Isolation and Analysis of Aortic Arch and Root Lesions in an Atherosclerotic Mouse Model

Summary

This protocol provides a comprehensive methodology for the quantitative analysis of atherosclerotic plaques to facilitate more in-depth research into the mechanisms and progression of atherosclerosis.

Abstract

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study introduces a comprehensive protocol for the isolation and histological analysis of aortic arch and root lesions in a widely used atherosclerotic mouse model, low-density lipoprotein receptor knock-out (Ldlr^-/-) mice. The aortic arch and root are key sites for atherosclerotic lesions, and their examination is critical for assessing the onset, progression, or regression of atherosclerosis, predicting cardiovascular event risks, and identifying potential therapeutic targets. This protocol outlines methods for quantifying atherosclerotic burden in the aortic arch and root, including tissue isolation, fixation, Oil Red O staining, aortic root sectioning, Hematoxylin and Eosin (HE) staining, Verhoeff-Van Gieson (VVG) staining, and image analysis. Oil Red O staining measures plaque area in the aortic arch, evaluating the severity of atherosclerosis, while HE staining of the aortic root reveals plaque components such as the lipid core and fibrous cap, facilitating the assessment of plaque stability and rupture risk. VVG staining can stain collagen fibers within tissues, providing further insights into plaque composition and related information. This thorough analysis offers valuable insights into the mechanisms of lesion development and may guide the creation of novel therapeutic strategies for preventing and treating atherosclerosis.

Introduction

Cardiovascular diseases, particularly atherosclerosis, have emerged as a significant health burden and a primary cause of death worldwide^1,2. Atherosclerosis is a chronic progressive inflammatory disease characterized by the gradual accumulation of lipids and the formation of plaques in the arterial wall, ultimately leading to the narrowing of the arterial lumen and potentially to the rupture of plaques, triggering acute cardiovascular events such as myocardial infarction and stroke^1,2,3. Given its profound impact on ....

Protocol

All animal protocols used in this study were approved by the Ethics Review Committee for Animal Experimentation of the Shanghai University of Sport.

1. Preparation of reagents and dissection tools

1. Sterilize dissection tools, including fine scissors, straight forceps, curved forceps, spring scissors, and pins, by autoclave in advance.
2. 75% ethanol: Mix 75 mL of Anhydrous ethanol with 25 mL of ddH₂O.
3. 1x Phosphate buffered saline (PBS): Dissolve 0.01 M PBS powder in 2 L of ddH₂O.
4. 4% paraformaldehyde (PFA) solution: Heat 800 mL of 1x PBS to approximately 60 °C ....

Results

The representative results demonstrate the application of the isolation and analysis technique for aortic arch and root lesions in an atherosclerotic mouse model. These results provide clear evidence of the technique's ability to identify and characterize atherosclerotic lesions. For example, histological images with specific stains (e.g., Oil Red O) highlight lipid accumulation, while hematoxylin and eosin (H&E) staining reveals the overall morphology of the atherosclerotic lesions, including the structure of the ar.......

Discussion

Here, we provide detailed information on the methods of aortic sampling in Ldlr knockout mice and quantitative analysis of plaques.

The precision of the dissection procedure is the biggest technical challenge to in vivo aortic stripping in the mouse model of atherosclerosis. Based on our experience, the key points are as follows: (1) use PBS to wash out all the blood in the artery to increase the comparison between aortic arch branches and perivascular fat. (2) Be patient. Due to the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesion microscope slides(25×75mm)	CITOTEST	Cat# 80312-3161
Embedding cassette	CITOTEST	Cat# 80106-1100-16
Eosin Staining Solution	Beyotime	Cat# C0109
Ethanol	Sinopharm Chemical Reagent Co.	Cat# 10009218
Hematoxylin Staining Solution	Beyotime	Cat# C0107
Low-profile disposable blades	Leica	Cat# 14035838925
Microscope cover glass(24×50mm)	CITOTEST	Cat# 10212450C
Neutral Balsam Mounting Medium	Sango Biotech	Cat# E675007-0100
Oil red o powder	Sigma-Aldrich	Cat# 1320-06-5
paraffin with ceresin	Sinopharm Chemical Reagent Co.	Cat# 69019461
Paraformaldehyde	Servicebio	Cat# G1101
Phosphate Buffered Saline (PBS, Powder)	Servicebio	Cat# G0002-2L
Ponceau S Staining Solution	EveryLab	Cat# FM024
Victoria Blue’B	Aladdin	Cat# 2580-56-5
Xylene	Sigma-Aldrich	Cat# 104-81-4