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

Published: December 10, 2021 doi: 10.3791/63184
Yuanmin Yang*1, Shuoqiu Deng*1, Shuiqing Qu1, Yu Zhang1, Zhongyuan Zheng1, Lina Chen1, Yujie Li1
* These authors contributed equally


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


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.


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, PM2.5) 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 event8,10. Tracheal infusion has been used in many research and stands out from exposure models11,12 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 severe15,16. 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.

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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 pure water. Store the solution at 4 °C and avoid exposure to light.
  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 anatomical observant directly17.
    NOTE: Anatomical observation is usually more reliable because ultrasound imaging could not be observed all plaque.
  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. Place the anesthetized mouse in a supine position on the mouse board and hook the upper incisors to the rubber band.
  4. 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.
  5. Put a small sterile cotton swab into the mouse's mouth, then roll the swab to stick the tongue out.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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 16th 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.
  4. Fix the mouse. 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.
  5. Remove the hair of the left chest and part of the adjacent right chest with hair removal cream before surgery.
  6. Perform orotracheal intubation in mice following the procedure described in steps 3.4-3.8.
  7. Link the pet indwelling needle with ananimal ventilator (see Table of Materials). Ventilator setting: respiratory rate- 120 times/min; inhalation/respiration ratio - 1:1.1; tidal volume - 1.7 mL.
  8. Wipe the skin with iodophor and alcohol to disinfect.
  9. 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.
  10. 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.
  11. At first, locate the LAD.
  12. 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.
  13. Place a short piece of sterile 5-0 silk between the ligature and myocardial tissues to prevent tissue breakage.
  14. 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.
  15. Gently squeeze out the air from the chest. Suture intercostal muscles and skin sequentially with sterile 5-0 silk.
    ​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 turning over is observed, 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.

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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 taken from the eyeballs. 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 pale due to decreased dehydrogenase activity18. The MI+PM group's heart has a larger infarct area than the MI group's.

Figure 2 shows plaques in the aorta by oil red O staining17,19. Oil red O can precisely color the neutral fats such as triglycerides in the tissues17. The red spots in the picture indicate plaques. The AS+PM group's aorta had more plaques than the AS group's. Figure 3 shows the homemade chest opening tools mentioned and its' usage.

Figure 1
Figure 1: TTC staining assay in mouse heart tissue. The infarct area shows white. PM exposure aggravated myocardial Ischemia. Sham: Suffered no MI surgery or PM exposure; MI: Suffered MI surgery but no PM exposure; MI+PM: Suffered both MI surgery and PM exposure. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Representative examples of Oil Red O staining of aortae of ApoE−/− mice. The plaque in the aorta was stained red. High-fat feeding led to atherosclerosis in ApoE-/- mice, and PM exposure aggravated atherosclerosis. Sham: wild-type mice with normal diet; AS: ApoE-/- mice with high-fat diet; AS+PM: ApoE-/- mice with a high-fat diet, suffered from PM exposure. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Homemade chest opening tools. Cross place the chest opening tools to open an operating window when in use. Please click here to view a larger version of this figure.

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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 inducing atherosclerosis according to the experiment's needs.

PM exposuremay exert little effect on the survival rate of the mice. But repeating tracheal intubation will cause intraoral bleeding and edema in mice20, which will increase the difficulty of subsequent experiments. Therefore, it is necessary to practice the intubation process diligently. Try to find the correct position in as few tries as possible. Since a long period is needed in this experiment, shortening the mouse's long incisors is necessary. Pruning the mouse's long incisors need to be avoided during the operations, Including endotracheal intubation; otherwise, the sharp incisors may scratch the mouse tongue and cause bleeding.

LAD ligation surgery affects the survival rate of the mice. Classic and conservative intrathoracic ligation of the LAD coronary artery has been prudently chosen rather than the 'Efficient Model'10 (a method that squeezes the heart out of the chest) to get better long-term survival after surgery with less training costs.

The most critical essentials in operation are anesthetizing, maintaining the mouse's breathing, and preventing bleeding. Compared with pentobarbital, tribromoethanol can significantly improve the survival rate of mice. The mouse will be unconscious 2-5 min after anesthesia, and this situation usually lasts until the end of the operation. If the mouse wakes up, an additional injection of 0.05 mL anesthetic is administered.

After the chest cavity is opened, the ventilator should be connected all the way. If the tracheal intubation falls off in the middle, the thoracic cavity should be sealed immediately with hemostatic forceps, and the experiment can be continued after reconnecting the ventilator. Bleeding should be avoided during the surgery. The bleeding process tends to occur in the open chest, pericardium removed, and LAD ligated. If bleeding occurs, remove the blood with cotton swabs. Exhaust should be entirely squeezed when closing the chest cavity, or use chest tube8 when the chest is closed.

The PM exposure method in mice mainly includes exposure tower21, tail vein injection22, and tracheal dripping23. Exposure tower costs huge (because of expensive equipment and the huge PM consumption), while the tail vein injection is quite different from the natural pattern of PM exposure. Tracheal drip is a compromise way. Compared to breathing under PM exposure, tracheal dripping is a passive exposure process. The distribution of PM in the trachea and lungs may be different from the natural state. But as a classic method, tracheal dripping is quantitatively accurate and easy to implement9.

The current protocol suffers from certain limitations. The raw materials of PM suspension used in tracheal instillation are a standard particulate matter from diesel engines. It mainly contains polycyclic aromatic hydrocarbons, which is one of the main components of PM. The chemical constituents of PM from theatmosphere include nitrates, sulfates, elemental, organic carbon, organic compounds (e.g., polycyclic aromatic hydrocarbons), biological compounds (e.g., endotoxin, cell fragments), and metals (e.g., iron, copper, nickel, zinc, and vanadium)24. The particulate matter standard may differ from the particulate matter in the air, which is also not a perfect choice. The composition of particulate matter varies by region, climate, and season. Therefore, the PM collected from the air is uncertain, causing the experiments to be challenging to repeat with the same results. Using PM standards could give the research better repeatability.

Altogether, a model of myocardial Ischemia occurring based on atherosclerosis following particulate matter exposure has been described. This model can be used to study the effect of air pollution on cardiovascular diseases and provide a reference for establishing an animal model of complex diseases.

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The authors have no competing financial interests to declare.


This model was developed with the support of the National Natural Science Foundation of China (Nos. 81673640, 81841001, and 81803814) and the Major National Science and Technology Program of China for Innovative Drug (2017ZX09301012002 and 2017ZX09101002001-001-3).


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



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Cite this Article

Yang, Y., Deng, S., Qu, S., Zhang, Y., Zheng, Z., Chen, L., Li, Y. Aggravation of Myocardial Ischemia upon Particulate Matter Exposure in Atherosclerosis Animal Model. J. Vis. Exp. (178), e63184, doi:10.3791/63184 (2021).More

Yang, Y., Deng, S., Qu, S., Zhang, Y., Zheng, Z., Chen, L., Li, Y. Aggravation of Myocardial Ischemia upon Particulate Matter Exposure in Atherosclerosis Animal Model. J. Vis. Exp. (178), e63184, doi:10.3791/63184 (2021).

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