This study was supported by a grant from the Dutch Kidney Foundation (KJPB 08.0003). 
Carolien Rothuizen is acknowledged for her contribution to the study. Hoang Pham is acknowledged for his assistance with the pathology work-up.

All experiments were approved by the committee on animal welfare of the Leiden University Medical Center.
1. Animal Preparation and Anaesthesia
<ol>
	<li>Anesthetize the mouse (1-3 months old) in a continuous anaesthetic induction chamber filled with 3-4% isoflurane.</li>
	<li>Shave the ventral side of the neck and the inside part of the left upper leg using an electrical razor and use a piece of tape to remove the hair.</li>
	<li>Apply ocular ointment on both eyes.</li>
	<li>Position the an...

<ol>
	<li>Rotmans, J. I. <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+Studying+Pathophysiology+of+Hemodialysis+Access.">Animal Models for Studying Pathophysiology of Hemodialysis Access.</a> The Open Urology &amp; Nephrology Journal. 7, 14-21 (2014).</li><li>Kwei, 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=Early+adaptive+responses+of+the+vascular+wall+during+venous+arterialization+in+mice.">Early adaptive responses of the vascular wall during venous arterialization in mice.</a> Am.J.Pathol. 164 (1), 81-89 (2004).</li><li>Castier, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neointima+lesions+associated+with+arteriovenous+fistulas+in+a+mouse+model.">Characterization of neointima lesions associated with arteriovenous fistulas in a mouse model.</a> Kidney Int. 70 (2), 315-320 (2006).</li><li>Krishnamoorthy, M. 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=Hemodynamic+wall+shear+stress+profiles+influence+the+magnitude+and+pattern+of+stenosis+in+a+pig+AV+fistula.">Hemodynamic wall shear stress profiles influence the magnitude and pattern of stenosis in a pig AV fistula.</a> Kidney Int. 74 (11), 1410-1419 (2008).</li><li>Ene-Iordache, B., Cattaneo, L., Dubini, G., Remuzzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anastomosis+angle+on+the+localization+of+disturbed+flow+in+'side-to-end'+fistulae+for+haemodialysis+access.">Effect of anastomosis angle on the localization of disturbed flow in 'side-to-end' fistulae for haemodialysis access.</a> Nephrol. Dial. Transplant. 28 (4), 997-1005 (2013).</li><li>Wong, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+remodeling+and+intimal+hyperplasia+in+a+novel+murine+model+of+arteriovenous+fistula+failure.">Vascular remodeling and intimal hyperplasia in a novel murine model of arteriovenous fistula failure.</a> J.Vasc.Surg. 59 (1), 192-201 (2014).</li><li>Rekhter, M., Nicholls, S., Ferguson, M., Gordon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+in+human+arteriovenous+fistulas+used+for+hemodialysis.">Cell proliferation in human arteriovenous fistulas used for hemodialysis.</a> Arterioscler. Thromb. 13 (4), 609-617 (1993).</li><li>Falk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+and+salvage+of+arteriovenous+fistulas.">Maintenance and salvage of arteriovenous fistulas.</a> J. Vasc. Interv. Radiol. 17 (5), 807-813 (2006).</li><li>Tordoir, 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=Prospective+evaluation+of+failure+modes+in+autogenous+radiocephalic+wrist+access+for+haemodialysis.">Prospective evaluation of failure modes in autogenous radiocephalic wrist access for haemodialysis.</a> Nephrol. Dial. Transplant. 18 (2), 378-383 (2003).</li><li>Dixon, B. S., Novak, L., Fangman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodialysis+vascular+access+survival:+upper-arm+native+arteriovenous+fistula.">Hemodialysis vascular access survival: upper-arm native arteriovenous fistula.</a> Am. J. Kidney Dis. 39 (1), 92-101 (2002).</li><li>Yang, B., Shergill, U., Fu, A. A., Knudsen, B., Misra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+arteriovenous+fistula+model.">The mouse arteriovenous fistula model.</a> J. Vasc. Interv. Radiol. 20 (7), 946-950 (2009).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+and+systemic+hemodynamic+responses+following+the+creation+of+a+murine+arteriovenous+fistula.">Regional and systemic hemodynamic responses following the creation of a murine arteriovenous fistula.</a> Am. J. Physiol Renal Physiol. 301 (4), F845-F851 (2011).</li><li>Wong, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin+is+a+key+regulator+of+outward+remodeling+in+arteriovenous+fistulas.">Elastin is a key regulator of outward remodeling in arteriovenous fistulas.</a> Eur. J. Vasc. Endovasc. Surg. 49 (4), 480-486 (2015).</li><li>Kennedy, 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=Does+renal+failure+cause+an+atherosclerotic+milieu+in+patients+with+end-stage+renal+disease.">Does renal failure cause an atherosclerotic milieu in patients with end-stage renal disease.</a> Am. J. Med. 110 (3), 198-204 (2001).</li><li>Cheung, A. 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=Atherosclerotic+cardiovascular+disease+risks+in+chronic+hemodialysis+patients.">Atherosclerotic cardiovascular disease risks in chronic hemodialysis patients.</a> Kidney Int. 58 (1), 353-362 (2000).</li><li>Lee, 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=Severe+venous+neointimal+hyperplasia+prior+to+dialysis+access+surgery.">Severe venous neointimal hyperplasia prior to dialysis access surgery.</a> Nephrol. Dial. Transplant. 26 (7), 2264-2270 (2011).</li><li>Kokubo, 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=CKD+accelerates+development+of+neointimal+hyperplasia+in+arteriovenous+fistulas.">CKD accelerates development of neointimal hyperplasia in arteriovenous fistulas.</a> J. Am. Soc. Nephrol. 20 (6), 1236-1245 (2009).</li></ol>

The AVF is considered to be the Achilles&#39; heel in hemodialysis treatment. Unfortunately, the AVF still suffers from a high number of failure8-10. Despite extensive research on the underlying mechanisms, the exact pathophysiology remains unknown. Numerous murine models for AVF failure have already been described in literature2,3,11,12. However, none of these models incorporate a venous end to arterial side anastomosis configuration that is most used in the clinical situation. This is very relevan...

A functional vascular access conduit is of vital importance for patients with renal failure that depend on chronic hemodialysis to stay alive. The construction of an arteriovenous fistula (AVF) is currently the preferred choice for vascular access. However, AVF related complications constitute a major cause of morbidity for patients on chronic hemodialysis. Despite extensive scientific efforts, none of the novel approaches to reduce AVF access-related complications did result in substantial improvement of AVF durability. Part of this disappointing progress relates to incomplete understanding of the underlying pathophysiology of hemodialysis access failure.
To unravel the pathophysiology of AV access failure, animal models that closely mimic human pathology are of utmost importance. In this respect, not only the animal species but also the anastomotic site, the required anti-coagulatory therapy and the duration of follow up after surgery should be taken into account1. While large animals are the most suitable for intervention studies aimed to develop new therapeutic strategies, murine models have the greatest potential to gain more insight in the molecular mechanisms underlying AV access failure due to the availability of transgenic mice. In addition, a large number of mice can be used for this purpose at lower costs compared to larger animal use.
The first murine model of AVF failure was described in 2004 by Kwei and et al.2 In this model, AVFs were constructed using the carotid artery and the jugular vein in an end-to-end manner using an intravascular catheter. This model could be useful to study early venous adaptation in AVFs although the end-to-end configuration and the presence of an intravascular catheter limit the validity of this model for human AVFs. An improved AVF model was introduced by Castier and et al.3 in which the end of the carotid artery is connected to the side of the jugular vein. However, AVFs in hemodialysis patients are usually constructed by anatomizing the end of a vein to the side of an artery. The exact configuration of the AVF is a crucial characteristic of an AV access model since it determines the hemodynamic profile within the conduit4. The latter is an important contributor to endothelial dysfunction and subsequent development of intimal hyperplasia (IH)5.
A novel murine model was recently developed with an identical anatomical configuration as is utilized in humans6. In this model, AVF are created in C57BL/6 mice by anastomosing the end of a branch of the external jugular vein to the side of the common carotid artery with interrupted sutures. In the present paper, we focus on the microsurgical procedure of this model in order to facilitate the widespread use of this murine model, aimed to unravel the complex pathophysiology of hemodialysis access failure.

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	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:247px;">Dissecting microsocpe</td>
			<td style="width:219px;">Leica</td>
			<td style="width:117px;">M80</td>
			<td style="width:115px;"></td>
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			<td>Medicon</td>
			<td>07.61.25</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular forceps</td>
			<td>S&#38;T</td>
			<td>JFL-3D.2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular forceps</td>
			<td>S&#38;T</td>
			<td>D-5a.2</td>
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		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Forceps</td>
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			<td>SS/45</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro scissor 5 mm blade</td>
			<td>Fine science tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro scissor 2 mm blade</td>
			<td>Fine science tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissor</td>
			<td>Medicon</td>
			<td>05.12.21</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Clip applier 1</td>
			<td>S&#38;T</td>
			<td>CAF-4</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular clamp 1</td>
			<td>S&#38;T</td>
			<td>B-1V</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Clip applier 2</td>
			<td>BBraun</td>
			<td>FE572K</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular clamp 2</td>
			<td>BBraun</td>
			<td>FE740K</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hemostatic forceps</td>
			<td>BBraun</td>
			<td>BH110</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10.0 sutures</td>
			<td>BBraun</td>
			<td>G1117041</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">6.0 sutures</td>
			<td>BBraun</td>
			<td>768464</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cauterizer</td>
			<td>Fine science tools</td>
			<td>18010-00</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle holder</td>
			<td>Medicon</td>
			<td>11.82.18</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ocular ointment</td>
			<td>Pharmachemie</td>
			<td>41821101</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Chlorhexidine tincture 0,5%</td>
			<td>Leiden University Medical Center</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heparin</td>
			<td>Leo Pharma</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Buprenorphin</td>
			<td>RB Pharmaceuticals&#160;</td>
			<td>283732</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Isoflurane</td>
			<td>Pharmachemie</td>
			<td>45,112,110</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia mask</td>
			<td>Maastricht university</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Midazolam</td>
			<td>Actavis</td>
			<td>AAAC6877</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dexmedetomidine</td>
			<td>Orion</td>
			<td>141-267</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fentanyl</td>
			<td>Bipharma</td>
			<td>15923002</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Continuous anaesthetic induction chamber</td>
			<td>Vet-tech solutions</td>
			<td>AN010R</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel murine model of arteriovenous fistula failure: the surgical procedure in detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

After the creation of the anastomosis (Figure 1), the patency should be assessed by shortly occluding the venous outflow tract with a vascular forceps. When the anastomosis is patent, the vascular tract proximal to the occlusion should clearly expand in a pulsatile manner. In addition, the patency is confirmed by using near infrared fluoroscopy (NIRF) that effectively functions as an angiography (Figure 2). A failure in the surgical procedure can lead to an occlusion of the anastomosis a...

Watch this Scientific Journal Video about A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail at JoVE.com

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

The overall goal of this procedure is to create an in vivo animal model that mimics the pathophysiology of arteriovenous fistula failure. This method can be used to unravel the pathophysiology of arteriovenous fistula failure and can help researchers from all over the world to develop new therapeutic strategies to prevent vascular access related complications. The main advantage of this technique is that it incorporates a veinous end to arterial site anastomosis that closely resembles the clinical situation that is utilized in humans.The procedure will be demonstrated by Chun Yu Wong, an excellent PhD student from my laboratory. After anesthetizing a one to three month old mouse with isoflurane, shave the hair off the ventral side of the neck and the inner region of the upper left leg. Use tape to clean up the loose hairs.Next, apply opthalmic ointment to both eyes then prepare a 37 degree Celsius heating blanket with a fixed nose ventilation mask and secure the head of the animal in position with adhesive tape. To the mask, send 1.5%to 2%isoflurane at a rate of 0.3 liters air per minute along with 100%oxygen at a rate of 0.2 liters per minute. Pinch the skin between the toes to assess the depth of anesthesia.Adjust the concentration of isoflurane if needed. Then, fix the mouse on the blanket in a supine position by taping down it's limbs. Now subcutaneouly inject a bolus of buprenorphine in a half milliliter of 0.9%sodium chloride into the left flank.Complete the preparation by cleaning the exposed skin for incision using 0.5%chlorhexidine. Under a dissection microscope start with a longitudinal incision along the midline of the neck, make it about 1.5 centimeters long exposing the salivary glands. Move the right salivary gland cranially until it is partially outside of the wound to uncover the right jugular vein.Next, find the dorsomedial branch of the vein and bluntly dissect it from the perivascular tissue by spreading the tissue just alongside the blood vessel. Then, place a loop of 10/0 suture around the dissected vein and add a loose knot. The next major step is to remove the sternocleidomastoid muscle.Begin by using a pair of forceps to bluntly dissect around the right sternocleidomastoid muscle. Then, ligate the muscle with two 6/0 suture loops and excise it using a cauterizer. Moving on, dissect and prepare the common carotid artery.Once identified, place a 6/0 suture around it and then, place vascular clamps as far distally and proximally as possible on to the artery. Then, with microscissors, make a one millimeter longitudinal incision into the artery. Once incised, rinse the artery with heparin solution to remove the blood then measure and adjust the length of the incision to match the width of the vascular clamp.Now position a thread around the vein and attach a vessel clamp proximally. Apply gentle traction caudally using a hemostat and ligate the vein as distally as possible using the already positioned 10/0 suture. Once ligated, cut the vein just proximal of the ligation to access the lumin.Gently dilate the lumin with the vascular forceps and rise the vein out with heparin and proceed with the anastomosis. To start the anastomosis connect the vein to the artery with an interrupted 10/0 suture, using two square knots at the 12:00 position followed by a suture at the 6:00 position. Make sure there is no torsion of the vein.Next, position a suture thread around the vein and apply gentle, lateral traction. Then, add three or four more interrupted sutures at the visible ventral side of the anastomosis to complete the connection. In order to create a patent arteriovenous fistula it is of utmost importance to prevent torsion of the vein and to prevent needle placement through both the anterior and posterior vessel wall during the suturing procedure.Now, rotate the heating blanket 180 degrees and focus on the left upper leg. Identify the femoral bundle by looking for a vascular structure that runs longitudinally in the medial part of the upper leg. It can be seen through the skin.Make an approximately one centimeter incision over the femoral vein in a lengthwise manner and carefully dissect the perivascular tissue of the femoral vein. Then, inject a dose of heparin into the femoral vein. Now, return to the neck area and remove the suture thread that was placed around the vein, follow this by placing a 6/0 suture thread below the carotid artery over the vein and again below the carotid artery.Now, remove the veinous vascular clamp. Next, twist the vascular clamps and apply traction to the suture thread to turn the half completed arteriovenous fistula 180 degrees around the carotid artery in a clockwise manner. Complete the anastomosis using interrupted sutures as previously demonstrated.Adequate of the posterior wall of the anastomosis is crucial in order to complete the second part. The use of a strategically placed suture thread eases this portion of the procedure. Now, remove the suture thread and remove the twist in the arteriovenous fistula.Following this, remove the distal vascular clamp and then remove the proximal vascular clamp. Assess the anastomosis by gently occluding the veinous outflow tract with vascular forceps. If it is patent, then the pre-occluding veinous section will expand in a pulsatile fashion.Patency can also be confirmed with near infrared fluoroscopy. A failure can lead to an occlusion caused by too narrow of an anastomotic area, torsion of the vessels, inadequate heparin dose, or an accidental suture placement that connects the front side of the anastomosis to the back side. To proceed with the surgery, relocate the salivary gland to it's original anatomical position and close up the two skin incisions using uninterrupted 6/0 suture.Allow the animal to recover in a darkened, heated cage after administration of 0.5 milliliters of 0.9%sodium chloride injected subcutaneously. Allow the animal to fully recover in a dark cage under a heat lamp. At a histological level, vascular remodeling in the AVF can be investigated elegantly using this model.Vascular remodeling in AVF occurs as a result of the increase in blood flow and pressure. In mice, this results in an increase in circumference and luminal area during the first two weeks after surgery. Immunostainin of the veinous outflow tract shows that the cellular compartment of the intima mainly consists of alpha smooth muscle actin positive cells.Two weeks ends up being the optimal time to harvest the AVF as after four weeks, approximately 50%of the AVFs are occluded. After watching this video you should have a good understanding of how to perform the surgical procdure and what the crucial steps are in this animal model. Once mastered this procedure can be performed in one hour.This technique offters new opportunities for researchers in the field of arteriovenous fistula failure to explore its pathophysiology. This model will also help to develop new therapeutic strategies to reduce the morbidity of patients with end stage renal disease that requite chronic hemodialysis.

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12.8K vistas.  Leiden University Medical Center. The overall goal of this procedure is to create an in vivo animal model that mimics the pathophysiology of arteriovenous fistula failure. This method can be used to unravel the pathophysiology of arteriovenous fistula failure and can help researchers from all over the world to develop new therapeutic strategies to prevent vascular access related complications. The main advantage of this technique is that it incorporates a veinous end to arterial site anastomosis that closely resembles the cli...