Name: a rabbit model of durable transgene expression in jugular vein to common carotid artery interposition grafts
Uploaded: 2018-09-10T12:30:00
Description: 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

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. 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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. 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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. 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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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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.

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			<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">
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		<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>
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			<td height="20" style="height:20px;width:215px;">Catheters, 24Ga x 3/4&#8221;</td>
			<td>Terumo Medical Products</td>
			<td>SROX2419V</td>
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			<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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Research

JoVE Journal

Medicine

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

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

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

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

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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. ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...