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

The protocol here is for building a compound animal model of myocardial ischemia. Combining with athero sclerosis and PM acute exposure. This is a more reliable animal model for the research on related disease.

This animal model can better simulate such diseases in a real situation. Besides this method has a high success rate and it’s easy to perform. The model can be used for pathological research and drug development of diseases with complex factors.

This model can provide a platform for cardiovascular and the environmental toxicology research, and it contributes ideas and references for research of the multifactor disease. Orotracheal intubation, cracked opening of the chest, and correct ligation position are the keys to the success of this model. It is essential to maintain the mouse briefing and prevent hemorrhage.

Begin by selecting two to three mice randomly and check whether there is a plaque in the aortic arch by ultrasound imaging, or by direct anatomical observation. Anesthetize the selected mouse and place it in a supine position on the mouse board. Hook the upper incisors to the rubber band.

Focus a small LED spotlight with a flexible pipe on the trachea, which is around the midpoint of the axillary line. Put a small sterile cotton swab into the mouse’s mouth and then roll the swab to stick the tongue out. 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, can be seen as a bright spot that opens and closes with each breath. Insert a 22 gauge canola into the trachea of the mouse by aiming at the glottis, and then pull out the needle core. Use a pipette gun with a small amount of normal saline to test whether the tube is correctly in the wheezend.

If the tube is at the correct position, the liquid column in the pipette gun will be bouncing with each breath. Drop 50 microliters of DPM suspension into the tube with a pipette gun. Remove the pet indwelling needle after PM exposure.

Keep the mouse on the heating pad until it regains consciousness and then place the mouse back into the home cage. Place the mouse in a supine position on the intubation platform and fix the mouse. Remove the hair of the left chest and part of the adjacent right chest with hair removal cream.

Then link the pet indwelling needle with an animal ventilator. Wipe the skin with iodoform and alcohol to disinfect. Make a skin cut for approximately one centimeter by ophthalmic scissors and brace the muscles to expose the ribs.

Clamp the rib with an ophthalmic tweezer with tooth and make a small cut at the third intercostal space. Make an operating window with homemade chest opening tools and then rip the pericardial membranes. Hold the sterile six zero silk suture with a needle using microvascular hemostatic forceps.

Pass the silk through a two millimeters width of myocardium in the area where the coronary artery is located. Place a short piece of sterile five zero silk between the ligature and the myocardial tissues to prevent tissue breakage. Tie the left anterior descending coronary artery and the small bundle of the myocardium around it tightly.

The legation is deemed successful when the anterior wall of the left ventricle turns pale. Gently squeeze out the air from the chest. Then suture the intercostal muscles and skin sequentially with sterile five zero silk.

Clean up all the blood stain after surgery and then place the mouse on a heating pad in a lateral recumbent position. Continually monitor the mouse signs for approximately five to 20 minutes until they recover from anesthesia. Once the righting reflex is recovered, transfer the mouse to a clean recovery cage on a heating pad with the food and water bottle.

Continue to monitor for 15 to 30 minutes to ensure the survival of the mouse. Finally, inject penicillin sodium intramuscularly according to the desired dose to prevent wound infection. The degree of ischemia was examined by TTC staining.

Normal tissues turn red when the TTC reacts with the succinate dehydrogenase. While the ischemic tissues remain pale due to decreased dehydrogenase activity. The representative images show the heart tissues that suffered no MI surgery or PM exposure, suffered MI surgery but no PM exposure, and suffered MI surgery and PM exposure.

PM exposure aggravated myocardial ischemia. Oil Red O can precisely color the neutral fats, such as triglycerides in the tissues. The red spots in the picture indicate plaques.

The plaques in the aorta of wild type mice with regular diet, APO E knockout mice with high fat diet, and APO E knockout mice with a high fat diet and suffered from PM exposure are shown here by oil red O staining. High fat feeding led to atherosclerosis in APO E knockout mice and PM exposure aggravated atherosclerosis. It is important to ensure that the endotracheal tube is properly insert during seromyotomy.

The operator need to complete the intubation operation quickly, accurately, and always pay attention to the breathing condition of the animal during the operation. Our method helps researchers who are concerned with the intersection of air pollution and cardiovascular disease. Here we provide an idea.

Of course, researchers can also directly carry out research based on the animal model we described.