Name: a rabbit venous interposition model mimicking revascularization surgery using vein grafts to assess intimal hyperplasia under arterial blood pressure
Uploaded: 2020-05-15T13:30:00
Description: Watch this Scientific Journal Video about A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure at JoVE.

This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (25462136).

NOTE: All the surgical procedures performed on animals should be carried out in accordance with the Guide for the Care and Use of Laboratory Animals (www.nap.edu/catalog/5140.html) or other appropriate ethical guidelines. Protocols should be approved by the animal welfare committee at the appropriate institution before proceeding.
1. Preparation of animals
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	<li>Purchase male Japanese white rabbits (or rabbits with equivalent body size) weighing 2.7&#8211;3.0 kg.</li>
	<li>Acclimate the ...

<ol>
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D., Dardik, 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+of+vein+graft+adaptation+to+the+arterial+circulation:+insights+into+the+neointimal+algorithm+and+management+strategies.">Mechanisms of vein graft adaptation to the arterial circulation: insights into the neointimal algorithm and management strategies.</a> Circulation Journal. 74 (8), 1501-1512 (2010).</li><li>Motwani, J. G., Topol, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortocoronary+saphenous+vein+graft+disease:+pathogenesis,+predisposition,+and+prevention.">Aortocoronary saphenous vein graft disease: pathogenesis, predisposition, and prevention.</a> Circulation. 97 (9), 916-931 (1998).</li><li>Schachner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+vein+graft+neointimal+hyperplasia.">Pharmacologic inhibition of vein graft neointimal hyperplasia.</a> Journal of Thoracic and Cardiovascular Surgery. 131 (5), 1065-1072 (2006).</li><li>Kulik, 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=Secondary+prevention+after+coronary+artery+graft+surgery:+a+scientific+statement+from+the+American+Heart+Association.">Secondary prevention after coronary artery graft surgery: a scientific statement from the American Heart Association.</a> Circulation. 131 (10), 927-964 (2015).</li><li>Gerhard-Herman, M. 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=2016+AHA/ACC+Guideline+on+the+Management+of+Patients+With+Lower+Extremity+Peripheral+Artery+Disease:+Executive+Summary:+A+Report+of+the+American+College+of+Cardiology/American+Heart+Association+Task+Force+on+Clinical+Practice+Guidelines.">2016 AHA/ACC Guideline on the Management of Patients With Lower Extremity Peripheral Artery Disease: Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines.</a> Circulation. 135 (12), 686-725 (2017).</li><li>Aboyans, 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=2017+ESC+Guidelines+on+the+Diagnosis+and+Treatment+of+Peripheral+Arterial+Diseases,+in+collaboration+with+the+European+Society+for+Vascular+Surgery+(ESVS):+Document+covering+atherosclerotic+disease+of+extracranial+carotid+and+vertebral,+mesenteric,+renal,+upper+and+lower+extremity+arteries+Endorsed+by:+the+European+Stroke+Organization+(ESO)+The+Task+Force+for+the+Diagnosis+and+Treatment+of+Peripheral+Arterial+Diseases+of+the+European+Society+of+Cardiology+(ESC)+and+of+the+European+Society+for+Vascular+Surgery+(ESVS).">2017 ESC Guidelines on the Diagnosis and Treatment of Peripheral Arterial Diseases, in collaboration with the European Society for Vascular Surgery (ESVS): Document covering atherosclerotic disease of extracranial carotid and vertebral, mesenteric, renal, upper and lower extremity arteries Endorsed by: the European Stroke Organization (ESO) The Task Force for the Diagnosis and Treatment of Peripheral Arterial Diseases of the European Society of Cardiology (ESC) and of the European Society for Vascular Surgery (ESVS).</a> European Heart Journal. 39 (9), 763-816 (2018).</li><li>Neumann, F. 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=2018+ESC/EACTS+Guidelines+on+myocardial+revascularization.">2018 ESC/EACTS Guidelines on myocardial revascularization.</a> European Heart Journal. 40 (2), 87-165 (2019).</li><li>Alexander, J. 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=Efficacy+and+safety+of+edifoligide,+an+E2F+transcription+factor+decoy,+for+prevention+of+vein+graft+failure+following+coronary+artery+bypass+graft+surgery:+PREVENT+IV:+a+randomized+controlled+trial.">Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery: PREVENT IV: a randomized controlled trial.</a> Journal of the American Medical Association. 294 (19), 2446-2454 (2005).</li><li>Carrel, A., Guthrie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uniterminal+and+biterminal+venous+transplantations.">Uniterminal and biterminal venous transplantations.</a> Surgery, Gynecology and Obstetrics. 2 (3), 266-286 (1906).</li><li>Nabatoff, R. A., Touroff, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maximal-size+vein+graft+feasible+in+the+replacement+of+experimental+aortic+defects,+long+term+observations+concerning+the+function+and+ultimate+fate+of+the+graft.">The maximal-size vein graft feasible in the replacement of experimental aortic defects, long term observations concerning the function and ultimate fate of the graft.</a> Bulletin of the New York Academy of Medicine. 28 (9), 616 (1952).</li><li>Kanar, E. 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=Experimental+vascular+grafts.+I.+The+effects+of+dicetyl+phosphate+on+venous+autografts+implanted+in+the+thoracic+aorta+of+growing+pigs:+a+preliminary+report.">Experimental vascular grafts. I. The effects of dicetyl phosphate on venous autografts implanted in the thoracic aorta of growing pigs: a preliminary report.</a> Annals of Surgery. 138 (1), 73-81 (1953).</li><li>Jones, T. I., Dale, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+peripheral+autogenous+vein+grafts.">Study of peripheral autogenous vein grafts.</a> AMA Archives of Surgery. 76 (2), 294-306 (1958).</li><li>Stehbens, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+production+of+aneurysms+by+microvascular+surgery+in+rabbits.">Experimental production of aneurysms by microvascular surgery in rabbits.</a> Vascular Surgery. 7 (3), 165-175 (1973).</li><li>Bannister, C. M., Mundy, L. A., Mundy, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+small+diameter+cervical+veins+grafted+into+the+common+carotid+arteries+of+growing+rabbits.">Fate of small diameter cervical veins grafted into the common carotid arteries of growing rabbits.</a> Journal of Neurosurgery. 46 (1), 72-77 (1977).</li><li>Bergmann, M., Walther, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+changes+of+venous+autologous+bypass+grafts+in+rabbits:+correlation+of+patency+and+development.">Ultrastructural changes of venous autologous bypass grafts in rabbits: correlation of patency and development.</a> Basic Research in Cardiology. 77 (6), 682-694 (1982).</li><li>Murday, A. 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=Intimal+hyperplasia+in+arterial+autogenous+vein+grafts:+a+new+animal+model.">Intimal hyperplasia in arterial autogenous vein grafts: a new animal model.</a> Cardiovascular Research. 17 (8), 446-451 (1983).</li><li>Quist, W. C., LoGerfo, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+smooth+muscle+cell+phenotypic+modulation+in+vein+grafts:+a+histomorphometric.">Prevention of smooth muscle cell phenotypic modulation in vein grafts: a histomorphometric.</a> Journal of Vascular Surgery. 16 (2), 225-231 (1992).</li><li>Davies, M. G., Dalen, H., Svendsen, E., Hagan, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+perioperative+catheter+injury+on+the+long-term+vein+graft+function+and+morphology.">Influence of perioperative catheter injury on the long-term vein graft function and morphology.</a> Journal of Surgical Research. 66 (2), 109-114 (1996).</li><li>Davies, M. G., Dalen, H., Svendsen, E., Hagan, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balloon+catheter+injury+and+vein+graft+morphology+and+function.">Balloon catheter injury and vein graft morphology and function.</a> Annals of Vascular Surgery. 10 (5), 429-442 (1996).</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+grat+model:+adaptation+to+differential+flow+environments.">A novel vein grat model: adaptation to differential flow environments.</a> American Journal of Physiology Heart and Circulatory Physiology. 286 (1), 240-245 (2004).</li><li>Ohnaka, M., 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=Effect+of+microRNA-145+to+prevent+vein+graft+disease+in+rabbits+by+regulation+of+smooth+muscle+cell+phenotype.">Effect of microRNA-145 to prevent vein graft disease in rabbits by regulation of smooth muscle cell phenotype.</a> Journal of Thoracic and Cardiovascular Surgery. 148 (2), 676-682 (2014).</li><li>Nishio, 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=MicroRNA-145-loaded+poly(lactic-co-glycolic+acid)+nanoparticles+attenuate+venous+intimal+hyperplasia+in+a+rabbit+model.">MicroRNA-145-loaded poly(lactic-co-glycolic acid) nanoparticles attenuate venous intimal hyperplasia in a rabbit model.</a> Journal of Thoracic and Cardiovascular Surgery. 157 (6), 2242-2251 (2019).</li><li>Ohno, 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=Accelerated+reendothelialization+with+suppressed+thrombogenic+property+and+neointimal+hyperplasia+of+rabbit+jugular+vein+grafts+by+adenovirus-mediated+gene+transfer+of+C-type+natriuretic+peptide.">Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide.</a> Circulation. 105 (14), 1623-1626 (2002).</li><li>Osgood, M. 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=Surgical+vein+graft+preparation+promotes+cellular+dysfunction,+oxydative+stress,+and+intimal+hyperplasia+in+human+saphenous+vein.">Surgical vein graft preparation promotes cellular dysfunction, oxydative stress, and intimal hyperplasia in human saphenous vein.</a> Journal of Vascular Surgery. 60 (1), 202-211 (2014).</li><li>Shintani, T., 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=Intraoperative+transfection+of+vein+grafts+with+the+NFkappaB+decoy+in+a+canine+aortocoronary+bypass+model:+a+strategy+to+attenuate+intimal+hyperplasia.">Intraoperative transfection of vein grafts with the NFkappaB decoy in a canine aortocoronary bypass model: a strategy to attenuate intimal hyperplasia.</a> Annals of Thoracic Surgery. 74 (4), 1132-1137 (2002).</li><li>Petrofski, J. 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=Gene+delivery+to+aortocoronary+saphenous+vein+grafts+in+a+large+animal+model+of+intimal+hyperplasia.">Gene delivery to aortocoronary saphenous vein grafts in a large animal model of intimal hyperplasia.</a> Journal of Thoracic and Cardiovascular Surgery. 127 (1), 27-33 (2004).</li><li>Steger, C. M., 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=Neointimal+hyperplasia+in+a+porcine+model+of+vein+graft+disease:+comparison+between+organ+culture+and+coronary+artery+bypass+grafting.">Neointimal hyperplasia in a porcine model of vein graft disease: comparison between organ culture and coronary artery bypass grafting.</a> European Surgery. 43 (3), 174-180 (2011).</li><li>Thim, T., 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=Oversized+vein+grafts+develop+advanced+atherosclerosis+in+hypercholesterolemic+minipigs.">Oversized vein grafts develop advanced atherosclerosis in hypercholesterolemic minipigs.</a> BMC Cardiovascular Disorders. 12 (24), (2012).</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> American Journal of Pathology. 153 (4), 1301-1310 (1998).</li><li>de Vries, M. R., Simons, K. H., Jukema, J. W., Braun, J., Quax, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vein+graft+failure:+from+pathophysiology+to+clinical+outcomes.">Vein graft failure: from pathophysiology to clinical outcomes.</a> Nature Reviews Cardiology. 13 (8), 451-470 (2016).</li><li>Dietrich, 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=Rapid+development+of+vein+graft+atheroma+in+ApoE-deficient+mice.">Rapid development of vein graft atheroma in ApoE-deficient mice.</a> American Journal of Pathology. 157 (2), 659-669 (2000).</li><li>Kumar, A., Lindner, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+with+neointima+formation+in+the+mouse+carotid+artery+after+cessation+of+blood+flow.">Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 17 (10), 2238-2244 (1997).</li><li>Lindner, V., Fingerie, J., Reidy, M. 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The present protocol is designed to provide an experimental platform to test various molecular or genetic interventions for VSMCs to control the phenotype from the proliferative to the contractile state and subsequently attenuate the progression of venous intimal hyperplasia in vivo. Using this model, we successfully prepared intimal hyperplasia at 2 weeks after surgery (Figure 1A) and indicated the therapeutic potential of microRNA-145 to control the VSMC phenotype26...

The number of patients experiencing ischemic diseases due to atherosclerosis is increasing worldwide1. Despite the current advances in medical and surgical therapies for cardiovascular diseases, ischemic heart diseases, such as myocardial infarction, remain a major cause of morbidity and mortality2. Furthermore, peripheral arterial diseases characterized by reduced blood flow to the limbs induces critical limb ischemia, wherein approximately 40% of the patients lose their legs within 6 months of diagnosis, and the mortality rate is up to 20%3.
Revascularization surgeries, such as coronary artery bypass grafting (CABG) and bypass surgery for peripheral arteries, are major therapeutic options for ischemic diseases. The purpose of these surgeries is to provide a new blood pathway to provide sufficient blood flow toward the distal site of the stenotic or occluded lesions of the atherosclerotic arteries. Although in situ arterial grafts, such as internal thoracic arteries for CABG, are preferred as the bypass grafts because of the expected longer patency, vein grafts, such as autologous saphenous veins, are commonly used because of the higher accessibility and availability4. The weak point of the vein grafts is the poor patency rate compared to that of artery grafts5 due to accelerated intimal hyperplasia when subjected to arterial pressure, which leads to vein graft disease6.
Vein graft disease develops through the following three steps: 1) thrombosis; 2) intimal hyperplasia; and 3) atherosclerosis7. In order to address vein graft disease, a lot of basic research has been conducted8. Thus far, no pharmacological strategy other than antiplatelet and lipid-lowering therapies are recommended for secondary prevention after coronary or peripheral revascularization surgeries in recent guidelines9,10,11,12. Thus, to overcome vein graft disease, especially intimal hyperplasia, the establishment of a relevant experimental platform for further studies is required.
Intimal hyperplasia is an adaptive phenomenon that occurs in response to the change in the surroundings, where vascular smooth muscle cells (VSMCs) proliferate, accumulate, and generate extracellular matrix in the intima. Consequently, it presents a foundation for graft atheroma7. In the hyperplastic intima, VSMCs bear proliferation, and production rather than contraction, termed &#8220;phenotypic change&#8221;8. It is a key research target to control the phenotype of the VSMCs of the vein grafts to prevent vein graft disease, and numerous basic studies have been conducted on this topic8. However, a randomized controlled clinical study that aimed to achieve pharmacological control of the VSMC phenotype showed limited results13. Further, there are no standardized therapies to prevent intimal hyperplasia. More basic research, including animal model studies, is necessary.
To promote research in this field, it is crucial to establish an animal model that recapitulates vein grafts under arterial blood pressure, allowing a long-term, postoperative observation. Carrel et al. established a canine model of implantation of the external jugular vein into the carotid artery14. Therafter, a variety of vein grafts have been employed to investigate the physiological and pathological effects of alterations in arterial blood pressure, including the inferior vena cava engrafted into the thoracic or the abdominal aorta, or the saphenous vein engrafted into the femoral artery15,16,17. These models were built in larger animals, such as pigs or dogs, that are suitable for mimicking a vein graft disease in a clinical case. However, the establishment of an animal model that can be prepared without special surgical techniques and at a lower cost would be ideal for researchers trying to develop a new therapeutic strategy for attenuating intimal hyperplasia through VSMC phenotype control in vivo. Initially, the interposition of the jugular vein into the carotid artery in a rabbit was introduced in the field of neurosurgery18,19. Thereafter, it was applied to research on intimal hyperplasia20,21. The initial model consists of venous interposition alone, thus saving time. Moreover, a subsequent study demonstrated that the preparation of a vein graft also affected the intimal hyperplasia22. Davies et al. evaluated the effect of balloon catheter injury on the intimal hyperplasia in a rabbit venous interposition model23,24. Although balloon catheter injury in addition to vein interposition was more relevant to a clinical setting, a more reproducible model was also desired. Thus, Jiang et al. examined the impact of differential flow environments on intimal hyperplasia and established a distal branch ligation procedure as a reproducible model25. However, they employed a cuff technique at the time of vein graft interposition that seems different from hand-sewn anastomosis in the clinical setting. In the present protocol, we report a reproducible, clinically relevant, and broadly available procedure for the preparation of a rabbit venous interposition model to assess intimal hyperplasia under arterial blood pressure.

<table>
	<tbody>
		<tr>
			<td>10% Povidone-iodine solution</td>
			<td>Nakakita</td>
			<td>872612</td>
			<td>Surgical expendables</td>
		</tr>
		<tr>
			<td>2-0 VICRYL Plus</td>
			<td>Johnson and Johnson</td>
			<td>VCP316H</td>
			<td>Surgical expendables</td>
		</tr>
		<tr>
			<td>4-0 Silk suture</td>
			<td>Alfresa pharma</td>
			<td>GA04SB</td>
			<td>Surgical expendables</td>
		</tr>
		<tr>
			<td>8-0 polypropylene suture</td>
			<td>Ethicon</td>
			<td>8741H</td>
			<td>Surgical expendables</td>
		</tr>
		<tr>
			<td>Cefazorin sodium</td>
			<td>Nichi-Iko Pharmaceutical</td>
			<td>6132401D3196</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Fogarty Catheter (2Fr)</td>
			<td>Edwards Lifesciences LLC</td>
			<td>E-060-2F</td>
			<td>Surgical expendables</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Nipro</td>
			<td>873334</td>
			<td>Anticoagulant</td>
		</tr>
		<tr>
			<td>Intravenous catheter (20G)</td>
			<td>Terumo</td>
			<td>SR-OT2051C</td>
			<td>Surgical expendables</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fujifilm</td>
			<td>095-06573</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Lidocaine hydrochloride</td>
			<td>MP Biomedicals</td>
			<td>193917</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Tokyo Chemical Industry</td>
			<td>P0776</td>
			<td>Anesthesia</td>
		</tr>
	</tbody>
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a rabbit venous interposition model mimicking revascularization surgery using vein grafts to assess intimal hyperplasia under arterial blood pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

Figure 1A shows a representative image of successful intimal hyperplasia at 2 weeks after venous interposition surgery (upper panel). The lower panel shows the therapeutic effects of microRNA-145-loaded poly(lactic-co-glycolic acid) nanoparticles that attenuated the intimal hyperplasia (lower panel). Figure 1B shows the comparison of intimal hyperplasia between the control group using phosphate buffered saline control (PBS), control microRNA (Cont-miR), and micr...

Watch this Scientific Journal Video about A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure at JoVE.

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Our protocol provides an easy, cost-effective, reproducible and clinical relevant model for researchers in the field of venous intimal hyperplasia. Our protocol is economically, clinically, and technically balanced. Therefore, it is highly recommended for researchers who want to apply their in vitro outcomes to an in vivo setting.We believe that our protocol can be applied to a larger animal model for the development of new treatment strategies in ischemic heart and lower extremity diseases. Our protocol is mainly relevant to the field of cardiovascular surgery. However, it may also be used to address degenerative changes in arteriovenous fistulas in patients with end stage renal disease.After local anesthetic, and prophylactic antibiotic analgesic administration, disinfect the surgical site with 10%povidone-iodine. And make a 50 to 60 millimeter incision with a surgical scalpel in the cervical region of the anesthetized male Japanese white rabbit. Bluntly dissect the subcutaneous tissues and fascia to expose a 20 to 30 millimeter segment of the jugular vein.And use 4-0 silk sutures to ligate all of the branches of the exposed vein. Place a 2-0 silk suture around the internal and external jugular veins and make a one millimeter incision in the venous wall of the distal side of the vein. Insert a 2-French balloon catheter from the cut toward the proximal side of the vein.And ligate the 2-0 silk suture at the distal sites of the jugular veins. Inflate the balloon with 200 microliters of air. And use the balloon to denude the intima of the vein three times for endothelial exfoliation.After the third passage, ligate the proximal end of the vein and cut the vein at the distal site. Then insert a 20 gauge intravenous catheter into the harvested vein from the distal to the proximal direction, and cut the vein at the proximal site. To interpose the carotid artery with the harvest jugular vein, expose a 20 to 30 millimeter segment of the ipsilateral carotid artery.And separate the artery carefully from the nearby vein and nerve. Ligate all the branches of the exposed vein with 4-0 silk suture. And intravenously administer 200 International Units per kilogram of heparin sodium.Clamp the proximal and distal ends of the artery with surgical rubber clamps. And cut the artery between the clamps. Inject normal saline into the incised carotid artery proximally and distally to distend the artery.And place the harvest vein near the artery to anastomose the vessel in a reversed end-to-end fashion. To anastomose the proximal end of the vein to the distal end of the artery, place two anchor stitches of 8-0 polypropylene at the site and the opposite site. Add stitches the upper side of the anastomosis line between the anchor stitches.Flip the artery and the vein graft upside down. And add stitches on the remaining part of the anastomosis line. Next, remove the intravenous catheter from the vein and clamp the vein graft proximally.Declamp the carotid artery distally. And observe whether the vein graft expands gradually. Using 8-0 polypropylene interrupted sutures, anastomose the distal end of the vein to the proximal end of the artery.And declamp the artery to check for bleeding from the anastomosis sites. Finally, ligate the internal carotid artery with a 4-0 silk suture to simulate a poor runoff condition and to facilitate intimal hyperplasia. Then clean the wound with saline.Close the incision with 3-0 polyglactin 910 in layers. And allow the rabbit to fully recover with monitoring before returning the animal to its home cage. In this representative image, a successful intimal hyperplasia at 2 weeks after venous interposition surgery can be observed.Here, the therapeutic effects of microRNA-145 loaded poly(lactic-co-glycolic acid)nanoparticles on the intimal hyperplasia morphology can be observed. Indeed, treatment with microRNAs loaded with poly(lactic-co-glycolic acid)nanoparticles results in a significant attenuation of intimal hyperplasia. In addition, immunostaining for Ki-67, a cell proliferative marker, reveals fewer Ki-67-positive cells within the microRNA-145 treated group indicating a phenotype change in the vascular smooth muscle cells from the immature proliferative state to the mature contractile state.The main goal of our protocol is to achieve patency of the implanted graft. It is also important to confirm that the graft expands after declamping the carotid artery.

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Research

JoVE Journal

Medicine

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2 To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8 Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11 
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12 The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13 These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16 Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.

An optimized technique for the microsurgical creation of arterial bifurcation aneurysms mimicking bifurcation human cerebral aneurysms is described. A venous pouch is sutured into an artificially created true bifurcation of both common carotid arteries. Facilitated microsurgical techniques and aggressive postoperative anticoagulation and analgesia lead to minimized morbidity rates and high aneurysm patency rates.

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

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. ...

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 ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.