Name: aggravation of myocardial ischemia upon particulate matter exposure in atherosclerosis animal model
Uploaded: 2021-12-10T14:00:00
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 co...

<ol>
	<li>Al-Kindi, S. G., Brook, R. D., Biswal, S., Rajagopalan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+determinants+of+cardiovascular+disease:+lessons+learned+from+air+pollution.">Environmental determinants of cardiovascular disease: lessons learned from air pollution.</a> Nature Reviews: Cardiology. 17 (10), 656-672 (2020).</li><li>Ambient (outdoor) Air Pollution. WHO Available from: <a target="_blank" href="https://www.who.int/news-room/fact-sheets/detail/ambient-(outdoor)">https://www.who.int/news-room/fact-sheets/detail/ambient-(outdoor)</a> (2021)</li><li>Kim, K. 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(122), e55353 (2017).</li><li>Lei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+effect+of+diesel+exhaust+particles+and+different+fractions+exposure+on+blood+coagulation+function+in+mice.">The acute effect of diesel exhaust particles and different fractions exposure on blood coagulation function in mice.</a> International Journal of Environmental Research and Public Health. 18 (8), 4136 (2021).</li><li>Gao, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+efficient+model+of+coronary+artery+ligation+and+myocardial+infarction+in+the+mouse.">A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse.</a> Circulation Research. 107 (12), 1445-1453 (2010).</li><li>Pei, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LncRNA+PEAMIR+inhibits+apoptosis+and+inflammatory+response+in+PM2.5+exposure+aggravated+myocardial+ischemia/reperfusion+injury+as+a+competing+endogenous+RNA+of+miR-29b-3p.">LncRNA PEAMIR inhibits apoptosis and inflammatory response in PM2.5 exposure aggravated myocardial ischemia/reperfusion injury as a competing endogenous RNA of miR-29b-3p.</a> Nanotoxicology. 14 (5), 638-653 (2020).</li><li>Jia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PM2.5-induced+pulmonary+inflammation+via+activating+of+the+NLRP3/caspase-1+signaling+pathway.">PM2.5-induced pulmonary inflammation via activating of the NLRP3/caspase-1 signaling pathway.</a> Environmental Toxicology. 36 (3), 298-307 (2021).</li><li>Vogel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ST-segment+elevation+myocardial+infarction.">ST-segment elevation myocardial infarction.</a> Nature Reviews Disease Primers. 5 (1), 39 (2019).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+landscape+of+atherosclerosis.">The changing landscape of atherosclerosis.</a> Nature. 592 (7855), 524-533 (2021).</li><li>Zhou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+neutrophil+extracellular+trap+formation+aggravates+acute+myocardial+infarction+injury+in+Apolipoprotein+E+deficiency+mice+via+the+ROS-dependent+pathway.">Excessive neutrophil extracellular trap formation aggravates acute myocardial infarction injury in Apolipoprotein E deficiency mice via the ROS-dependent pathway.</a> Oxidative Medicine and Cellular Longevity. 2019, 1209307 (2019).</li><li>Pluijmert, N. J., Bart, C. I., Bax, W. H., Quax, P. H. A., Atsma, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+on+cardiac+function,+remodeling+and+inflammation+following+myocardial+ischemia-reperfusion+injury+or+unreperfused+myocardial+infarction+in+hypercholesterolemic+APOE*3-Leiden+mice.">Effects on cardiac function, remodeling and inflammation following myocardial ischemia-reperfusion injury or unreperfused myocardial infarction in hypercholesterolemic APOE*3-Leiden mice.</a> Scientific Reports. 10 (1), 16601 (2020).</li><li>Centa, M., Ketelhuth, D. F. J., Malin, S., Gistera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+atherosclerosis+in+mice.">Quantification of atherosclerosis in mice.</a> Journal of Visualized Experiments. (148), e59828 (2019).</li><li>Benedek, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+TTC+staining+for+the+evaluation+of+tissue+injury+in+the+early+phases+of+reperfusion+after+focal+cerebral+ischemia+in+rats.">Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats.</a> Brain Research. 1116 (1), 159-165 (2006).</li><li>Mehlem, A., Hagberg, C. E., Muhl, L., Eriksson, U., Falkevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+neutral+lipids+by+oil+red+O+for+analyzing+the+metabolic+status+in+health+and+disease.">Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease.</a> Nature Protocols. 8 (6), 1149-1154 (2013).</li><li>Nelson, A. M., Nolan, K. E., Davis, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+orotracheal+intubation+in+mice.">Repeated orotracheal intubation in mice.</a> Journal of Visualized Experiments. (157), e60844 (2020).</li><li>Zheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+fine+airborne+particulate+matters+induces+hepatic+fibrosis+in+murine+models.">Exposure to fine airborne particulate matters induces hepatic fibrosis in murine models.</a> Journal of Hepatology. 63 (6), 1397-1404 (2015).</li><li>Bai, N., van Eeden, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+and+vascular+effects+of+circulating+diesel+exhaust+particulate+matter.">Systemic and vascular effects of circulating diesel exhaust particulate matter.</a> Inhalation Toxicology. 25 (13), 725-734 (2013).</li><li>Furuyama, A., Kanno, S., Kobayashi, T., Hirano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrapulmonary+translocation+of+intratracheally+instilled+fine+and+ultrafine+particles+via+direct+and+alveolar+macrophage-associated+routes.">Extrapulmonary translocation of intratracheally instilled fine and ultrafine particles via direct and alveolar macrophage-associated routes.</a> Archives of Toxicology. 83 (5), 429-437 (2009).</li><li>Brunekreef, B., Holgate, S. 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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&#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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;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 pal...

Watch this Scientific Journal Video about Aggravation of Myocardial Ischemia upon Particulate Matter Exposure in Atherosclerosis Animal Model at JoVE.com

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 ...

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.

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Medicine

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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. ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...