Name: aggravation of myocardial ischemia upon particulate matter exposure in atherosclerosis animal model
Uploaded: 2021-12-10T14:00:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Aggravation of Myocardial Ischemia upon Particulate Matter Exposure in Atherosclerosis Animal Model at JoVE.com

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).

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&#8722;/&#8722;&#160;mice (C57BL/6 background) of 6-8 weeks old were used for the study.
1. Experimental preparation
<ol>
	<li>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 &#176;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.</li>
	<li>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.</li>
</ol>
2. Induction of atherosclerosis in mice
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Once atherosclerosis has judged formed, prepare the mice for the next step.</li>
</ol>
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.
<ol>
	<li>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&#176; angle from the table plane.</li>
	<li>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.</li>
	<li>Disinfect the dissection board with alcohol wipes.</li>
	<li>Place the anesthetized mouse in a supine position on the board and hook the upper incisors to the rubber band.</li>
	<li>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.</li>
	<li>Put a small sterile cotton swab into the mouse&#39;s mouth, then roll the swab to stick the tongue out.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Drop 50 &#181;L 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 &#181;L for once), 10 s apart, is better.</li>
	<li>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.</li>
</ol>
4. Coronary artery ligation
NOTE: Myocardial Ischemia modeling operation (coronary artery ligation) is performed at the 16th week.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Anesthetize the mice following the procedure described in step 3.2. 
	NOTE: A 1-week interval must be ensured between each tribromoethanol administration.</li>
	<li>Disinfect the platform with alcohol wipes.</li>
	<li>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.</li>
	<li>Remove the hair of the left chest and part of the adjacent right chest with hair removal cream before surgery.</li>
	<li>Perform orotracheal intubation in mice following the procedure described in steps 3.4-3.8.</li>
	<li>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.</li>
	<li>Wipe the skin with iodophor and alcohol to disinfect.</li>
	<li>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.</li>
	<li>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.</li>
	<li>At first, locate the LAD.</li>
	<li>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.</li>
	<li>Place a short piece of sterile 5-0 silk between the ligature and myocardial tissues to prevent tissue breakage.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Recovery
<ol>
	<li>Clean up all the bloodstain after surgery, or the mouse would be attacked by others.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the mouse back into the home cage. Keep monitoring for the next 24 h before sample collection. Administer analgesics for long-term experiments.</li>
</ol>

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The authors have no competing financial interests to declare.

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&#8722;/&#8722; 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&#39;s needs.
PM exposure may 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&#39;s long incisors is necessary. Pruning the mouse&#39;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 &#39;Efficient Model&#39;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&#39;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 towers have huge costs (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. Although nasal instillation is less harmful, upon nasal instillation, some of the suspension may enter the lungs, some may enter the digestive system, and some will remain in the nasal cavity. Since the PM suspension will not all enter the lungs, a nasal instillation cannot simulate exposure to air pollution. In contrast, injecting the particulate matter into the trachea ensures that all particulate matter enters the lungs directly. In addition, the nasal cavity is smaller and requires a higher concentration of the suspension to achieve the desired dose, making it more difficult to control the average dose administered.
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.

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) (&#8804;2.5 &#181;m 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&#8722;/&#8722; (apolipoprotein E knocked out) mouse is a traditional mouse model used in atherosclerosis studies. The ability to clear plasma lipoproteins in ApoE&#8722;/&#8722; 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&#8722;/&#8722; 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.

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

aggravation of myocardial ischemia upon particulate matter exposure in atherosclerosis animal model

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&#8722;/&#8722; 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&#39;s survival rate and reduce the difficulty of the operation. This animal model can reasonably simulate the patient&#39;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.

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 pale due to decreased dehydrogenase activity18. The MI+PM group&#39;s heart has a larger infarct area than the MI group&#39;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&#39;s aorta had more plaques than the AS group&#39;s. Figure 3 shows the homemade chest opening tools mentioned and its&#39; usage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63184/63184fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63184/63184fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63184/63184fig02.jpg" /> 
Figure 2: Representative examples of Oil Red O staining of aortae of ApoE&#8722;/&#8722; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63184/63184fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63184/63184fig03.jpg" /> 
Figure 3: Homemade chest opening tools. Cross place the chest opening tools to open an operating window when in use. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63184/63184fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

aggravation-myocardial-ischemia-upon-particulate-matter-exposure

Nanyang Technological University

Research

JoVE Journal

Medicine

1.9K Views.  China Academy of Chinese Medical Sciences. Het protocol hier is voor het bouwen van een samengesteld diermodel van myocardiale ischemie. Gecombineerd met athero sclerose en PM acute blootstelling. Dit is een betrouwbaarder diermodel voor het onderzoek naar verwante ziekten.Dit diermodel kan dergelijke ziekten beter simuleren in een echte situatie. Bovendien heeft deze methode een hoog slagingspercentage en is het gemakkelijk uit te voeren. Het model kan worden gebruikt voor pathologisch onderzoek en medicijnontwikkeling van zie...

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

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Immunology and Infection

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

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

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