An Atherosclerosis Model in Mice Induced by Both Partial Carotid Ligation and Overexpression of PCSK9

Summary

A reproducible mouse model induces carotid atherosclerosis by combining PCSK9 adeno-associated virus (PCSK9-AAV)- high-fat diet mediated hypercholesterolemia and partial left carotid ligation (PCL), recapitulating human pathophysiology. This enables rapid induction of human-like carotid atherosclerotic lesions in wild-type mice without genomic editing, providing a standardized tool to facilitate carotid atherosclerosis research.

Abstract

The lack of rapid, reproducible carotid-specific atherosclerosis models in wild-type mice limits translational studies of plaque biology and therapies. This protocol describes a non-genomically edited, time-efficient method to induce reproducible carotid atherosclerosis in C57BL/6 mice by combining adeno-associated virus (AAV)-mediated PCSK9 overexpression, an atherogenic high-fat diet, and left partial carotid ligation (PCL). The objective is to provide a standardized workflow that reproduces key pathophysiological features of human carotid disease-hyperlipidemia plus disturbed flow-without requiring genetically modified animals. Methods include PCSK9-AAV dosing and administration, perioperative preparation, step-by-step surgical ligation of the left carotid, postoperative care, dietary regimen, tissue collection, and histological assessment including Oil Red O and Verhoeff-Van Gieson staining. The results indicate elevated circulating cholesterol and low-density lipoprotein levels compared with reference values. Pronounced atherosclerotic plaques develop at the left ligated carotid artery, with significantly increased lipid accumulation and significant arterial intima-media thickening relative to the right non-ligated control artery. This standardized protocol improves reproducibility and accessibility for investigators studying carotid plaque formation, progression, intervention strategies, and facilitates cross-laboratory comparisons of experimental therapies.

Introduction

Cardiovascular diseases (CVDs) remain the leading cause of global mortality, with atherosclerosis serving as their common pathological basis¹. The carotid artery is a critical site for atherosclerotic lesions; studying carotid atherosclerosis is essential for understanding the pathogenesis of CVDs and developing therapeutic strategies². Animal models are indispensable tools for in vivo atherosclerosis research, yet existing models often have limitations in terms of convenience and their ability to recapitulate human pathophysiological features.

Compared to atherosclerosis in other arterial sites (e.g., aorta, femoral artery), the formation of carotid atherosclerosis is more strongly influenced by hemodynamics. The carotid artery experiences high shear stress (10-15 dyn/cm², dynes per square centimeter), second only to the coronary artery (6-45 dyn/cm²)³. Particularly at the carotid bifurcation, vortex flow occurs, which is characterized by disturbed, oscillatory shear stress. This abnormal hemodynamic environment triggers sustained pro-inflammatory activation of local endothelial cells, characterized by upregulated expression of adhesion molecules and chemokines that facilitate monocyte recruitment and the initiation of atherosclerosis³. Additionally, high-risk human carotid atherosclerotic lesions often exhibit a stenosis degree exceeding 70%, which is defined as the percentage reduction in luminal diameter caused by the plaque⁴. Thus, animal models capable of inducing severe atherosclerotic stenosis are important.

The partial carotid ligation (PCL) model is an atherosclerosis model induced by persistent blood flow disturbance, which has the advantages of short modeling time and severe lesions⁵. Apolipoprotein E (ApoE) is a key protein involved in the metabolism and clearance of circulating cholesterol and triglyceride-rich lipoproteins, playing a critical role in maintaining plasma lipid homeostasis. However, the traditional PCL model using ApoE-knockout (ApoE⁻/⁻) mice is difficult and costly to create⁵. When a target gene knockout is required, double-gene knockout (of both the target gene and ApoE gene) is necessary, further complicating the modeling process. In contrast, when only wild-type C57BL/6 mice are used for PCL, the model lacks hypercholesterolemia, a core risk factor for human atherosclerosis, and rarely form prominent lipid-rich atherosclerotic lesions^6,7. Other models, such as ApoE⁻/⁻ or LDLR-knockout (LDLR⁻/⁻) mice fed a high-fat diet (HFD), are widely used in atherosclerosis research for studying aortic atherosclerosis. Nevertheless, these models fail to incorporate the effect of disturbed blood flow and cannot form severe atherosclerosis in a short period of time. Previous studies have demonstrated that after 3 months of HFD feeding, ApoE⁻/⁻ or LDLR⁻/⁻ mice only develop mild atherosclerotic plaques in the aortic or brachiocephalic artery regions, whereas no obvious plaques lesions are formed in the carotid arteries^8,9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein cholesterol (LDL-C) metabolism: it promotes the degradation of LDL receptors, thereby increasing serum LDL-C levels¹⁰. Recombinant adeno-associated virus expressing PCSK9 (PCSK9-AAV) enables dose-controllable hypercholesterolemia in WT mice without the need for genetic modifications¹¹. Combining PCSK9-AAV-induced hypercholesterolemia, HFD (which further exacerbates lipid metabolism disorders), and PCL (which induces local hemodynamic disturbance) allows the construction of an animal model that incorporates two key risk factors for human atherosclerosis: hypercholesterolemia and flow disturbance.

Building on previous research progress¹², this protocol establishes a standardized method for inducing carotid atherosclerosis in C57BL/6 mice. Compared with existing model protocols, it offers three key advantages: (1) Pathophysiological relevance: It simultaneously recapitulates hypercholesterolemia and flow disturbance, resulting in carotid lesions that exhibit features similar to mid-to-late stage human atherosclerosis; (2) Reproducibility: Detailed parameterization of AAV dosage, surgical procedures, and dietary control reduces inter-laboratory variability; (3) Timeliness: The total modeling period is 5 weeks, significantly shortening the time required for model establishment.

This protocol is suitable for researchers studying vascular wall biology, evaluating the efficacy of anti-atherosclerotic drugs, and exploring the interaction between hemodynamics and lipid metabolism in atherosclerosis. Notably, this protocol is a carotid-focused model, which cannot fully replicate all aspects of systemic human risk factors for atherosclerosis, including but not limited to smoking, diabetes mellitus, hypertension and obesity. Below is a step-by-step guide for model establishment and lesion assessment.

Protocol

All procedures were approved by the Animal Ethics Committee of Committee of Ethics on Animal Experiments at Peking Union Medical College (ACUC-A02-2024-006) and comply with the Guide for the Care and Use of Laboratory Animals.

1. Experimental animal preparation

1. Purchase Male C57BL/6 mice aged 6-8 weeks (body weight: 22-25 g).
2. House mice in a specific pathogen-free (SPF) environment under strictly controlled conditions: temperature (23 ± 1°C), humidity (50 ± 5 %), and a 12 h light/dark cycle (lighting: 07:00-19:00).
3. Before the experiment, provide mice a standard chow diet (10% fat energy ratio) and sterile water, with free access to food and water, for a 1 week acclimatization period.

2. Preparation and administration of PCSK9 -AAV

CAUTION: Treat AAV as a biosafety hazard and conduct all procedures under the appropriate institutional biosafety level (typically BSL-2). Wear double gloves, eye protection, and protective clothing. Avoid aerosol generation during handling. Decontaminate AAV-contaminated materials with appropriate disinfectants (e.g., 10% bleach) and follow clearly defined responsibility and incident response protocols. All virus-contaminated waste must be inactivated by immersion in chlorine-containing disinfectants.

1. Purchase recombinant PCSK9-AAV (rAAV8-HCRApoE/hAAT-D377Y-mPCSK9).
2. Verify AAV titer by quantitative real-time PCR targeting the CMV promoter: the final titer was 6.10×10¹³ vg/mL (vector genomes per milliliter). Store AAV at -80°C and avoid repeated freeze-thaw cycles (≤3 freeze-thaw cycles).
3. Thaw AAV on ice for 15-20 min, then gently invert the centrifuge tube 3-5 times to mix (avoid vortexing).
4. Dilute PCSK9-AAV with sterile phosphate-buffered saline (PBS) to a final concentration of 2×10¹¹ vg/200 µL per mouse.
5. Administer via tail vein injection (i.v.): Fix the mouse using a vein-visualizing mouse tail injection holder and slowly inject 200 µL of diluted AAV using an insulin syringe (injection rate: 10 µL/s). The injection time point is 1 week before PCL (Figure 1A).

3. Preoperative preparation for partial carotid ligation

CAUTION: Tribromoethanol is a controlled reagent with a risk of overdose. Administer strictly according to body weight, avoid repeated injections, and accurately document dosing time and volume. Store in a locked container and ensure preparation and use are performed by trained personnel only. In cases of suspected overdose, initiate emergency management and document the incident.

1. Anesthetize mice via intraperitoneal injection (i.p.) of tribromoethanol (2.5% Avertin, 0.18 mL/10 g) until the righting reflex is lost (approximately 2-3 min). Then, transfer the mouse to a heating pad (37 ± 0.5°C). Check the anesthetic effect by pinching the toes.
   NOTE: Anesthesia is critical for successful surgery. To minimize anesthetic risks, multiple anesthetic injections should be avoided. Prior to surgery initiation, the anesthetic dose should be determined based on the anticipated surgical duration. Additionally, the surgical duration for each mouse is recommended to be <30 min to mitigate risks associated with prolonged procedures.
2. During surgery, check the mouse's respiratory rate (normal: 40-60 breaths/min) and plantar reflex by gently pinching the hind paw pad with forceps every 5 min.
3. Apply depilatory cream thoroughly to the anterior cervical skin of the mouse. After 2-3 min, remove the anterior cervical hair with cotton swabs and clean residual hair with normal saline. Disinfect the exposed skin with 5% povidone-iodine three times.
4. Perform surgery in a biosafety cabinet in an aseptic mouse room. Sterilize the surgical space with ultraviolet light to maintain asepsis as much as possible and autoclave all metal instruments.
   NOTE: An aseptic environment and thorough disinfection are essential for ensuring post-operative survival and mouse physiological stability.

4. Partial carotid ligation- surgical procedure

1. Make a midline incision (approximately 1.0-1.5 cm in length) on the mouse's neck using sterile surgical scissors; separate subcutaneous tissues and the sternocleidomastoid muscle with blunt forceps to expose the left common carotid artery (LCCA).
   NOTE: The operative area should be continuously moistened with warmed normal saline to prevent additional trauma caused by vascular/tissue desiccation.
2. Bluntly separate the LCCA from the vagus nerve (located adjacent to the artery) to avoid damaging the vagus nerve (which may cause respiratory depression).
3. Dissect upward along the LCCA to expose the internal carotid artery (ICA), external carotid artery (ECA), occipital artery (OA), and superior thyroid artery (STA) (Figure 1B).
4. Use 6-0 sterile silk sutures to ligate the left external carotid artery above the STA. Then, use another 6-0 sterile silk suture to ligate the roots of the ICA and OA simultaneously.
   NOTE: Avoid ligating nerves and other surrounding tissues. Completely block the shunt flow of ECA/ICA/OA while preserving a certain degree of retrograde flow in LCCA. The ligation effect shall be evaluated by observing the bleeding/blood flow status and distal arterial pulsation.
5. Check for bleeding at the surgical site. If bleeding occurs, gently press with sterile cotton swabs for 30-60 s to achieve hemostasis. Suture the skin with tissue adhesive or surgical suture.
   NOTE: Avoid excessive tissue adhesive from seeping into the subcutaneous tissue.

5. Post-partial carotid ligation care and high fat diet intervention

1. Administer 0.5 mL of warm normal saline via intraperitoneal injection for fluid replacement post-operatively.
2. Relieve pain by subcutaneous injection of meloxicam (0.1 mg/kg) immediately after surgery and 12 h post-operation.
3. Place the mouse in a heated recovery cage (37°C) until the righting reflex is restored (approximately 15-30 min). Monitor the mouse for abnormal behaviors (e.g., lethargy, dyspnea) within 24 h post-operation.
4. Provide antibiotics to prevent infection post-surgery as advised by veterinarians, such as antibiotic-containing drinking water for 7 days post-surgery.
5. Feed with Western-style HFD (with 40% fat energy ratio and 1.25% cholesterol content) until lesion assessment (4 weeks post-operation).
   NOTE: Store the HFD at 4°C to prevent fat oxidation and use it within 2 weeks of opening. Replace the HFD every 2 days to ensure freshness and monitor the mice's food intake daily (normal food intake: approximately 4-5 g per mouse per day). Synchronizing HFD feeding with PCSK9-AAV-induced hypercholesterolemia maximizes lipid deposition in the carotid artery; delaying the initiation of HFD will reduce lesion severity.

6. Sample collection and processing

CAUTION: Paraformaldehyde emits toxic vapors. Preparation, use, and removal must be performed in a chemical fume hood while wearing appropriate PPE (gloves, safety goggles, and lab coat). Dispose of waste using established neutralization and chemical waste collection procedures. fixatives should be neutralized and centrally collected in accordance with institutional liquid waste disposal protocols.

NOTE: The human carotid atherosclerotic specimens in this study were obtained from patients with carotid artery stenosis who underwent carotid endarterectomy. This study complies with the Declaration of Helsinki and was approved by the Institutional Ethics Review Board of Peking Union Medical College Hospital (PUMCH) (No. JS-2966). All study participants provided written informed consent.

1. At 4 weeks post-operation, anesthetize mice via intraperitoneal injection of tribromoethanol.
2. Perform bilateral thoracotomy on anesthetized mice and puncture the heart to collect blood samples for blood lipids tests. Subsequently, perfuse the left ventricle with 20 mL of ice-cold PBS to flush residual blood from the blood vessels in 3-6 mL/min. Observe changes in tissue color as a visual indicator of perfusion completion.
   NOTE: If permitted by institutional ethics/regulations, alternative methods may be used if they are clearly stated and performed in compliance with the requirements of the local Institutional Animal Care and Use Committee (IACUC). Animal carcasses must be incinerated by the institutional veterinary team or authorized contractors following biowaste regulations or processed via approved biohazard disposal procedures.
3. Dissect and harvest the carotid arteries together with the heart, isolate the bilateral carotid artery under a microscope, and fix it in 4% paraformaldehyde (PFA) at 4°C for 24 h.
4. Transfer the fixed carotid artery to a 30% sucrose solution (dissolved in PBS) and incubate at 4°C for 24 h (until the tissue sinks). Embed the artery in optimal cutting temperature (OCT) compound and store at -80°C.
5. Use a cryostat to cut 5 µm thick cross-sections of the left common carotid artery (section location: 1 mm proximal to the ligation site; cutting temperature: -20°C ~ -22°C, Fine-tune based on the tissue water content). Using a pre-chilled fine brush, gently transfer the section onto the cryostat's anti-roll plate to ensure it lies flat. Hold a poly-L-lysine-coated slide at a near-vertical angle and bring it slowly into contact with the section.
   NOTE: The perfusion step is critical for removing residual blood; otherwise, it will interfere with lesion staining. Complete arterial fixation within 30 min after euthanasia to preserve tissue morphology.

7. Atherosclerotic lesion assessment 1 - Oil Red O staining

1. Air-dry the sections at room temperature (20-25 °C) for 30 min; soak in 60% isopropanol for 1 min, then incubate in Oil Red O working solution (diluted 3:2 with 60% isopropanol) at room temperature (20-25 °C) for 10 min.
2. Differentiate by fully immersing the slide in 60% isopropanol at room temperature for ~5-10 s with gentle agitation to remove unbound dye; stain with hematoxylin for 30 s; rinse with tap water for 5 s (to blue the cell nuclei).
3. Capture section images using slide scanner or optical microscope with magnification 5x, 10x and 20x; quantitatively analyze the lipid deposition area using ImageJ software (Version 1.54p)- calculate the percentage of Oil Red O-positive area relative to the total intimal area.
4. Image J quantification procedures
   1. Open the image; select the Freehand Selection tool to manually outline the intima-media area. Navigate to Analysis > Tools > ROI Manager and click Add.
   2. Go to Image > Adjust > Color Threshold; adjust the Hue and Brightness parameters to match the Oil Red O-positive regions. Click Select, then Add to the ROI Manager.
   3. Select both the intima-media ROI and the stained ROI, click AND to obtain the Oil Red O-positive area within the intima-media region.
   4. Click Add and Measure to calculate the areas of the intima-media and Oil Red O-positive regions.
   5. Calculation formula:
      Oil Red O area (%) = (Oil Red O_positive_area / intimal_media_area) × 100

8. Atherosclerotic lesion assessment 2 - Verhoeff's Van Gieson staining

1. Retrieve OCT compound-embedded frozen sections on slides.
2. Immerse slides in distilled water at room temperature (20-25°C) for 10-15 min with gentle agitation (2-3 times) to dissolve OCT compound completely.
3. Discard OCT compound-containing water, then rinse sections with fresh distilled water for 2 min to eliminate residual OCT compound.
4. Incubate the sections in Verhoeff stain (containing hematoxylin, iodine, and ferric chloride) at room temperature (20-25°C) for 15 min, gently shaking the slide rack 2-3 times during incubation to ensure uniform staining.
5. Rinse the sections quickly with distilled water twice, 10 s each time.
6. Differentiate in 2% ferric chloride solution for 30-60 s; observe under a microscope until elastic fibers appear blue-black and the background appears gray. Rinse with distilled water twice, 1 min each time.
7. Treat the sections in 5% sodium thiosulfate solution for 1-2 min to remove residual iodine.
8. Rinse with distilled water for 2 min, then counterstain with Van Gieson stain (acid fuchsin + picric acid) for 5 min to stain collagen fibers bright red.
9. Dehydrate sequentially in 95% ethanol (twice, 1 min each) and 100% ethanol (twice, 1 min each); clear in xylene for 5 min; Sections were cover slipped using a synthetic resin mounting medium.
10. Capture section images; Use digital image analysis software to measure the intima-media thickness or intimal thickness of plaques in four directions (anterior, posterior, left, right) using the length measurement tool, then calculate the average value.
    1. Open the slide, zoom to the target vessel cross-section, and click the Measurement Tool.
    2. Click the inner of the intima and outer edges of the intima/media in the anterior, posterior, left, and right directions sequentially, and record the four thickness values.
    3. Calculate the average value and record the measurements.
       ​Average value = (anterior + posterior + left + right) ÷ 4

Results

Oil Red O staining results showed that the ligated arteries exhibited severe atherosclerotic plaque formation, obvious lipid cores, and necrotic areas. The proportion of lipid regions was significantly higher in the left carotid artery than that of the right carotid artery control (p < 0.001) (Figure 2A). VVG staining revealed destruction or disappearance of elastic fibers in the intima, along with significant thickening of the plaque intima. Both the intima-media thickness (IMT) and inti...

Discussion

This protocol describes a standardized method for establishing a carotid atherosclerosis model in C57BL/6 mice by combining PCSK9-AAV-induced hypercholesterolemia, high-fat diet (HFD), and partial ligation of the common carotid artery (PCL).

Compared with traditional model like ApoE -/- or LDLR-/- model or PCL models using WT mouse⁵, this model offers unique advantages in atherosclerosis research. First, mice are administered PCSK9-AAV and fed a HFD to...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-0 sterile nylon suture	Jinhuan Medical	F602	NA
C57BL/6 mice	Shanghai Model Organisms Center, Inc. (SMOC)	SM-001	NA
Chow deit	Xietong Bio	No. XT93G	NA
Cryostat	Leica	CM1950	NA
Depilatory cream	Veet	RB Health LLC	NA
High-fat diet	Xietong Bio	XT108c	NA
OCT	Sakura	4582	NA
Oil Red O Solution	servicebio	G1017-100mL	NA
Optical microscope	Leica	DM4B	NA
Paraformaldehyde	Servicebio	G1101-500mL	NA
PCSK9-AAV	WZBioscience	AV208001-AV8	NA
Poly-L-lysine-coated slides	Sigma	P0425	NA
Slide scanner	3DHISTECH	Pannoramic SCAN	NA
Sulfamethoxazole	Selleck	S1915	NA
Tissue adhesive	3M Vetbond	1469SB	NA
Vein-visualizing mouse tail injection holder	Jinan Yiyan Science & Technology Development Co., Ltd	YLS-Q9G	NA