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 attent....

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....

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