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 health³, especially to the people who live in cities of low-income and middle-income countries.

The adverse effects of air pollution on cardiovascular diseases deserve more attention. Previous studies have shown that PM leads to an increased risk of cardiovascular disease (CVDs)⁴. Exposure to high concentrations of ultrafine particles for several hours can lead to increased myocardial infarction mortality. For people with a history of myocardial infarction, exposure to ultrafine particles can significantly increase the risk of recurrence⁵. Moreover, it is generally accepted that PM exposure accelerates the progression of atherosclerosis⁶.

For medical research, it is crucial to select a suitable animal model. Simple atherosclerosis animal models⁷, myocardial ischemia animal models⁸, and PM exposure animal models⁹ already exist. ApoE^−/− (apolipoprotein E knocked out) mouse is a traditional mouse model used in atherosclerosis studies. The ability to clear plasma lipoproteins in ApoE^−/− mice is severely impaired. The high-fat diet feeding would cause severe atherosclerosis, resembling the diet dependency of atherosclerotic heart disease observed in humans⁷. Ligation of the left anterior descending coronary artery (LAD) is a classic method to induce the ischemic event^8,10. Tracheal infusion has been used in many research and stands out from exposure models^11,12 because of its better simulation and lower cost.

However, animal models of single disease have significant limitations in scientific research. The myocardial ischemia induced merely by LAD ligation is not simulated in the actual situation. In the natural state, myocardial ischemia is usually caused by plaque rupture and blocked coronary arteries¹³. Patients with ischemic cardiomyopathy usually have atherosclerotic basic lesions¹³. There are also abnormal lipid metabolism and inflammatory reactions in the body¹⁴. Therefore, ischemia caused by physical factors or under natural conditions has different pathological manifestations.Existing studies have shown that the infarction and inflammation in myocardial ischemia models with atherosclerosis are more severe^15,16. PM exposure can aggravate atherosclerosis and myocardial ischemia further by inducing inflammation and oxidative stress¹. Three factors usually coexist in the natural state, so the actual situation could be better simulated by using a compound model.

This protocol describes developing an animal model of myocardial ischemia (MI) combining atherosclerosis (AS) and PM acute exposure. ApoE^−/− mice were fed with a high-fat diet to induce atherosclerosis. Pulmonary exposure of PM was imitated by dripping PM suspension through the trachea. Ligation of the LAD in mice was used to induce myocardial ischemia. These methods were combined and optimized to simulate the disease state better and improve the survival rate of animals. No large exposure unit or gas anesthesia machine is needed, making the experiment easy to perform. This model can be used to study the impact of PM exposure in air pollution on atherosclerosis and ischemic cardiomyopathy and conduct research on new drugs developed to treat diseases with such complex factors.

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 Prepare Tribromoethanol anesthetics (15 mg/mL): dissolve 0.75 g of tribromoethanol in 1 mL of tert-amyl alcohol (see Table of Materials). After co…

Representative 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

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