Aggravation of Myocardial Ischemia upon Particulate Matter Exposure in Atherosclerosis Animal Model

Summary

This protocol describes a composite animal model with exposure to particulate matter (PM) that aggravates myocardial ischemia with atherosclerosis.

Abstract

The health problems caused by air pollution (especially particulate pollution) are getting more and more attention, especially among cardiovascular disease patients, which aggravates complicated disorders and causes poor prognosis. The simple myocardial ischemia (MI) or particulate matter (PM) exposure model is unsuitable for such studies of diseases with multiple causes. Here, a method for constructing a composite model combining PM exposure, atherosclerosis, and myocardial ischemia has been described. ApoE^−/− mice were fed with a high-fat diet for 16 weeks to develop atherosclerosis, tracheal instillation of PM standard suspension was performed to simulate the pulmonary exposure of PM, and the left anterior descending coronary artery was ligated one week after the last exposure. Tracheal instillation of PM can simulate acute lung exposure while significantly reducing the cost of the experiment; the classic left anterior descending artery ligation with noninvasive tracheal intubation and a new auxiliary expansion device can ensure the animal's survival rate and reduce the difficulty of the operation. This animal model can reasonably simulate the patient's pathological changes of myocardial infarction aggravated by air pollution and provide a reference for the construction of animal models related to studies involving diseases with multiple causes.

Introduction

Air pollution has been associated with high all-cause mortality and contributed a significant burden of disease more than the sum of water pollution, soil pollution, and occupational exposure¹. A report from WHO revealed that outdoor air pollution caused 4.2 million premature deaths in both cities and rural areas worldwide in 2016². 91% of people worldwide live in places where air quality exceeds WHO guideline limits². Further, the fine particulate matter (PM) (≤2.5 µm in diameter, PM_2.5) is recognized as the most significant air pollution threat to global public health

Protocol

All animal activities described here were approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Male ApoE^−/− mice (C57BL/6 background) of 6-8 weeks old were used for the study.

1. Experimental preparation

1. Prepare Tribromoethanol anesthetics (15 mg/mL): dissolve 0.75 g of tribromoethanol in 1 mL of tert-amyl alcohol (see Table of Materials). After complete dissolution, dilute it to 50 mL with sterile saline. Store the solution at 4 °C in a sterile container and avoid exposure to light.
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Results

The mice were euthanized 24 h after the coronary artery ligation, and the blood was collected after anesthesia. Mice were anesthetized by tribromoethanol (as per step 3.2), and the blood sample was collected from the retroorbital sinus. The heart was harvested, and the degree of Ischemia was examined by 2,3,5-Triphenyltetrazolium Chloride (TTC) staining (Figure 1). Normal tissues turn red when the TTC reacts with succinate dehydrogenase, while the ischemic tissues remain pal.......

Discussion

The establishment of a composite animal model is slightly different from the single MI model. Maintaining a high survival rate is challenging in the development of the composite model. The severity of atherosclerosis in ApoE^−/− mice will become more severe with the extension of high-fat feeding time⁷, and the weakness of mice leads to increased mortality. Therefore, it is necessary to monitor the condition of the mice during the experiment continually and adjust the time for.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,2,2-Tribromoethanol	Sigma-Aldrich	T48402
75% alcohol disinfectant
Animal ventilator	Shanghai Alcott Biotech	ALC-V8S
Cotton swabs			Sterile
Cotton swabs for babies			Sterile , Approximately 3 mm in diameter
Culture Dish	Corning	430597	150 mm x 25 mm
Diesel Particulate Matter	National Institute of Standards Technology	1650b
Dissection board			About 25 x 17 cm. The dissecting board can be replaced with a wooden board of the same size
High-fat diet for mice			Prescription: egg yolk powder 10%, lard 10%, sterol 1%, maintenance feed 79%
Iodophor disinfectant
LED spotlight			5 V, 3 W,with hoses and clamps
Medical silk yarn ball	Shanghai Medical Suture Needle Factory Co., Ltd.	-	0-0
Medical tape	3M	1527C-0
Micro Vascular Hemostatic Forceps	Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory	W40350
Needle Holders	Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory	JC32010
Normal saline
Ophthalmic Scissors	Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory	Y00040
Ophthalmic tweezer, 10cm, curved, with hooks	Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory	JD1080
Ophthalmic tweezer, 10cm, curved, with teeth	Shanghai Medical Instruments (Group) Ltd., Corp. Surgical Instruments Factory	JD1060
Pipet Tips	Axygen	T-200-Y-R-S	0-200 μL
Pipette	eppendorf	3121000074	100 uL
Safety pin			Approximately 4.5 cm in length , for making chest opening tools
Small Animal I.V. Cannulas	Baayen healthcare suzhou	BAAN-322025	I.V CATHETER 22FG x 25 MM
Suture needle with thread	Shanghai Medical Suture Needle Factory Co., Ltd.	-	6-0,Nylon line
Suture needle with thread	JinHuan Medical	F503	5-0
Syringe			1 mL
Tert-amyl alcohol
Zoom-stereo microscope	Mshot	MZ62