces significant atherosclerotic lesion formation in mice, and that en face Oil Red O staining is a reliable and efficient technique for assessing lipid deposition and plaque burden. Furthermore, although this protocol is optimized for the AAV8-PCSK9-induced model in C57BL/6J mice, it can be readily adapted for use in other models and species, such as Apoe^-/- mice, Ldlr^-/- mice, and Ldlr^-/- golden Syrian hamsters.

Introduction

Atherosclerosis, marked by the deposition of lipids and inflammation within the arterial intima, represents the pathological foundation of coronary artery disease and a broad range of cardiovascular disease (CVD), including myocardial infarction and stroke^1,2,3,4. CVD remains the leading cause of morbidity and mortality worldwide, posing a significant global health and economic burden^3,5. As the primary pathological basis of CVD, atherosclerosis has garnered extensive research attention aimed at uncovering its underlying mechanisms and developing effective therapeutic strategies. Mouse models play a central role in elucidating the mechanisms underlying atherogenesis and in evaluating potential therapeutic interventions⁶. The development of animal models has been instrumental in advancing our understanding of atherogenesis, allowing researchers to dissect the cellular and molecular mechanisms involved and to evaluate emerging therapeutic strategies^6,7. Classical murine models of atherosclerosis, such as apolipoprotein E-deficient (Apoe^−/−) and low-density lipoprotein receptor-deficient (Ldlr^−/−) mice, have long been employed to induce hyperlipidemia and robust plaque formation. However, the generation and maintenance of these genetically modified lines require extensive breeding, which can be time-consuming and limit experimental flexibility^8,9,10. To overcome these limitations, an alternative approach utilizing adeno-associated virus serotype 8 encoding a gain-of-function mutation in proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9^D377Y) has gained prominence. When administered to wild-type C57BL/6J mice in combination with a western-type high-fat, high-cholesterol diet, this method efficiently induces hypercholesterolemia and atherosclerotic lesions within a relatively short timeframe-without the need for complex genetic crosses or backcrossing into atherosclerosis-prone backgrounds^9,11. Despite its advantages, the AAV8-PCSK9 model has certain limitations that warrant consideration. Notably, the extent of hypercholesterolemia and atherosclerotic lesion development can be influenced by variables such as viral dose and means of administration, which may introduce inter-animal variability and affect the reproducibility of experimental outcomes¹¹.

To assess disease burden in such models, en face Oil Red O staining of the aorta provides a well-established, sensitive technique for visualizing and quantifying lipid-rich atherosclerotic lesions along the luminal surface of the vasculature^12,13. This approach offers both qualitative and quantitative data and is widely used in preclinical studies to evaluate the effects of genetic manipulation, pharmacological intervention, or dietary modulation¹⁴.

In this study, we present a practical and reproducible protocol for the isolation, perfusion, longitudinal opening, Oil Red O staining, imaging, and semi-automated quantification of atherosclerotic lesions in whole-mount mouse aortas. In this protocol, AAV8-PCSK9^D377Y is administered at a dose of 5 × 10¹¹ vector genomes (vg) per mouse via tail vein injection. When combined with a western-type diet (WD) containing 40% kcal from fat and 1.25% cholesterol, robust atherosclerotic lesion development can be observed within 12-16 weeks. The protocol here streamlines each step of the process, eliminating the need for pinning tissues onto wax surfaces and instead employing a simplified flattening technique that reduces variability and technical demand. The method is not only optimized for the AAV8-PCSK9^D377Y-induced model in C57BL/6J mice, but is also readily applicable to other widely used models of atherosclerosis, including Apoe^-/- and Ldlr^-/- mice, as well as Ldlr^-/- Syrian golden hamsters^{15,16,17,18,19}, which have emerged as a promising model due to their human-like lipid metabolism.

Protocol

All animal procedures were performed in accordance with the guidelines outlined in the NIH and were approved by the Animal Ethics Committee of the University of Science and Technology of China (Ethics Approval No. USTCACUC27120124102).

NOTE: All procedures involving AAV8-PCSK9 should be conducted in a certified Class II biological safety cabinet under Biosafety Level 2 (BSL-2) conditions. Personnel must wear appropriate personal protective equipment (PPE), including a lab coat, disposable gloves, and safety goggles.

1. Induction of atherosclerosis in mice

1. Inject an 8-week-old C57BL/6J mouse intravenously with AAV8-PCSK9^D377Y (5 × 10¹¹ vg/mouse via tail vein).
2. Place the mouse on WD (containing 40% kcal from fat and 1.25% cholesterol) immediately after injection.
3. Feed the mouse WD for 12-16 weeks to induce atherosclerotic lesion formation.
   NOTE: A 12-week feeding period is commonly used and has been shown to induce evident atherosclerotic plaque formation, particularly in AAV8-PCSK9-induced models. Longer durations (e.g., 16 weeks) may result in more advanced lesions, and the optimal time point should be selected based on the specific experimental goals.
4. Regularly monitor body weight, general health, and plasma lipid levels throughout the feeding period. For lipid profiling, collect blood samples at baseline and selected time points (e.g., every 4 weeks) via submandibular or tail vein bleeding under brief isoflurane anesthesia.

2. Euthanasia and perfusion

1. Administer sodium pentobarbital (200 mg/kg, intraperitoneal injection) to euthanize the mouse.
2. Open the chest cavity to expose the heart.
3. Make an incision in the right atrial appendage and perfuse cold Phosphate-Buffered Saline (PBS, about 10 mL) through the left ventricle using a 21 G needle to flush out blood.

3. Aorta dissection

1. Remove excess tissues such as the lungs and thymus to expose the thoracic aorta from the heart to the diaphragm.
2. Use fine dissecting scissors and precision forceps to remove the surrounding adipose tissue around the aortic arch. Gently elevate the adipose tissue surrounding the base of the thoracic aorta near the diaphragm. Excise along the root of the adipose tissue using fine dissecting scissors (ensuring that the vascular structure is not damaged), continuing the dissection upwards to the aortic arch to fully expose the aortic vessel.
   NOTE: The dissociation of perivascular fat tissue from the aorta is critical, and this step is suggested to be performed with the aorta in situ (before being cut from the mouse) as dissection of perivascular fat from the aorta is technically difficult once the aorta is dissected and placed in PBS.
3. Transect the vessel at the base of the thoracic aorta using precision scissors. Elevate the cut end with forceps and dissect upwards along the vessel toward the aortic arch. Sever the three primary branches of the aorta at an appropriate distance.
4. Transect at the aortic root to separate the thoracic aorta from the heart, isolating the complete segment for further analysis.
5. Transfer the excised vessel into pre-chilled 4% paraformaldehyde (PFA) solution and incubate at 4 °C overnight (12-16 h). To standardize handling and facilitate batch processing, place individual aortas into wells of a 24-well or 48-well plate during fixation.
   NOTE: Avoid prolonged fixation (over 24 h), as excessive incubation in PFA may cause vessel hardening, hindering longitudinal sectioning and further analysis.
   CAUTION:4% PFA is a toxic and volatile fixative that poses risks of respiratory and dermal exposure. All steps involving PFA fixation must be carried out in a fume hood while wearing PPE, including nitrile gloves, lab coat, and safety glasses. Avoid inhalation and skin contact. The used PFA solution should be collected in designated formaldehyde waste containers, clearly labeled, and disposed of as hazardous chemical waste by an institutional or licensed chemical waste disposal service.

4. Oil Red O staining

1. Wash the fixed aortas three times with PBS to remove residual PFA.
2. As shown in Figure 1, open the aorta longitudinally under a dissecting microscope using fine scissors, starting from the aortic root alongside the inner curvature. Alternatively, open the aorta before fixation, which may facilitate easier unfolding and alignment of the vessel during mounting. If needed, longitudinally open the major branches of the aortic arch-such as the brachiocephalic artery (BCA), right carotid artery (RCA), and left subclavian artery-to expose additional regions¹¹. Take care during this step, as plaques in these areas may detach or become damaged.
   NOTE: Either approach can be selected based on operator preference and the condition of the tissue.
3. Prepare the Oil Red O working solution by diluting the 0.5% stock in isopropanol with deionized water at a 3:2 ratio (v/v, final concentration 0.3%). Incubate in a 37 °C water bath for 5 min to dissolve the dye completely.
4. Filter through a 0.22-0.45 µm membrane to remove precipitates. Keep the solution in the 37 °C water bath until use.
5. Remove PBS from each well. Add Oil Red O working solution to fully immerse the aortas. Incubate at room temperature (RT) for 10 min.
6. After incubation, aspirate the solution and immediately add 1 mL of 60% isopropanol to each well. Place the plate on a shaker and gently agitate to wash.
7. After approximately 30 s, when the non-lesion areas of the vessel appear translucent, stop the wash by aspirating the isopropanol. Rinse the samples twice with PBS.
8. After Oil Red O staining, trim the residual perivascular adipose tissue further, if necessary. At this stage, the adipose tissue is typically well-stained and visually distinguishable, which allows for more accurate identification and removal without compromising the integrity of the vascular wall.
9. After Oil Red O staining, open the aorta longitudinally and carefully mount it flat on a glass slide for immediate imaging under a stereomicroscope.
10. Alternatively, store the samples for short-term in PBS at 4 °C before imaging. For arteries that are difficult to flatten, pin the vessels onto a black wax plate using fine needles to ensure optimal positioning and clarity. For long-term preservation, mount the stained vessels on glass slides, seal them with glycerol gelatin, and then image them directly.
    NOTE: Transfer stained aortas to a new plate for washing to avoid contamination from residual Oil Red O.
    CAUTION: Oil Red O is a lipophilic dye typically prepared in isopropanol, which is flammable and an irritant. Handle ORO solutions in a chemical fume hood to avoid inhalation of fumes. Avoid contact with skin and eyes.Oil Red O staining solutions should be collected separately in flammable chemical waste containers. Dispose of according to local hazardous waste disposal regulations.

5. Imaging

1. Mount the stained aortas on a white background to enhance contrast. Maintain vessel flexibility.
   NOTE: A high-contrast background facilitates more efficient plaque recognition using image analysis software. Other background colors may also be used based on user preference.
2. Image the whole aorta using a flatbed scanner or dissecting microscope with a high-resolution camera.
3. Ensure uniform lighting and consistent magnification across samples (e.g., 6 to 8 times magnification). If the aorta dries during imaging, rehydrate using saline- or PBS-dipped kiwi wipes.

6. Quantification of atherosclerotic lesions

1. Open the image in ImageJ. The following steps describe the analysis procedure using ImageJ as an example.
   NOTE: Other image analysis software (e.g., Zeiss Zen, Fiji) can also be used depending on user preference and availability.
2. Set the scale using a known reference (e.g., scale bar or ruler). Use the Straight-Line Tool to draw over the known distance. Go to Analyze > Set Scale, input the distance and units, select Global, and click OK.
3. Use the Polygon or Freehand Selection Tool to outline the entire aortic region.
4. Measure the area using Analyze > Measure.
5. Identify atherosclerotic plaque areas using Image > Adjust > Color Threshold. Adjust Saturation and Brightness to isolate Oil Red O-positive regions.
6. Click Select to generate binary selection of stained areas. Measure the selected regions using Analyze > Measure. Apply the same threshold settings across all images in a batch.
   NOTE: Given that residual fat or artifacts may result in false-positive staining, users may choose to manually or semi-automatically define plaque regions based on their specific experimental needs and image quality. Manual tracing of plaque area can be performed using the same method as for defining the total vessel area.
7. Export results (File > Save As > Results) as .csv for further analysis.
8. Express plaque area as plaque area/total aortic area × 100%. To obtain a more accurate assessment of atherosclerotic plaque burden, quantify plaque area by anatomical regions (e.g., aortic arch, especially the lesser curvature, and total aorta), and express the results as the percentage of plaque area relative to the total surface area of each respective region: plaque area (%) = (plaque area / total area of the region) × 100%.
   NOTE: To minimize potential bias, blinded analysis is recommended. If analyzing a large number of images, the entire workflow from thresholding to measurement can be automated using the Macro Recorder (Plugins > Macros > Record) and processed in bulk using Process > Batch > Macro. This enables efficient and reproducible high-throughput analysis.

Results

To assess the extent of lipid deposition in the aortic wall, en face Oil Red O staining was performed on aortas isolated from male C57BL/6J mice injected with AAV8-PCSK9^D377Y and fed a Western diet for 12 weeks, along with age-matched control mice. After systemic perfusion with PBS and careful dissection, the entire aortas were carefully longitudinally dissected and stained with Oil Red O to visualize lipid-laden atherosclerotic lesions. The overall experimental procedure is illustrated in

Discussion

The en face Oil Red O staining method described in this study offers a robust, reproducible, and time-efficient approach for evaluating atherosclerotic lesion burden in the whole aorta. In this study, we specifically selected the aortic arch and thoracic aorta as the regions of interest for plaque analysis, rather than the entire aorta. This decision was guided by the nature of the AAV8-PCSK9^D377Y model²⁰, which typically produces less extensive atherosclerotic plaque formation compared...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J mice	GemPharmatech	NO. N000013
4% paraformaldehyde	Beyotime	P0099	To fix tissues
AAV8-PCSK9D377Y viral particles	Wzbio	pAV208001	To induce atherosclerosis
Fine Forceps	Fine Science Tools	No. 11254-20	For aorta dissection
ImageJ	NIH	1.47
Isopropanol	Sangon biotech	A600918	To clean the excess oil red
Oil Red O 0.5% Solution in isopropanol	Poly Scientific R&D Corp	S1849-32OZ	For oil red O staining
Phosphate-buffered saline	Servicebio	G4202	For perfusion and cleaning of aortas
Spring Scissors	Fine Science Tools	No. 15003-08	For aorta dissection
Stereoscopic Microscope	Mshot	MZ62	For aorta dissection and imaging
Western diet	Research Diet	D12108C	To induce atherosclerosis