All animal protocols and studies were approved by the University of Washington Office of Animal Welfare.
1. Pre-operation (for All Surgeries)
<ol>
	<li>Anesthetize the rabbit with 30 mg/kg ketamine and 1.5 mg/kg xylazine by intramuscular injection (IM) in the paraspinous muscles. 
	&#8203;NOTE: Food and water are not restricted before surgery.</li>
	<li>While waiting for sufficient depth of anesthesia, set up the tables in the preparation room and operating room (OR).
	<ol>
		<li>Prepar...

<ol>
	<li>Libby, P., Bornfeldt, K. E., Tall, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis:+Successes,+Surprises,+and+Future+Challenges.">Atherosclerosis: Successes, Surprises, and Future Challenges.</a> Circ Res. 118, 531-534 (2016).</li><li>Fowkes, 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=Comparison+of+global+estimates+of+prevalence+and+risk+factors+for+peripheral+artery+disease+in+2000+and+2010:+a+systematic+review+and+analysis.">Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis.</a> Lancet. 382, 1329-1340 (2013).</li><li>Mohr, F. 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=Coronary+artery+bypass+graft+surgery+versus+percutaneous+coronary+intervention+in+patients+with+three-vessel+disease+and+left+main+coronary+disease:+5-year+follow-up+of+the+randomised,+clinical+SYNTAX+trial.">Coronary artery bypass graft surgery versus percutaneous coronary intervention in patients with three-vessel disease and left main coronary disease: 5-year follow-up of the randomised, clinical SYNTAX trial.</a> Lancet. 381, 629-638 (2013).</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> Nat Rev Cardiol. 13 (8), 451-470 (2016).</li><li>Harskamp, R. E., Lopes, R. D., Baisden, C. E., de Winter, R. J., Alexander, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saphenous+vein+graft+failure+after+coronary+artery+bypass+surgery:+pathophysiology,+management,+and+future+directions.">Saphenous vein graft failure after coronary artery bypass surgery: pathophysiology, management, and future directions.</a> Ann Surg. 257, 824-833 (2013).</li><li>Sabik, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+saphenous+vein+graft+patency.">Understanding saphenous vein graft patency.</a> Circulation. 124 (3), 273-275 (2011).</li><li>Owens, C. D., Ho, K. J., Conte, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+extremity+vein+graft+failure:+a+translational+approach.">Lower extremity vein graft failure: a translational approach.</a> Vasc Med. 13, 63-74 (2008).</li><li>Robertson, K. E., McDonald, R. A., Oldroyd, K. G., Nicklin, S. A., Baker, A. H. <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+coronary+in-stent+restenosis+and+vein+graft+failure:+does+vascular+gene+therapy+have+a+role.">Prevention of coronary in-stent restenosis and vein graft failure: does vascular gene therapy have a role.</a> Pharmacol Ther. 136, 23-34 (2012).</li><li>Yla-Herttuala, S., Baker, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+Gene+Therapy:+Past,+Present,+and+Future.">Cardiovascular Gene Therapy: Past, Present, and Future.</a> Mol Ther. 25, 1095-1106 (2017).</li><li>Schwartz, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+of+a+constitutively+active+form+of+the+retinoblastoma+gene+product+attenuates+neointimal+thickening+in+experimental+vein+grafts.">Adenoviral-mediated gene transfer of a constitutively active form of the retinoblastoma gene product attenuates neointimal thickening in experimental vein grafts.</a> J Vasc Surg. (5), 874-881 (1999).</li><li>Eefting, 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=Local+lentiviral+short+hairpin+RNA+silencing+of+CCR2+inhibits+vein+graft+thickening+in+hypercholesterolemic+apolipoprotein+E3-Leiden+mice.">Local lentiviral short hairpin RNA silencing of CCR2 inhibits vein graft thickening in hypercholesterolemic apolipoprotein E3-Leiden mice.</a> J Vasc Surg. 50, 152-160 (2009).</li><li>Handa, 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=Adventitial+delivery+of+platelet-derived+endothelial+cell+growth+factor+gene+prevented+intimal+hyperplasia+of+vein+graft.">Adventitial delivery of platelet-derived endothelial cell growth factor gene prevented intimal hyperplasia of vein graft.</a> J Vasc Surg. 48 (6), 1566-1574 (2008).</li><li>Kloppenburg, G. T., Grauls, G. E., Bruggeman, C. A., Stassen, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+activin+A+expression+prevents+vein+graft+intimal+hyperplasia+in+a+rat+model.">Adenoviral activin A expression prevents vein graft intimal hyperplasia in a rat model.</a> Interact Cardiov Th. 8, 31-34 (2009).</li><li>Eefting, 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=A+novel+urokinase+receptor-targeted+inhibitor+for+plasmin+and+matrix+metalloproteinases+suppresses+vein+graft+disease.">A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease.</a> Cardiovasc Res. 88, 367-375 (2010).</li><li>Eichstaedt, H. C., 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+transfer+of+COX-1+improves+lumen+size+and+blood+flow+in+carotid+bypass+grafts.">Gene transfer of COX-1 improves lumen size and blood flow in carotid bypass grafts.</a> J Surg Res. 161, 162-167 (2010).</li><li>Kritz, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+modulation+of+Nogo-B+attenuates+neointima+formation.">In vivo modulation of Nogo-B attenuates neointima formation.</a> Mol Ther. 16 (11), 1798-1804 (2008).</li><li>Peroulis, 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=The+role+of+ex-vivo+gene+therapy+of+vein+grafts+with+Egr-1+decoy+in+the+suppression+of+intimal+hyperplasia.">The role of ex-vivo gene therapy of vein grafts with Egr-1 decoy in the suppression of intimal hyperplasia.</a> Eur J Vasc Endovasc. 40, 216-223 (2010).</li><li>Kochanek, S., 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+new+adenoviral+vector:+Replacement+of+all+viral+coding+sequences+with+28+kb+of+DNA+independently+expressing+both+full-length+dystrophin+and+b-galactosidase.">A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and b-galactosidase.</a> Proc Natl Acad Sci U S A. 93, 5731-5736 (1996).</li><li>Parks, R. 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=A+helper-dependent+adenovirus+vector+system:+Removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> P Natl Acad Sci USA. 93, 13565-13570 (1996).</li><li>Chen, H. -. 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=Persistence+in+muscle+of+an+adenoviral+vector+that+lacks+all+viral+genes.">Persistence in muscle of an adenoviral vector that lacks all viral genes.</a> P Natl Acad Sci USA. 94, 1645-1650 (1997).</li><li>Wen, S., Graf, S., Massey, P. G., Dichek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vascular+gene+transfer+with+a+helper-dependent+adenoviral+vector.">Improved vascular gene transfer with a helper-dependent adenoviral vector.</a> Circulation. 110, 1484-1491 (2004).</li><li>Du, L., Zhang, J., Clowes, A. W., Dichek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+and+durable+transgene+expression+in+grafted+rabbit+veins.">Efficient gene transfer and durable transgene expression in grafted rabbit veins.</a> Hum Gene Ther. 26, 47-58 (2015).</li><li>Channon, K. 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=Efficient+adenoviral+gene+transfer+to+early+venous+bypass+grafts:+comparison+with+native+vessels.">Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels.</a> Cardiovasc Res. 35, 505-513 (1997).</li><li>Flynn, R., 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=Expression+of+apolipoprotein+A-I+in+rabbit+carotid+endothelium+protects+against+atherosclerosis.">Expression of apolipoprotein A-I in rabbit carotid endothelium protects against atherosclerosis.</a> Mol Ther. 19, 1833-1841 (2011).</li><li>George, S. 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=Sustained+Reduction+of+Vein+Graft+Neointima+Formation+by+Ex+Vivo+TIMP-3+Gene+Therapy.">Sustained Reduction of Vein Graft Neointima Formation by Ex Vivo TIMP-3 Gene Therapy.</a> Circulation. 124, 135-142 (2011).</li><li>Chiu-Pinheiro, C. 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=Gene+transfer+to+coronary+artery+bypass+conduits.">Gene transfer to coronary artery bypass conduits.</a> Ann Thorac Surg. 74, 1161-1166 (2002).</li><li>Byrom, M. J., Bannon, P. G., White, G. H., Ng, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+assessment+of+novel+vascular+conduits.">Animal models for the assessment of novel vascular conduits.</a> J Vasc Surg. 52, 176-195 (2010).</li><li>Ribichini, F., 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=Effects+of+oral+prednisone+after+stenting+in+a+rabbit+model+of+established+atherosclerosis.">Effects of oral prednisone after stenting in a rabbit model of established atherosclerosis.</a> J Am Coll Cardiol. 50, 176-185 (2007).</li><li>Langheinrich, A. C., 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=Quantification+of+in-stent+restenosis+parameters+in+rabbits+by+Micro-CT.">Quantification of in-stent restenosis parameters in rabbits by Micro-CT.</a> Rofo. 177 (4), 501-506 (2005).</li><li>Wacker, B. K., Dronadula, N., Zhang, J., Dichek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+Vascular+Gene+Therapy+With+Apolipoprotein+A-I+to+Promote+Regression+of+Atherosclerosis.">Local Vascular Gene Therapy With Apolipoprotein A-I to Promote Regression of Atherosclerosis.</a> Arterioscler Thromb. 37, 316-327 (2017).</li><li>Zwolak, R. M., Kirkman, T. R., 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=Atherosclerosis+in+rabbit+vein+grafts.">Atherosclerosis in rabbit vein grafts.</a> Arteriosclerosis. 9, 374-379 (1989).</li><li>Qiang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statin+therapy+prevents+expansive+remodeling+in+venous+bypass+grafts.">Statin therapy prevents expansive remodeling in venous bypass grafts.</a> Atherosclerosis. 223, 106-113 (2012).</li><li>Casa, L. D. C., Ku, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombus+Formation+at+High+Shear+Rates.">Thrombus Formation at High Shear Rates.</a> Annu Rev Biomed Eng. 19, 415-433 (2017).</li><li>Chen, C., Coyle, K. A., Hughes, J. D., Lumsden, A. B., Ku, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+blood+flow+accelerates+intimal+hyperplasia+in+endarterectomized+canine+arteries.">Reduced blood flow accelerates intimal hyperplasia in endarterectomized canine arteries.</a> Cardiovasc Surg. 5 (2), 161-168 (1997).</li><li>Binns, R. L., Ku, D. N., Stewart, M. T., Ansley, J. P., Coyle, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+graft+diameter:+effect+of+wall+shear+stress+on+vascular+healing.">Optimal graft diameter: effect of wall shear stress on vascular healing.</a> J Vasc Surg. 10, 326-337 (1989).</li><li>Oka, K., Mullins, C. E., Kushwaha, R. S., Leen, A. M., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+rhesus+monkeys+heterozygous+for+LDL+receptor+deficiency+by+balloon+catheter+hepatic+delivery+of+helper-dependent+adenoviral+vector.">Gene therapy for rhesus monkeys heterozygous for LDL receptor deficiency by balloon catheter hepatic delivery of helper-dependent adenoviral vector.</a> Gene Ther. 22, 87-95 (2015).</li><li>Miyake, 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=Prevention+of+neointimal+formation+after+angioplasty+using+nuclear+factor-kappaB+decoy+oligodeoxynucleotide-coated+balloon+catheter+in+rabbit+model.">Prevention of neointimal formation after angioplasty using nuclear factor-kappaB decoy oligodeoxynucleotide-coated balloon catheter in rabbit model.</a> Circ Cardiovasc Interv. 7, 787-796 (2014).</li><li>Chorny, 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=Site-specific+gene+delivery+to+stented+arteries+using+magnetically+guided+zinc+oleate-based+nanoparticles+loaded+with+adenoviral+vectors.">Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors.</a> FASEB J. 27, 2198-2206 (2013).</li><li>Hoshino, 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=Three+catheter-based+strategies+for+cardiac+delivery+of+therapeutic+gelatin+microspheres.">Three catheter-based strategies for cardiac delivery of therapeutic gelatin microspheres.</a> Gene Ther. 13, 1320-1327 (2006).</li><li>Nouri, F., Sadeghpour, H., Heidari, R., Dehshahri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+transfection+efficiency+of+low+molecular+weight+polyethylenimine-based+nanoparticles+for+delivery+of+the+plasmid+encoding+CD200+gene.">Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene.</a> Int J Nanomed. , 5557-5569 (2017).</li><li>Jia, S. 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The authors have nothing to disclose.

Critical steps in this protocol include the management of anesthesia, anticoagulation, surgical manipulation of the artery/grafted vein, and hemodynamic measurements of the grafted vein. Proper management of anesthesia is critical in this multiple survival surgery model that includes two relatively long operations (typically 3-3.5 h for bilateral vein grafting and 1.5-2.5 h for bilateral graft transduction). We have administered anesthesia both via a nosecone and by endotracheal intubation and found that the intubation i...

Atherosclerosis is a chronic inflammatory disease in which lipid accumulation and inflammation in the blood vessel wall lead to narrowing of the vessel lumen, heart attacks, strokes, and loss of limbs1,2. Percutaneous interventions (e.g., angioplasty and stenting) and medical therapy (e.g., statins and antiplatelet agents) are useful treatments for atherosclerosis; however, they are often ineffective in treating severe obstructive disease both in the coronary and peripheral circulations. Bypass grafting, using autogenous vein segments, remains a common procedure for treating patients with severe, diffuse coronary and peripheral vascular disease3,4. However, vein grafts placed in both the coronary and peripheral circulations have poor long-term patency rates. In the coronary circulation, approximately 10-20% of vein grafts are occluded at 1 year and 50% are occluded by 10 years5,6.In the peripheral circulation, vein graft failure rates are 30-50% at 5 years7.
Gene therapy is an attractive approach for the prevention of vein graft failure because it can deliver a therapeutic gene product precisely at the site of the disease. Accordingly, numerous preclinical studies have tested vein graft gene therapy8,9. However, essentially all of these studies have examined the efficacy at early time points (2-12 weeks)10,11,12,13,14,15,16,17. We are aware of no evidence that gene-therapy interventions can provide durable (years) protection against late vein graft failure that typically results from neointimal hyperplasia and atherosclerosis4. We developed a method that allows durable transgene expression in grafted veins, and thereby allows the testing of gene-therapy interventions at late as well as early time points. To achieve durable transgene expression, the method incorporates HDAd vectors and a delayed transduction strategy. HDAd vectors provide prolonged transgene expression because they lack viral genes, preventing the recognition (and rejection) of transduced cells by the immune system18,19,20,21. Delayed transduction (performed 28 days after the graft placement) prevents the loss of transduced cells during the arterialization process that occurs early after the grafting22.
Other methods that achieve therapeutic transgene expression in the vein graft wall rely on the transduction of the vein graft at the time of the graft placement10,11,12,15,16,17. When measured serially, transgene expression using this approach declines quickly after the transduction22,23. Accordingly, studies using this approach have not examined the efficacy beyond 12 weeks after the vein grafting, with most not assessing efficacy beyond 4 weeks. In contrast, our method achieves vein graft transgene expression that persists stably for at least 24 weeks and-based on similar studies performed in arteries-likely continues far longer22,24. We are aware of no other vein graft gene therapy intervention that achieves stable transgene expression of this duration.
We used a rabbit model to develop our method. Others have used rodents, rabbits, or larger animals to test vein graft gene therapy10,11,12,15,16,17,25,26. Compared to rodent models, rabbits are more expensive and are subject to more stringent regulatory requirements. However, compared to larger animals (e.g., pigs and dogs), rabbits are far less expensive to purchase and house and much easier to handle. Moreover, rabbit vessels resemble human vessels physiologically27, they are sufficiently large that they can be used for testing percutaneous interventions28,29, and they provide sufficient tissue that multiple endpoints (e.g., histology, protein, RNA) can be examined using a single blood vessel specimen22,30. In addition, when the rabbits with vein grafts are fed with a high-fat diet, they develop vein graft atherosclerosis31,32, which is a common cause of coronary artery bypass vein graft failure4,5. These atherosclerotic rabbit vein grafts can serve as a substrate for testing gene-therapy interventions delivered with this method. The provided protocol can help investigators to master the technical skills required to achieve durable transgene expression in rabbit vein grafts.

<table border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="707">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Disposables</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">3mL syringe with 24G needle</td>
			<td>Becton Dickinson</td>
			<td>309571</td>
			<td style="width:207px;">2x for vein graft surgery; 2x for gene transfer&#160; surgery</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">1 mL syringe with 27G needle</td>
			<td>Becton Dickinson</td>
			<td>309623</td>
			<td style="width:207px;">2x for vein graft surgery, 5x for gene transfer surgery</td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:215px;">19G needle</td>
			<td rowspan="2">Becton Dickinson</td>
			<td rowspan="2">305187</td>
			<td rowspan="2" style="width:207px;">Gene transfer surgery</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">20 mL syringe, luer lock</td>
			<td>Nipro Medical Corp</td>
			<td>JD+20L</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Catheters, 24Ga x 3/4&#8221;</td>
			<td>Terumo Medical Products</td>
			<td>SROX2419V</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">21G needle</td>
			<td>Becton Dickinson</td>
			<td>305165</td>
			<td style="width:207px;">Gene transfer surgery and for 20 mL syringe of saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Gauze 4&#8221; x 4&#8221;</td>
			<td>Dynarex</td>
			<td style="width:119px;">3242</td>
			<td style="width:207px;">~10-15 per surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">3-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-244</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">5-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-182</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8703P</td>
			<td style="width:207px;">Vein graft surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8648P</td>
			<td style="width:207px;">Gene transfer surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">5-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>421A</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">3-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>398A</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Alcohol swabs</td>
			<td>Covidien Ltd.</td>
			<td>6818</td>
			<td style="width:207px;">For the placement of I.V. line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Catheter plug</td>
			<td>Vetoquinol</td>
			<td>411498</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Ketamine HCl, 100 mg/mL</td>
			<td>Vedco Inc.</td>
			<td>5098916106</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Xylazine, 100 mg/mL</td>
			<td>Akorn Inc.</td>
			<td>4821</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Lidocaine, 20 mg/mL</td>
			<td>Pfizer</td>
			<td>409427702</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Marcaine 0.5%</td>
			<td>Pfizer</td>
			<td>409161050</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Beuthanasia D-Special</td>
			<td>Intervet Inc.</td>
			<td>NDC 00061047305</td>
			<td style="width:207px;">Harvest surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Buprenorphine HCl, 0.3 mg/mL</td>
			<td>Patterson Veterinary</td>
			<td>12496075705</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Saline IV bag, 0.9% sodium chloride</td>
			<td>Baxter</td>
			<td>2B1309</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Heparin&#160; (5000 U/mL)</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-047-10</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Papaverine (3.5 mg/ml)</td>
			<td>American Reagent Inc.</td>
			<td>NDC 0517-4002-25</td>
			<td style="width:207px;">Diluted from 30mg/mL stock; Use 1 mL maximum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Fentanyl patch, 25 mcg/h</td>
			<td>Apotex Corp.</td>
			<td>NDC 60505-7006-2</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Isoflurane</td>
			<td>Multiple vendors</td>
			<td style="width:119px;">Catalog number not available</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">&#160;Viral vector</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:207px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Surgical Instruments</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Metzenbaum needle holder 7&#34; straight</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7900</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Operating scissors 6.5&#34; straight blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6828</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Needle holder /w suture scissors</td>
			<td>Miltex</td>
			<td style="width:119px;">8-14-IMC</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Castroviejo scissors</td>
			<td>Roboz</td>
			<td>RS-5658</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Castroviejo needle holder, 5.75&#34; straight with lock</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6412</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Stevens scissors 4.25&#34; curved blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5943</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Alm retractor 4&#34; 4X4 5mm blunt prongs</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6514</td>
			<td style="width:207px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Backhaus towel clamp 3.5&#34;</td>
			<td>Roboz</td>
			<td style="width:119px;"></td>
			<td style="width:207px;">4x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Micro clip setting forceps 4.75&#34;</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-6496</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Micro vascular clips, 11 mm</td>
			<td>Roboz</td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Surg-I-Loop</td>
			<td>Scanlan International</td>
			<td style="width:119px;">1001-81M</td>
			<td style="width:207px;">5 cm length</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:215px;">Bonaccolto forceps, 4&#8221; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5210</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Dumont #3 forceps Inox tip size .17 &#215; .10 mm</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5042</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Graefe forceps, 4&#8221; (10 cm) long serrated straight, 0.8 mm tip</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-5280</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Halstead mosquito forceps,&#160; 5&#34; straight, 1.3 mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7110</td>
			<td style="width:207px;">2x</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Halstead mosquito forceps,&#160; 5&#34; curved, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7111</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Jacobson mosquito forceps 5&#34; curved extra delicate, 0.9 mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7117</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Kantrowitz forceps, 7.25&#34; 90 degree delicate, 1.7 mm tips</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-7305</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Tissue forceps 5&#34;, 1X2 teeth, 2 mm tip width</td>
			<td>Roboz</td>
			<td style="width:119px;">RS-8162</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">11092-12</td>
			<td style="width:207px;">2x</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Adson forceps, 12 cm, serrated, straight</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">11006-12</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Veterinary electrosurgery handpiece and electrode</td>
			<td>MACAN Manufacturing</td>
			<td style="width:119px;">HPAC-1; R-F11</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Surgical Suite Equipment</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Circulating warm water blanket and pump</td>
			<td>Multiple vendors</td>
			<td style="width:119px;">Catalog number not available</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Bair hugger warming unit</td>
			<td>3M</td>
			<td style="width:119px;">Model 505</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">IV infusion pump</td>
			<td>Heska</td>
			<td style="width:119px;">Vet IV 2.2</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Isoflurane vaporizer and scavenger</td>
			<td>Multiple vendors</td>
			<td style="width:119px;">Catalog number not available</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Veterinary multi-parameter monitor</td>
			<td>Surgivet</td>
			<td style="width:119px;">Surgivet Advisor</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Veterinary electrosurgery unit</td>
			<td>MACAN Manufacturing</td>
			<td style="width:119px;">MV-9</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Surgical microscope</td>
			<td style="width:167px;">D.F. Vasconcellos</td>
			<td style="width:119px;">M900</td>
			<td style="width:207px;">25X magnification for vein graft surgery; 16X magnification for gene transfer surgery</td>
		</tr>
	</tbody>
</table>

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.

The technical proficiency of a new operator must be validated before the operator can use this method to generate experimental data. The first milestone that a new operator must achieve is consistent vein graft patency after both the initial vein-grafting surgery and the subsequent delayed-transduction surgery. Over 90% patency after each of the surgeries is desirable and achievable. The patency can be assessed non-invasively using transcutaneous ultrasound, which we typically perform on ...

Watch this Scientific Journal Video about A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts at JoVE.com

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

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, ...

This method can answer key questions about the pathogenesis of vein graft failure. For example, it could identify mediators of graft failure and test strategies aimed at preventing graft failure. The main advantage of this technique is its ability to achieve durable, stable transgene expression in the vein graft endothelium.After anesthetizing a rabbit according to the text protocol, shave the animal from the sternal notch to the edge of the mandible. Upon completion of the surgical setup use electrocautery to cut the skin along the midline between the sternal notch and the mandible, approximately seven to nine centimeters in length. Then attach towel clamps to hold open the incision.Bluntly dissect under the fascia along the entire midline. Then use electrocautery to cut through the dissected fascial layer along the midline. Next, beginning on the right side, dissect between the sternal hyoid muscle overlying the trachea and the sternoephalic muscle to expose the common carotid artery.Then carefully dissect a four to five centimeter segment of the common carotid artery and its larger branches free from surrounding tissues. Use 5-0 silk sutures to ligate the larger branches of the common carotid artery before cutting each branch distal to the suture. After repeating the dissection on the left common carotid, use tissue holding forceps to temporarily close the subcutaneous tissue layer.Then dissect the superficial fascia from the skin on the right side until the external jugular vein is exposed. Dissect a four to five centimeter segment of the external jugular vein and free its branches from the surrounding tissue. Use 5-0 silk sutures to ligate the branches, then cut the branches.Next ligate the isolated vein at both ends, cut the cranial end, and flush the lumen with heparinized saline. To carry out vein grafting, use a vascular clip to clamp the right common carotid artery at the cranial end of the isolated segment. Then place a vascular clip on the caudal end.Now perform a caudal arteriotomy just cranial to the caudal clamp equal in length to the diameter of the cranial end of the vein segment. Then insert an IV catheter connected to a syringe of heparinized normal saline through the caudal arteriotomy and flush the carotid artery lumen. The fluid will exit via the arteriotomy.Returning to the jugular vein, excise the vein and lay it next to the carotic artery. Using single stitches of 7-0 polypropylene suture the caudal end of the caudal arteriotomy to the cranial end of the vein segment. Then place a second stitch 180 degrees from the first stitch to suture the cranial edge of the caudal arteriotomy to the cranial end of the vein segment.Place a third stitch at a point equidistant from the first two stitches on the lateral side of the anastomosis. Then place a fourth stitch on the medial side of the anastomosis equidistant from the first two stitches and across from the third stitch. Place four additional stitches between each of stitches one through four.Perform the cranial arteriotomy on the caudal side of the distal clip. The distance between the cranial end of the cranial arteriotomy and the caudal end of the caudal arteriotomy should be the same as the length of the vein segment. The length of the cranial arteriotomy is equal to the diameter of the caudal end of the vein segment.With the vein extended cranially from the anastomosis along the carotid, ensure the vein is not twisted. Then use heparinized normal saline to flush the carotid artery and vein via the cranial arteriotomy. Next place a first stitch to suture the caudal end of the cranial arteriotomy to the caudal end of the vein segment.Then place a second stitch 180 degrees from the first stitch to suture the cranial edge of the cranial arteriotomy to the caudal end of the vein segment. Complete the anastomosis as demonstrated earlier in the video. Temporarily release the cranial clip to allow the vein graft to fill with blood and apply gentle pressure with dry gauze to stop any bleeding.Repeat this step until the anastomosis no longer bleeds. Then remove both the cranial and caudal clips. Use gauze to stop any additional bleeding at the anastomoses.Use a 3-0 silk suture to ligate both ends of the common carotid artery segment between the anastomoses. Then divide the carotid halfway between the ligatures. Brisk pulsations should be seen in the vein graft.Repeat the vein graft placement on the left side. With a 5-0 PGA suture, close the subcutaneous tissue with a continuous pattern. Then use a 3-0 PGA suture to close the skin with a intradermal pattern and bury the knots on both ends.Carry out post operative care and transcutaneous ultrasound according to the text protocol. To perform gene transfer surgery, carefully isolate the right vein graft including 1.5 to two centimeters of the carotid artery adjacent to the graft on both the cranial and caudal sides. Repeat the dissection on the left side in the same manner.Use normal saline to fill the surgical wound cavity on the right side to enable the transmission of sound waves. Connect a two millimeter perivascular flow probe to a volume flow meter and immerse the probe in the saline in the wound cavity. Set the flow rate to zero.Place the flow probe around the carotid artery either caudal or cranial to the vein graft. Then use an electronic data acquisition system to record data from the flow probe. After injecting heparin into the rabbit via the the IV catheter and flushing it with saline, use a vascular clip to clamp the common carotid artery at the cranial end of the vein graft.And then apply a second clip to the carotid approximately 10 millimeters caudal to the caudal anastomosis to leave room for the arteriotomy. Then place two silk ties loosely around the carotid artery just caudal to the vein graft. Next using a previously prepared bent 21 gauge needle, puncture the carotid caudal to the graft and just cranial to the caudal vascular clip.Advance the needle into the lumen back and forth twice to ensure that the lumen is clear. Then carefully withdraw the needle. After preparing a syringe and IV catheter with DMEM according to the text protocol, insert it into the common carotid arteriotomy up to the bend point and wash the vein graft lumen by filling it with 0.5 milliliters of DMEM two times.Remove the DMEM from the graft by using a gloved finger to lightly press at the cranial end of the graft. Then carefully slide the finger along the graft toward the caudal end of the graft to flush the luminal contents out via the arteriotomy. Remove the DMEM syringe from the catheter leaving the catheter in the vessel.Connect the syringe containing the virus solution making sure that no air enters the catheter. Move the loosely knotted silk ties down the artery until they are around the catheter tip but do not tighten them. Infuse 0.03 milliliters of virus solution to push the remaining DMEM out of the catheter.Then using a finger, remove all fluid from the vein graft lumen pressing gently from cranial to caudal. Tighten the two ties around the artery and catheter tip to seal the lumen. Then infuse the virus solution until the vein is distended.Gently lay the syringe down on a nest of gauze. After 20 minutes, use an empty syringe to gently aspirate the virus containing solution from the graft until the vessel collapses. Remove the syringe leaving the catheter in place.Then cut and untie the silk sutures and withdraw the catheter from the vessel. Now use 7-0 polypropylene suture in an X pattern to close the arteriotomy. Then gently pull the suture ends and tie them together with two square knots.After restoring blood flow in the right vein graft as described in the text protocol, repeat the virus infusion on the left side. To harvest the transduced grafts, dissect free the vein grafts and common carotid arteries and make flow measurements on both the left and right sides as demonstrated earlier. Use 3-0 silk suture to ligate the right common carotid artery cranial to the graft.Then use 3-0 silk suture to ligate the carotid caudal to the graft. Excise the right vein graft by dividing the adjacent carotid artery between each of the ligations and the adjacent anastomosis. Then remove the vein graft from the rabbit and use saline to flush the lumen.The first milestone that a new operator must achieve is consistent vein graft patency after both the initial vein grafting surgery and the subsequent delayed transduction surgery. When a vein graft is patent, the ultrasound exam will reveal a large blood vessel with brisk caudal to cranial blood flow. The grafts were harvested three days after the transduction, cut into sections and stained with X-gal.En-face imaging of the luminal surfaces showed vein grafts with efficient transduction and poor transduction. As shown in this plot, the levels of beta galactosidase mRNA in vein grafts transduced by the new operator, were not significantly different from the levels of beta galactosidase mRNA in vein grafts transduced by an experienced operator. When performing this protocol, it is important to remember to reverse the vein graft cranial to caudal before contracting the anastomosis.This is necessary to avoid obstruction of blood flow by venous bolus. Once mastered these surgeries can be completed in about three hours for the vein grafting procedure, two hours for the vein graft gene transfer procedure and 45 minutes for the harvest procedure. Don't forget that because this protocol requires working with viral vectors and sharp objects, proper biosafety and sharps procedures must be followed.

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10.2K vues.  University of Washington School of Medicine. This method can answer key questions about the pathogenesis of vein graft failure. For example, it could identify mediators of graft failure and test strategies aimed at preventing graft failure. The main advantage of this technique is its ability to achieve durable, stable transgene expression in the vein graft endothelium.After anesthetizing a rabbit according to the text protocol, shave the animal from the sternal notch to the edge of the mandible. Upon completion of the surgical se...