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

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.
   NOTE: In this protocol, tribromoethanol was used due to optimal anesthesia recovery time and survival rate of animals. Follow local animal ethics committee recommendations when selecting the anesthesia regimen.
2. Prepare PM suspension: measure 5 mg of DPM (Diesel particulate matter, see Table of Materials) in 10 mL centrifuge tubes. Add 5 mL of normal saline and turn the tube upside down to mix well. Use paraffin film to seal the tube and then put it into ultrasonic cleaner for 2-3 h (40KHz, 80w) for ultrasonic breaking.
   NOTE: The suspension should be homogeneous and free of particles agglomerates. Shake well before use.

2. Induction of atherosclerosis in mice

1. Feed the mice with a high-fat diet (egg yolk powder 10%, lard 10%, sterol 1%, maintenance feed 79%, see Table of Materials) for 12 weeks.
2. To estimate the progress of atherosclerosis, select 2-3 mice randomly and check whether there is a plaque in the aortic arch by ultrasound imaging or direct anatomical observation¹⁷.
   NOTE: For anatomical observation, animals were selected via random sampling and euthanized after anesthetization. Then, their chest cavity was opened, and blood vessels were directly visualized. Anatomical observation is usually more reliable because ultrasound imaging may not detect all plaques.
3. Once atherosclerosis has judged formed, prepare the mice for the next step.

3. Orotracheal intubation and particulate matter acute exposure

NOTE: PM will be exposed once a week for 4 weeks after 12 weeks of high-fat feeding and continually given a high-fat diet.

1. Prepare a dissection board (see Table of Materials) with a rubber band securing 1.5 cm from the top edge. Fix the dissection board at a 60° angle from the table plane.
2. Anesthetize the mouse using tribromoethanol anesthetic by intraperitoneal injection (0.1 mL for every 10 g of body weight). After 2-3 min, flip the mouse to check if there is a righting reflex. Perform a toe pinch to confirm sedation. Drop sterile lubricating on the eyes.
3. Disinfect the dissection board with alcohol wipes.
4. Place the anesthetized mouse in a supine position on the board and hook the upper incisors to the rubber band.
5. Use a small LED spotlight (see Table of Materials) with a flexible pipe. Focus the light on the trachea, which is at around the midpoint of the axillary line.
6. Put a small sterile cotton swab into the mouse's mouth, then roll the swab to stick the tongue out.
7. Hold the tongue and gently pull it up to make the oral cavity, pharynx, and trachea in the same longitudinal direction. The glottis, which is the entrance of the trachea, will be shown as a bright spot, which opens and closes with each breath.
8. Keep holding the tongue gently. Insert the cannula (22 G) into the trachea of the mouse by aiming at the glottis, pulling out the needle core after the cannula is inserted in the trachea.
9. Use a pipette gun with a small amount of normal saline to test whether the tube is correctly in the weasand. If the tube is at the right position, the liquid column in the pipette gun will be bouncing with each breath.
10. Drop 50 µL of DPM suspension (prepared in step 1.2) into the tube with a pipette gun. The suspension will be naturally inhaled into the lungs of the mouse as it breathes.
    NOTE: To ensure smooth breathing, giving the mouse two times the DPM suspension (25 µL for once), 10 s apart, is better.
11. Remove the pet indwelling needle after PM exposure. Wait for the mouse to remain on the heating pads until they regained consciousness (10-20 min) and then place back into the home cage.

4. Coronary artery ligation

NOTE: Myocardial Ischemia modeling operation (coronary artery ligation) is performed at the 16^th week.

1. Prepare surgical instruments. After autoclaving, store all the surgical tools in a sealed instrument box. Soak them in 75% alcohol for 20-30 min before surgery.
2. Construct the surgery platform. To achieve the proper platform slope, use a cell culture dish cover (150 mm x 25 mm). Fold 0-0 silk (10-15 cm length) in half and attach the ends of the thread to the top of the inclined platform using tape to create a suspension loop.
3. Anesthetize the mice following the procedure described in step 3.2.
   NOTE: A 1-week interval must be ensured between each tribromoethanol administration.
4. Disinfect the platform with alcohol wipes.
5. Place the mouse in a supine position on the intubation platform and hook the upper incisors at the suspension loop described in step 4.2. Tape the tail, limbs, and whiskers.
6. Remove the hair of the left chest and part of the adjacent right chest with hair removal cream before surgery.
7. Perform orotracheal intubation in mice following the procedure described in steps 3.4-3.8.
8. Link the pet indwelling needle with an animal ventilator (see Table of Materials). Ventilator setting: respiratory rate- 120 times/min; inhalation/respiration ratio - 1:1.1; tidal volume - 1.7 mL.
9. Wipe the skin with iodophor and alcohol to disinfect.
10. Expose the heart. Make a skin cut for 0.5-1 cm by ophthalmic scissors and brace the muscles (pectoral superficialis muscle and serratus anterior muscle) to expose the ribs. Clamp the rib with an ophthalmic tweezer (with hooks) and then make a small cut at the third intercostal space (see Table of Materials). Make an operating window with homemade chest opening tools.
    NOTE: The skin cut is located at about one-third of the xiphoid process and axilla line.
11. Rip the pericardial membranes. Then it is possible to ligate LAD by following steps 4.11-4.14.
    NOTE: If the pulmonary lobes are blocking the view, push it behind the heart using a small sterile cotton swab.
12. At first, locate the LAD.
13. Hold the sterile 6-0 silk suture with a needle using microvascular hemostatic forceps (see Table of Materials). Pass the silk through a 2 mm width of myocardium in the area where the coronary artery is located.
    NOTE: Do not try to ligate the LAD only, which may cause major intraoperative hemorrhage.
14. Place a short piece of sterile 5-0 silk between the ligature and myocardial tissues to prevent tissue breakage.
15. Tie the LAD and the small bundle of the myocardium around it tightly. The ligation is deemed successful when the anterior wall of the left ventricle (LV) turns pale; ST-segment elevation can be observed simultaneously if an electrocardiogram machine is connected.
16. Gently squeeze out the air from the chest. Suture intercostal muscles and skin sequentially with sterile 5-0 silk.
    NOTE: To squeeze the air from the chest, close the chest at the moment of lung expansion and use the index and middle fingers to gently squeeze the ribcage in the middle and allow the air to escape from beyond the last stitch. Syringes can also be used to extract chest gas.
    NOTE: Simple interrupted suture is recommended, for the mice may gnaw the silk when they are awake.

5. Recovery

1. Clean up all the bloodstain after surgery, or the mouse would be attacked by others.
2. Place the mouse on a heating pad in a lateral recumbent position. Continuously monitor mouse signs for 5-20 min until they recover from anesthesia. The monitoring time depends on the state of the body.
   NOTE: Mice breathe easier in the lateral recumbent position.
3. Once the righting reflex is recovered, transfer the mice to clean recovery cages on a heating pad with food and water bottle. Continue to monitor for 15-30 min to ensure the survival of the mouse. Keep the mouse away from others before it can move entirely autonomously.
4. To prevent wound infection, inject penicillin sodium intramuscularly according to the desired dose (1,00,000-1,50,000 U/kg). For details, please refer to the drug labeling for dosage conversion.
5. Place the mouse back into the home cage. Keep monitoring for the next 24 h before sample collection. Administer analgesics for long-term experiments.

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