Name: establishment and evaluation of a porcine vein graft disease model
Uploaded: 2022-07-25T14:00:00
Description: Watch this Scientific Journal Video about Establishment and Evaluation of a Porcine Vein Graft Disease Model at JoVE.com

The authors thank Guangdong Laboratory Animals Monitoring Institute for technical support, animal care, and sample collection. They also thank Shenzhen Mindray Bio-Medical Electronics Co., Ltd, for technical support in the ultrasonic examination. This work was supported by Guangdong Science and Technology Program, China, and Jinan University Central Universities Basic Scientific Research Business Expenses Project (2017A020215076, 2008A08003, and 21621409).

The procedures for the care and use of laboratory animals were approved by the Institutional Animal Care and Use Committee of the Guangdong Laboratory Animals Monitoring Institute. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th Ed., 2011, National Research Council, USA). The surgical procedure is shown in Figure 1.
1. Preoperative preparation of animals
<ol>
	<li>Randomly divide 10 3-m...

<ol>
	<li>Lloyd-Jones, D., 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=Executive+summary:+Heart+disease+and+stroke+statistics--2010+update:+A+report+from+the+American+Heart+Association.">Executive summary: Heart disease and stroke statistics--2010 update: A report from the American Heart Association.</a> Circulation. 121 (7), 948-954 (2010).</li><li>Taggart, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contemporary+coronary+artery+bypass+grafting.">Contemporary coronary artery bypass grafting.</a> Frontiers of Medicine. 8, 395-398 (2014).</li><li>Wolny, R., Mintz, G. S., Pregowski, J., Witkowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms,+prevention+and+treatment+of+saphenous+vein+graft+disease.">Mechanisms, prevention and treatment of saphenous vein graft disease.</a> The American Journal of Cardiology. 154, 41-47 (2021).</li><li>Schachner, T., Laufer, G., Bonatti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+(animal)+models+of+vein+graft+disease.">In vivo (animal) models of vein graft disease.</a> European Journal of Cardio-thoracic Surgery: Official Journal of the European Association for Cardio-thoracic Surgery. 30 (3), 451-463 (2006).</li><li>Suggs, W. D., 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=Antisense+oligonucleotides+to+c-fos+and+c-jun+inhibit+intimal+thickening+in+a+rat+vein+graft+model.">Antisense oligonucleotides to c-fos and c-jun inhibit intimal thickening in a rat vein graft model.</a> Surgery. 126 (2), 443-449 (1999).</li><li>Jiang, 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=A+novel+vein+graft+model:+Adaptation+to+differential+flow+environments.">A novel vein graft model: Adaptation to differential flow environments.</a> American Journal of Physiology. Heart and Circulatory Physiology. 286 (1), H240-H245 (2004).</li><li>O'Brien, J. 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=Early+injury+to+the+media+after+saphenous+vein+grafting.">Early injury to the media after saphenous vein grafting.</a> The Annals of Thoracic Surgery. 65 (5), 1273-1278 (1998).</li><li>Zou, Y., 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=Mouse+model+of+venous+bypass+graft+arteriosclerosis.">Mouse model of venous bypass graft arteriosclerosis.</a> The American Journal of Pathology. 153 (4), 1301-1310 (1998).</li><li>Klyachkin, M. L., 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=Postoperative+reduction+of+high+serum+cholesterol+concentrations+and+experimental+vein+bypass+grafts.+Effect+on+the+development+of+intimal+hyperplasia+and+abnormal+vasomotor+function.">Postoperative reduction of high serum cholesterol concentrations and experimental vein bypass grafts. Effect on the development of intimal hyperplasia and abnormal vasomotor function.</a> The Journal of Thoracic and Cardiovascular Surgery. 108 (3), 556-566 (1994).</li><li>Tan, W., 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+porcine+model+of+heart+failure+with+preserved+ejection+fraction+induced+by+chronic+pressure+overload+characterized+by+cardiac+fibrosis+and+remodeling.">A porcine model of heart failure with preserved ejection fraction induced by chronic pressure overload characterized by cardiac fibrosis and remodeling.</a> Frontiers in Cardiovascular Medicine. 8, 677727 (2021).</li><li>Hocum Stone, ., L, L., 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=Magnetic+resonance+imaging+assessment+of+cardiac+function+in+a+swine+model+of+hibernating+myocardium+3+months+following+bypass+surgery.">Magnetic resonance imaging assessment of cardiac function in a swine model of hibernating myocardium 3 months following bypass surgery.</a> The Journal of Thoracic and Cardiovascular Surgery. 153 (3), 582-590 (2017).</li><li>Gedik, N., 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=Proteomics/phosphoproteomics+of+left+ventricular+biopsies+from+patients+with+surgical+coronary+revascularization+and+pigs+with+coronary+occlusion/reperfusion:+Remote+ischemic+preconditioning.">Proteomics/phosphoproteomics of left ventricular biopsies from patients with surgical coronary revascularization and pigs with coronary occlusion/reperfusion: Remote ischemic preconditioning.</a> Scientific Reports. 7 (1), 7629 (2017).</li><li>Tsirikos Karapanos, ., N, , 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+impact+of+competitive+flow+on+distal+coronary+flow+and+on+graft+flow+during+coronary+artery+bypass+surgery.">The impact of competitive flow on distal coronary flow and on graft flow during coronary artery bypass surgery.</a> Interactive Cardiovascular and Thoracic Surgery. 12 (6), 993-997 (2011).</li><li>Meng, X., 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=Competitive+flow+arising+from+varying+degrees+of+coronary+artery+stenosis+affects+the+blood+flow+and+the+production+of+nitric+oxide+and+endothelin+in+the+internal+mammary+artery+graft.">Competitive flow arising from varying degrees of coronary artery stenosis affects the blood flow and the production of nitric oxide and endothelin in the internal mammary artery graft.</a> European Journal of Cardio-thoracic Surgery: Official Journal of the. 43 (5), 1022-1027 (2013).</li><li>Thankam, F. G., 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=Association+of+hypoxia+and+mitochondrial+damage+associated+molecular+patterns+in+the+pathogenesis+of+vein+graft+failure:+A+pilot+study.">Association of hypoxia and mitochondrial damage associated molecular patterns in the pathogenesis of vein graft failure: A pilot study.</a> Translational Research: The Journal of Laboratory and Clinical. , 38-52 (2021).</li><li>Li, X., 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+surgical+model+of+heart+failure+with+preserved+ejection+fraction+in+Tibetan+minipigs.">A surgical model of heart failure with preserved ejection fraction in Tibetan minipigs.</a> Journal of Visualized Experiments: JoVE. 180 (180), 63526 (2022).</li><li>Rueda, A. l. c. a. l. &. #. 2. 2. 5. ;., I, , 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+live+porcine+model+for+surgical+training+in+tracheostomy,+neck+dissection,+and+total+laryngectomy.">A live porcine model for surgical training in tracheostomy, neck dissection, and total laryngectomy.</a> European Archives of Oto-Rhino-Laryngology: Official Journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS): Affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery. 278 (8), 3081-3090 (2021).</li><li>Proudfit, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+value+of+coronary+arteriography.">Prognostic value of coronary arteriography.</a> Cardiovascular Clinics. 12 (2), 1-8 (1981).</li><li>Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fibroplasia+in+cutaneous+wound+repair.">Regulation of fibroplasia in cutaneous wound repair.</a> TheAmerican Journal of the Medical Sciences. 306 (1), 42-48 (1993).</li><li>Darby, I., Skalli, O., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-smooth+muscle+actin+is+transiently+expressed+by+myofibroblasts+during+experimental+wound+healing.">Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing.</a> Laboratory Investigation. 63 (1), 21-29 (1990).</li><li>Sterpetti, A. V., 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=Formation+of+myointimal+hyperplasia+and+cytokine+production+in+experimental+vein+grafts.">Formation of myointimal hyperplasia and cytokine production in experimental vein grafts.</a> Surgery. 123 (4), 461-469 (1998).</li><li>Shannon, A. 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=Porcine+model+of+infrarenal+abdominal+aortic+aneurysm.">Porcine model of infrarenal abdominal aortic aneurysm.</a> Journal of Visualized Experiments: JoVE. 153 (153), (2019).</li><li>Langille, B. L., O'Donnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductions+in+arterial+diameter+produced+by+chronic+decreases+in+blood+flow+are+endothelium-dependent.">Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent.</a> Science. 231 (4736), 405-407 (1986).</li><li>Zwolak, R. M., Adams, M. C., Clowes, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+vein+graft+hyperplasia:+Association+with+tangential+stress.">Kinetics of vein graft hyperplasia: Association with tangential stress.</a> Journal of Vascular Surgery. 5 (1), 126-136 (1987).</li><li>Kotani, K., 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+subacute+hypoxic+model+using+a+pig.">A subacute hypoxic model using a pig.</a> Surgery Today. 35 (11), 951-954 (2005).</li><li>Liu, D., 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=Comparison+of+ketamine-pentobarbital+anesthesia+and+fentanyl-pentobarbital+anesthesia+for+open-heart+surgery+in+minipigs.">Comparison of ketamine-pentobarbital anesthesia and fentanyl-pentobarbital anesthesia for open-heart surgery in minipigs.</a> Lab Animal. 38 (7), 234-240 (2009).</li><li>Geovanini, G. R., Pinna, F. R., Prado, F. A., Tamaki, W. T., Marques, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+anesthesia+in+swine+for+experimental+cardiovascular+surgeries.">Standardization of anesthesia in swine for experimental cardiovascular surgeries.</a> Revista Brasileira de Anestesiologia. 58 (4), 363-370 (2008).</li><li>Alhomary, M., Ramadan, E., Curran, E., Walsh, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Videolaryngoscopy+vs.+fibreoptic+bronchoscopy+for+awake+tracheal+intubation:+A+systematic+review+and+meta-analysis.">Videolaryngoscopy vs. fibreoptic bronchoscopy for awake tracheal intubation: A systematic review and meta-analysis.</a> Anaesthesia. 73 (9), 1151-1161 (2018).</li><li>Parang, P., Arora, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+vein+graft+disease:+Pathogenesis+and+prevention.">Coronary vein graft disease: Pathogenesis and prevention.</a> Canadian Journal of Cardiology. 25 (2), e57-e62 (2009).</li><li>Egan, T. D., 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=Fentanyl+pharmacokinetics+in+hemorrhagic+shock:+a+porcine+model.">Fentanyl pharmacokinetics in hemorrhagic shock: a porcine model.</a> Anesthesiology. 91 (1), 156-166 (1999).</li></ol>

In this study, we described in detail the protocol for animal selection, instrument preparation, surgical procedures, and post-operative evaluation when developing a CABG-induced VGD model. We performed ultrasonic examination of the venous graft before and after CABG surgery and histological examination of the graft 30 days after the surgery. The blood flow in the internal mammary vein was normal before the CABG surgery, while retrograde flow was observed in the graft of the internal mammary vein. Compared with the sham ...

Although coronary heart disease mortality has declined significantly in recent years, half of middle-aged adults in the United States develop ischemic heart-related symptoms each year, and one-third of older adults die from coronary heart disease1. Coronary artery bypass grafting (CABG) is an effective surgical modality to improve myocardial ischemia, and more importantly, it is an irreplaceable surgical modality for the treatment of multivessel coronary artery disease2. Over time, however, vascular grafts develop inflammation, intimal hyperplasia, and progressive atherosclerosis, which is known to lead to vein graft failure or vein graft disease (VGD)3. In patients after CABG, if restenosis occurs, only the diseased blood vessel can be replaced in some cases2. Older patients and added comorbidities make redoing coronary artery bypass grafting quite challenging. Delaying or controlling the pathological problems associated with grafted blood vessels is an urgent problem to be solved. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. Researchers have successfully established animal VGD models in small and large animals such as mice4, rats5, rabbits6, and pigs7. Compared with small animals, large animals such as pigs have anatomical structures and physiological characteristics similar to humans and have longer lifespans8,9. Thus, large animals are more suitable for exploring long-term pathological changes in venous graft disease and for preclinical testing of drugs or devices. We and our collaborating team have successfully applied surgical techniques to establish a porcine heart failure model and described the cardiac pathological changes in this model10.
CABG surgery has been standardized in clinical practice, but when it is applied to the establishment of VGD animal models, the differences between species, the acquisition of animal equipment and facilities, animal surgical operations, and animal feeding and nursing are huge challenges for researchers. As in clinical practice, the approaches for CABG surgery used to establish VGD animal models include midline sternotomy11 and left lateral thoracotomy12. Midline sternotomy is more commonly used13,14. However, this approach has high risks for both humans and animals. In the study reported by Thankam et al., two of the six pigs used for modeling died during surgery15. High model mortality increases study costs and affects the accuracy of results. A study showed earlier that a left chest wall incision was feasible to establish CABG-induced VGD in pigs11. Here, this study aims to describe a step-by-step protocol to establish a reproducible surgery for a CABG-induced VGD model in minipigs and to evaluate the phenotype of this model. The experimental protocol was jointly designed by the cardiac surgery and anesthesia teams. The surgical approach for the left third intercostal space was determined according to the cadavers of other minipigs in the laboratory before surgery, and the anesthesia method was performed according to the method used at the center16. Blood biochemical tests, ultrasonic examination, and histology examination were conducted to evaluate animal models.

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	<tbody>
		<tr>
			<td>Aortic Punch</td>
			<td>Medtronic Inc. , America</td>
			<td>3.0mm, 3.5mm, 4.0mm</td>
			<td>Used for proximal coronary bridge anastomosis</td>
		</tr>
		<tr>
			<td>Automatic biochemical analyzer</td>
			<td>IDEXX Laboratories, Inc. America</td>
			<td>Catalyst One</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac coronary artery bypass grafting instrument kit</td>
			<td>LANDANGER, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiogram monitor</td>
			<td>Shenzhen Mindray Bio-Medical Electronics Co, Ltd</td>
			<td>MEC-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coronary Shunt</td>
			<td>AXIUS&#160;</td>
			<td>OF-1500, OF-2500, OF-3000</td>
			<td>The product temporarily blocks the coronary artery during arteriotomy to reduce the amount of bleeding in the surgical field and provide blood flow to the distal end during anastomosis.&#160; The Axius shunt plug is not an implant and should be removed prior to completion of the anastomosis.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Defibrillator</td>
			<td>MEDIANA</td>
			<td>Mediana D500</td>
			<td></td>
		</tr>
		<tr>
			<td>Diazepam</td>
			<td>Nanguo pharmaceutical Co. LTD, Guangdong, China</td>
			<td>H37023039&#160;</td>
			<td>Narcotic inducer</td>
		</tr>
		<tr>
			<td>Disposable manual electric knife</td>
			<td>Covidien, America</td>
			<td>E2516H</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric negative pressure suction machine</td>
			<td>Shanghai Baojia Medical Instrument Co, Ltd</td>
			<td>YX932D</td>
			<td></td>
		</tr>
		<tr>
			<td>Esmolol</td>
			<td>Guangzhou Wanzheng Pharmaceutical Co. LTD</td>
			<td>H20055990</td>
			<td>Emergency drugs</td>
		</tr>
		<tr>
			<td>Ice machine&#160;</td>
			<td>Local suppliers, Guangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine&#160;</td>
			<td>Chengdu First Pharmaceutical Co. LTD</td>
			<td>H51021662</td>
			<td>Emergency drugs</td>
		</tr>
		<tr>
			<td>Luxtec headlight system</td>
			<td>Luxtec, America</td>
			<td>AX-1375-BIF</td>
			<td>Used for lighting fine parts during operation</td>
		</tr>
		<tr>
			<td>Medical operation magnifier (glasses)</td>
			<td>Germany Lista co, LTD</td>
			<td>SuperVu Galilean 3.5&#215;</td>
			<td>Used for fine site operation during operation</td>
		</tr>
		<tr>
			<td>Multi-function high-frequency electrotome</td>
			<td>Shanghai Hutong Electronics Co, Ltd</td>
			<td>GD350-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen canister</td>
			<td>Local suppliers, Guangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nonabsorbable surgical suture (polypropylene suture)</td>
			<td>Johnson &#38; Johnson, America</td>
			<td>6-0, 7-0</td>
			<td>Used to suture blood vessels.</td>
		</tr>
		<tr>
			<td>Nonabsorbable suture (cotton thread)</td>
			<td>Covidien, America</td>
			<td>1-0</td>
			<td>Used for skin and muscle tissue tugging</td>
		</tr>
		<tr>
			<td>Open heart surgery instrument kit</td>
			<td>Shanghai Medical Instrument (Group) Co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol injection</td>
			<td>Xi &#39;an Libang Pharmaceutical Co. LTD</td>
			<td>H19990282</td>
			<td>Anesthetic sedative</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>Local suppliers, Guangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Respiratory anesthesia machine for animal</td>
			<td>Shenzhen Reward Life Technology Co, Ltd, China</td>
			<td>R620-S1</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-occlusion clamp</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZL1701RB</td>
			<td>Temporarily cut off the aortic flow</td>
		</tr>
		<tr>
			<td>vecuronium bromide</td>
			<td>Richter, Hungary&#160;</td>
			<td>JX20090127</td>
			<td>Muscle relaxant</td>
		</tr>
		<tr>
			<td>Veterinary ultrasound system&#160;</td>
			<td>Royal Philips, Netherlands</td>
			<td>CX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac, France</td>
			<td>Zoletil 50&#160;</td>
			<td>Animal narcotic</td>
		</tr>
	</tbody>
</table>

establishment and evaluation of a porcine vein graft disease model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

BMI and serum biochemical indices 
The BMI between the sham and VGD groups was not significantly different (sham vs. VGD, 22.05 kg/cm2 &#177; 0.46 kg/cm2 vs. 21.14 kg/cm2 &#177; 0.39 kg/cm2, p = 0.46). The serum biochemical results&#160;are listed in Table 1. Statistically significant changes between the groups were found in four biochemical indexes, including aspartate aminotransferase (AST, sham vs. VGD, 25.25 IU/L &#177; 1.88 I...

Watch this Scientific Journal Video about Establishment and Evaluation of a Porcine Vein Graft Disease Model at JoVE.com

Establishment and Evaluation of a Porcine Vein Graft Disease Model

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

This model provides a standard protocol for establishing animal models of coronary graft disease. The procedure of animal model established by this method is standard, and the result is stable. To begin, divide three-month-old, male minipigs weighing 30 to 35 kilograms into the sham and venous graft disease groups of five animals each.After anesthetizing the animal, establish ear access by inserting an indwelling venous catheter into the marginal ear vein. Now, transfer the pig onto the operation table in the supine position. Perform tracheal intubation with a 7.0 to 7.5 French gauge tube, and connect it to the anesthesia breathing circuit.Immobilize the limbs with bandages, and elevate the head with a sterile drape. After injecting vecuronium bromide intravenously for muscle relaxation, monitor heart rate, blood oxygen levels, and body temperature using an electrocardiogram. After shaving and sterilizing the surgical area, place a sterile surgical drape around it.After making the incision and exposing the internal mammary vein as described in the text, locate the internal mammary vein and the left internal mammary artery on the left side of the sternum, and perform blunt dissection of the internal mammary vein with vascular forceps. Perform hemostasis by electrocoagulation of the left internal mammary vein branches with an electric knife. If hemostasis is incomplete, using cotton thread, ligate and mark the two ends of the vein while it is being harvested.Once the vein is removed, inject heparin, normal saline into the vein for pre-treatment. Then, put the vein into the normal saline, keep it for backup, and similarly incise the internal mammary vein of the sham group. Then, open the pericardium, close the chest wall, and use the internal mammary vein for pathological control without coronary artery bypass grafting.Make an approximately seven-centimeter incision with an electric knife on the pericardium to expose the right coronary artery trunk. Suspend the pericardium, and sew the skin on the ipsilateral side with 1-0 surgical sutures. Now, separate the right coronary artery trunk from the surrounding tissues.Next, using a wire hook, bypass the blocking band under the proximal end of the isolated right coronary artery near the aorta. Then, treat the myocardium with three cycles of two minutes ischemia and five minutes reperfusion by tightening and relaxing the blocking band. Also, monitor the heart's electrical activity with the electrocardiogram during the ischemia reperfusion pre-conditioning.Next, cut the epicardium covering the blood vessels after tightening the band to block the right coronary blood flow. Exposing the coronary artery wall, cut longitudinally with the tip of a surgical blade against the center of the anterior wall of the blood vessels. After cutting the lumen and enlarging the incision and placing a coronary shunt, insert one end of the shunt with a coil into the distal coronary artery through the tear.Now, shunt the blood in the coronary arteries into the hollow coronary shunt to ensure a clear operative field, and perform an end-to-side continuous suture between the internal mammary vein and the aortic wall with the 6-0 polypropylene suture. In the middle of the ascending aorta, occlude the left anterolateral wall of the ascending aorta with a semi-occlusion clamp. After making a small incision with the surgical blade in the aortic wall where the adventitia has been cut, insert the head end of the sliding shaft at the head end of the punch into the aortic cavity through this incision.Then, contract the sliding shaft outward and the circular knife cuts off a piece of the arterial wall. Once the shunt is pulled out, perform an end-to-side continuous suture between the internal mammary vein and the right coronary artery with the 6-0 polypropylene suture. After opening the semi-occlusion clamp, record the bypass flow of the right coronary artery trunk proximal to the anastomosis site using ultrasound.Prepare the animal for surgery as demonstrated earlier. And with an electric knife, make a 10-centimeter median sternal incision to split the sternum and harvest the vein 30 days post-surgery. While separation, avoid the main blood vessels, heart, and separate the grafted blood vessels layer by layer.Quickly cut off the large blood vessels connecting to the heart, and place the heart and ascending aorta on ice chips. Once the graft vascular bridge, the connected aorta, and the right coronary artery are removed, rinse all the samples with normal saline at four degrees Celsius. After taking the entire graft vessel of about three to four centimeters and dividing it into four to five equal parts, transfer it to CryoTubes to quickly flash-freeze in liquid nitrogen.For analysis, fix the ice-cold, saline-rinsed graft in 4%paraformaldehyde solution. After 12 hours of tissue fixation, stain the sections in 50 milliliters of an aqueous solution of hematoxylin for three minutes. Then, separate the sections by washing with 50 milliliters of 0.5%hydrochloric acid-ethanol and 0.2%ammonia water for 10 seconds each.Once the sections are rinsed for one hour with running water, clean them by soaking them in distilled water for three minutes. After dehydrating the sections in 70%and 90%ethanol for 10 minutes, place them in 50 milliliters of 0.5%alcohol eosin staining solution for two to three minutes. Once the stained sections are dehydrated in pure ethanol, soak the samples in pure xylene for 10 minutes to make them transparent.Finally, drip the transparent sections with neutral glue before covering them with a cover slip. Observe pathological sections under a light microscope at 40 times magnification. Ultrasonic examination showed that the overall blood flow direction was normal in the proximal end, vascular cavity, and the distal end of the graft vessel.However, the proximal and distal ends of the graft vessel showed some retrograde flow of blood. The histological analysis of the internal mammary vein revealed that in the sham group the tunica intima, tunica media, and the venous wall of the venous graft appeared normal. However, 30 days after coronary artery transplantation, the intima of the vessel was thickened and the lumen was narrowed.Among the sham and venous graft disease groups, four biochemical indexes, aspartate aminotransferase, serum bilirubin, total bilirubin, and creatinine, showed statistically significant changes. The surgeon should completely expose the internal mammary vein and make sure it is not attached to the surrounding tissue. The technique provides a standardized modeling process for studying venous graft diseases in animal models.

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Research

JoVE Journal

Medicine

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the Trinity Clip, is developed based on the excimer laser-assisted nonocclusive anastomosis technique. The Trinity Clip connector enables simplified, sutureless, and nonocclusive connection of the graft to the coronary artery, and an excimer laser catheter laser-punches the opening of the anastomosis. Consequently, owing to the complete nonocclusive anastomosis construction, coronary conditioning (i.e., occluding or shunting) is not necessary, in contrast to the conventional anastomotic technique, hence simplifying the off-pump bypass procedure. Prior to clinical application in coronary artery bypass grafting, the safety and quality of this novel connector will be evaluated in a long-term experimental porcine off-pump coronary artery bypass (OPCAB) study. In this paper, we describe how to evaluate the coronary anastomosis in the porcine OPCAB model using various techniques to assess its quality. Representative results are summarized and visually demonstrated.

This paper describes a novel nonocclusive coronary anastomotic connector in a porcine off-pump coronary artery bypass (OPCAB) model. This easy-to-use coronary connector has intrinsic potential to facilitate minimally invasive OPCAB surgery.

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the ...

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for treatment efficacy&#160;in a multicenter randomized study comparing edoxaban monotherapy with a heparin/warfarin regimen for acute, symptomatic lower extremities deep vein thrombosis (DVT) treatment. We also used a direct thrombus imaging approach (DTHI, without the use of a contrast agent) to quantify fresh thrombus. We then sought to evaluate the reproducibility of the analysis methodology and applicability of using 3D magnetic resonance venography and direct thrombus imaging for the quantification of DVT in a multicenter trial setting. From 10 randomly selected subjects participating in the edoxaban Thrombus Reduction Imaging Study (eTRIS), total thrombus volume in the entire lower extremity deep venous system was quantified bilaterally. Subjects were imaged using 3D-T1W gradient echo sequences before (direct thrombus imaging, DTHI) and 5 min after injection of 0.03 mmol/kg of gadofosveset trisodium (magnetic resonance venography, MRV). The margins of the DVT on corresponding axial, curved multi-planar reformatted images were manually delineated by two observers to obtain volumetric measurements of the venous thrombi. MRV was used to compute total DVT volume, whereas DTHI was used to compute volume of fresh thrombus. Intra-class correlation (ICC) and Bland Altman analysis were performed to compare inter and intra-observer variability of the analysis. The ICC for inter and intra-observer variability was excellent (0.99 and 0.98, p &#60;0.001, respectively) with no bias on Bland-Altman analysis for MRV images. For DTHI images, the results were slightly lower (ICC = 0.88 and 0.95 respectively, p &#60;0.001), with bias for inter-observer results on Bland-Altman plots. This study showed feasibility of thrombus volume estimation in DVT using MRV with gadofosveset trisodium, with good intra- and inter-observer reproducibility in a multicenter setting.

The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined.

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

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 ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Modified Arteriovenous Fistula Surgery to Establish the RV Volume Overload Mouse Model

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Author Spotlight: Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.