This work was supported in part by the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program Merit Review Award I01-BX002336, the National Institute of Health grant R56-HL095498, as well as with the resources and the use of facilities at the VA Connecticut Healthcare System, West Haven, CT.

Approval by the appropriate Institutional Animal Care and Use Committee is obtained.
1. Anesthesia and Pre-operative Procedures
<ol>
	<li>Anesthetize male C57Bl/6 ice, aged 8 weeks, with vaporized 3% isoflurane and 0.8 L/min oxygen administered into an acrylic induction chamber.</li>
	<li>Confirm adequate anesthesia by lack of reaction to toe pinch. Position the mouse supine on the operation table and position a silicone mask to deliver vaporized 2 - 3% isoflurane by continuous inhalation.</...

<ol>
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N., Quintanar-Guerrero, D., Ganem-Quintanar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+thermo-reversible+pluronic+F-127+gels+in+pharmaceutical+formulations.">Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations.</a> J. Pharm. Pharm. Sci. 9 (3), 339-358 (2006).</li><li>Almeida, H., Amaral, M. H., Lob&#227;o, P., Lobo, J. 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=Pluronic&#174;+F-127+and+Pluronic+Lecithin+Organogel+(PLO):+main+features+and+their+applications+in+topical+and+transdermal+administration+of+drugs.">Pluronic&#174; F-127 and Pluronic Lecithin Organogel (PLO): main features and their applications in topical and transdermal administration of drugs.</a> J. Pharm. Pharm. Sci. 15 (4), 592-605 (2012).</li><li>Karper, J. 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=Toll-like+receptor+4+is+involved+in+human+and+mouse+vein+graft+remodeling,+and+local+gene+silencing+reduces+vein+graft+disease+in+hypercholesterolemic+APOE*3Leiden+mice.">Toll-like receptor 4 is involved in human and mouse vein graft remodeling, and local gene silencing reduces vein graft disease in hypercholesterolemic APOE*3Leiden mice.</a> Arterioscler., Thromb., Vasc. Biol. 31 (5), 1033-1040 (2011).</li><li>Redmond, E. M., Hamm, K., Cullen, J. P., Hatch, E., Cahill, P. A., Morrow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+patched-1+prevents+injury-induced+neointimal+hyperplasia.">Inhibition of patched-1 prevents injury-induced neointimal hyperplasia.</a> Arterioscler., Thromb., Vasc. 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Surg. 129 (6), 1405-1413 (2005).</li><li>Ohta, 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=Intraluminal+gene+transfer+of+endothelial+cell-nitric+oxide+synthase+suppresses+intimal+hyperplasia+of+vein+grafts+in+cholesterol-fed+rabbit:+a+limited+biological+effect+as+a+result+of+the+loss+of+medial+smooth+muscle+cells.">Intraluminal gene transfer of endothelial cell-nitric oxide synthase suppresses intimal hyperplasia of vein grafts in cholesterol-fed rabbit: a limited biological effect as a result of the loss of medial smooth muscle cells.</a> Surg. 131 (6), 644-653 (2002).</li><li>Baldwin, Z. 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=Modulation+of+vascular+remodeling+induced+by+a+brief+intraluminal+exposure+to+the+recombinant+R7020+strain+of+Herpes+simplex-1.">Modulation of vascular remodeling induced by a brief intraluminal exposure to the recombinant R7020 strain of Herpes simplex-1.</a> J. Vasc. Surg. 41 (1), 115-121 (2005).</li><li>Eslami, M. 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=Gene+delivery+to+in+situ+veins:+differential+effects+of+adenovirus+and+adeno-associated+viral+vectors.">Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors.</a> J. Vasc. Surg. 31 (6), 1149-1159 (2000).</li><li>Globerman, A. 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=Efficient+transgene+expression+from+naked+DNA+delivered+into+an+arterio-venous+fistula+model+for+kidney+dialysis.">Efficient transgene expression from naked DNA delivered into an arterio-venous fistula model for kidney dialysis.</a> J. Gene Med. 13 (11), 611-621 (2011).</li><li>Yang, 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=Adventitial+transduction+of+lentivirus-shRNA-VEGF-A+in+arteriovenous+fistula+reduces+venous+stenosis+formation.">Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation.</a> Kidney Int. 85 (2), 289-306 (2014).</li><li>Brahmbhatt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Iex-1+in+the+pathogenesis+of+venous+neointimal+hyperplasia+associated+with+hemodialysis+arteriovenous+fistula.">The role of Iex-1 in the pathogenesis of venous neointimal hyperplasia associated with hemodialysis arteriovenous fistula.</a> PLoS ONE. 9 (7), e102542 (2014).</li><li>Karram, 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=Induction+of+cardiac+hypertrophy+by+a+controlled+reproducible+sutureless+aortocaval+shunt+in+the+mouse.">Induction of cardiac hypertrophy by a controlled reproducible sutureless aortocaval shunt in the mouse.</a> J. Investig. Surg. 18 (6), 325-334 (2005).</li><li>Perry, G. 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=Genetic+variation+in+angiotensin-converting+enzyme+does+not+prevent+development+of+cardiac+hypertrophy+or+upregulation+of+angiotensin+II+in+response+to+aortocaval+fistula.">Genetic variation in angiotensin-converting enzyme does not prevent development of cardiac hypertrophy or upregulation of angiotensin II in response to aortocaval fistula.</a> Circ. 103 (7), 1012-1016 (2001).</li><li>Guzman, R. J., Krystkowiak, A., Zarins, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+and+sustained+medial+cell+activation+after+aortocaval+fistula+creation+in+mice.">Early and sustained medial cell activation after aortocaval fistula creation in mice.</a> J. Surg. Res. 108 (1), 112-121 (2002).</li><li>Alexander, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+edifoligide,+an+E2F+transcription+factor+decoy,+for+prevention+of+vein+graft+failure+following+coronary+artery+bypass+graft+surgery:+PREVENT+IV:+a+randomized+controlled+trial.">Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery: PREVENT IV: a randomized controlled trial.</a> JAMA. 294 (19), 2446-2454 (2005).</li><li>Khaleel, M. 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This modification of the murine AVF model incorporates intraluminal drug delivery to the venous endothelium at the time of AVF creation. An AVF was created by puncturing the infra-renal aorta with a 25 G needle and extending&#160;the puncture through the opposite aortic wall into the IVC, followed by injection of drug solution through the same needle. The solution is maintained intra-caval, i.e., in the venous limb of AVF, until de-clamping. What distinguishes this model from other murine AVF models17-19</s...

The murine aortovenous fistula (AVF) puncture model between the aorta and the inferior vena cava (IVC) is now an established technique.1 In this model, both walls of the infra-renal aorta are punctured with a 25 G needle, exiting into the adjoining infra-renal vena cava; the anterior aortic entrance hole is repaired with simple compression, and does not require suture repair. Serial follow-up examination by high-resolution Doppler ultrasound and histological analysis shows the AVF to have a maturation phase and then a failing phase, recapitulating the known pathophysiology of human AVF.2
To explore mechanisms that modulate AVF maturation, improved methods for delivery of therapeutic agents to the maturing AVF endothelium are needed. Delivery of therapeutic agents to vessels can be either via endovascular delivery to the lumen, or via external delivery to the adventitia. One example of external delivery is the commonly used adventitial application of Pluronic gel. This copolymer is thermo-reversible and transformed from liquid to solid gel when warmed to body temperature. Prior studies have shown sustained drug delivery is achieved when drug mixed in pluronic gel is applied topically in vivo.3,4 Adventitial application of viral vectors or siRNA with Pluronic gel has been reported to be effective as a perivascular delivery system.5,6 We have also reported that treatment of explanted human saphenous veins with adventitial stimulation by peptides resulted in phosphorylation of endothelial receptor proteins.7
On the other hand, investigators have also used intraluminal delivery of both viral and nonviral vectors in canine8-10 and rabbit11,12 models of vein grafts. In these reports, gene transfer was performed ex vivo after vein harvest. Eslami et al. reported endovascular viral gene delivery to carotid veins in situ without creating a bypass.13 Gloverman et al. reported intraluminal and adventitial delivery of naked DNA in rat femoral artery-superficial epigastric vein fistulae.14 The Mayo group reported adventitial drug delivery in mouse carotid artery-jugular vein fistulae.15,16 However, these previously reported models required a sutured anastomosis to create the AVF. In this report, intraluminal drug delivery with simultaneous AVF creation in mice is described, using a suture-less model of AVF creation. By using this modified murine AVF model a simple method for intraluminal drug delivery to the venous limb of the fistula can be performed.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Pluronic F-127</td>
			<td style="width:259px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2443-250G</td>
			<td style="width:231px;">Used as 30% solution in d-water</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">GFP antibody</td>
			<td style="width:259px;">NOVUS BIOLOGICALS INC</td>
			<td style="width:119px;">NB100-1770</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Ad-CMV-GFP</td>
			<td style="width:259px;">VECTOR BIOLABS</td>
			<td style="width:119px;">1060</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">0.9% Sodium Chloride Irrigation, USP</td>
			<td style="width:259px;">Baxter</td>
			<td style="width:119px;">2F7122</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">BD PrecisionGlide Needle 25G x 5/8</td>
			<td style="width:259px;">BD</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">BD 1ml Syringe Tuberculin Slip Tip</td>
			<td style="width:259px;">BD</td>
			<td style="width:119px;">309659</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Scarpel</td>
			<td style="width:259px;">Surgical Design Inc</td>
			<td style="width:119px;">22079707</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">6-0 ETHILON P-1 11mm 3/8c Reverse Cutting</td>
			<td style="width:259px;">ETHICON INC</td>
			<td style="width:119px;">697G</td>
			<td></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:343px;">Vevo 770 ultrasound machine&#160;</td>
			<td style="width:259px;">Visualsonics&#160;</td>
			<td style="width:119px;">20-60 Mhz scan head; RMV-704</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Vascular clamp&#160;</td>
			<td style="width:259px;">Roboz Surgical Instrument Co.</td>
			<td style="width:119px;">RS-5424</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:343px;">Clamp applying forceps&#160;</td>
			<td style="width:259px;">Roboz Surgical Instrument Co.</td>
			<td style="width:119px;">RS-5410</td>
			<td></td>
		</tr>
	</tbody>
</table>

intraluminal drug delivery to the mouse arteriovenous fistula endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

In a series of 33 mice, survival on the first post-operative day was 97.0%; AVF patency, as determined by ultrasound,&#160;was 84.9%.
Gene transduction efficiency of this endovascular delivery route with the traditional external route was compared. For intraluminal delivery (ILD), immediately after the puncture, 200 &#181;l of Adenovirus-GFP (Ad-GFP) vector solution (1 &#215; 109 PFU/ml) was infused into the IVC via t...

Watch this Scientific Journal Video about Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium at JoVE.com

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

The overall goal of this procedure is to modify the murine arteriovenous fistula, or AVF model, into a simple and intergrated method for drug delivery to the fistula endothelium. This method can help answer key questions about the role of the endothelium in venous remodeling. After exposure to the arterial enivroment and in the development of neointimal hyperplasia.The main advantage of this technique is that it uses a single needle to simultaneously create an AVF while delivering the drug of interest to the endothelium. Before beginning the procedure, autoclave all surgical instruments and vascular clamps. Equip a one-milliliter syringe with a 25 gauge needle and load the syringe with the drug of interest.Then bend the needle to a 60-degree angle approximately four millimeters from the needle tip and grasp the needle with the curved needle holder. Next, clean the incision site of an eight-week old anesthetized C57 black 6 mouse with a topical antiseptic and place a surgical drape over the animal. Using a scalpel make a mid-line abdominal incision extending from the lower liver edge to just above the pubis and insert a retractor.Then, transfer the bowels from the abdominal cavity to the right side and wrap them in saline soaked gauze Dissect the membrane connecting the retroperitoneum and lower colon to obtain a full view of the aorta and IVC. To prepare the tissue for the proximal and distal clamp, dissect the infrarenal aorta and IVC from the surronding tissues and place a single microsurgery clip across both the proximal aorta and IVC just below the left renal vein. Place a second microsurgery clip across both the distal aorta and IVC.Then grasp the connective tissue surrounding the aorta and rotate it medially so that the dorsal surface of the aorta is slightly exposed for the arterial puncture. Keeping the aorta in a rotated position with the left hand, dissect the left lateral margin of the aorta for three quarters of the distance from the left renal vein to the aortic bifurcation so there is ample room to make the puncture with the right hand. Maintaining the aorta in the rotated position, puncture the vessel into the IVC with a curved 25 gauge needle and infuse 100 to 200 microliters of the drug of interest.The needle will be visible through the dilated thin wall of the IVC as the transparent drug solution displaces the venous blood out of the IVC. Carefully maintain the needle in position for 15 minutes without moving, then remove the distal microsurgery clip from the distal aorta and IVC only. When the distal vessel have been declamped, remove the needle and cover the puncture site of the aorta with the adjacent retroperitoneal tissue.Remove the proximal microsurgery clip from the proximal aorta and IVC. The arterial blood will be observed flowing into the IVC instead of the usual dark venous blood flow. Take care to provide careful compression when covering the puncture site to avoid occlusion of the AVF and thrombosis.Finally, after observing 30 seconds of blood flow without compression return the bowels to the abdominal cavity and close the abdomen with a running suture. After closure of the abdomen, apply post-operative care including analgesia, thermal support, and monitoring every 15 minutes until sternal recumbency. In these images, the gene transduction efficiency of this endovascular delivery route with the traditional external route was compared.As evident, en face observation demonstrated a stronger expression of GFP in the IVC wall in mice treated by intraluminal compared to adventitial delivery of the virus with a characteristic augmentation observed around the fistula at 24 hours post adminstration. Cross sectional histological analysis with anti-GFP antibody showed more robust and site-specific GFP expression along the endothelium of the IVC in mice treated with ILD compared with AD.To confirm these results, infrarenal IVCs treated with adenovirus GFP by either route of delivery were harvested for western blood analysis. Indeed, GFP protein expression was sufficient for detection only after intraluminal delivery, suggesting a superior viral drug delivery to the IVC wall via the intraluminal route at 24 hours post transfection.Once mastered, this technique can be completed in 25 minutes, including the 15 minute incubation time if it's performed properly.

intraluminal-drug-delivery-to-mouse-arteriovenous-fistula

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9.2K visualizações.  Yale University. The overall goal of this procedure is to modify the murine arteriovenous fistula, or AVF model, into a simple and intergrated method for drug delivery to the fistula endothelium. This method can help answer key questions about the role of the endothelium in venous remodeling. After exposure to the arterial enivroment and in the development of neointimal hyperplasia.The main advantage of this technique is that it uses a single needle to simultaneously create an AVF while delivering the ...