Name: a modified technique for arteriovenous fistula construction in rabbits
Uploaded: 2023-02-10T13:33:00
Description: Watch this Scientific Journal Video about A Modified Technique for Arteriovenous Fistula Construction in Rabbits at JoVE.com

This study was supported by grants from the Suzhou Science and Technology Plan Project (SYS2020077), Suzhou High-tech Zone Medical and Health Science and Technology Plan Project (2020z001), Suzhou Science and Technology Development Plan Project-Medical and Health Science and Technology Innovation (SYK2021030), Nanjing Medical University Science and Technology Development Fund-General Project (NMUB20210253), Suzhou Science and Technology Bureau of the application of the basic research project (No.SYSD2019205, No.SYS2020119), Jiangsu Province Traditional Chinese Medicine Science and Technology Development Plan Project (No.MS2021098), the Ministry of Education Industry-University Cooperation Collaborative Education Project (No. 202102242003),&#160;the Sixth &#34;333 High-level Talent Cultivation&#34; Project in Jiangsu Province, Suzhou Science and Technology City Hospital 2022 Hospital-level Pre-research Fund Project (SZKJCYY2022014), and Suzhou &#34;KeJiaoXingWei&#34; Youth Science and Technology Project (KJXW2022086).

The experimental procedures using laboratory animals were approved by the Experimental Animal Welfare Ethics Committee of Nanjing Medical University. New Zealand rabbits aged 10 months (of both sexes; body weight, 3.18 &#177; 0.24 kg) were used for this study. The animals were obtained from a commercial source (see Table of Materials).
1. Animal preparation
<ol>
	<li>Anesthetize the rabbits using a mixed intravenous injection of tiletamine hydrochloride and ...

<ol>
	<li>Lok, C. E., 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=KDOQI+Clinical+Practice+Guideline+for+Vascular+Access:+2019+update.">KDOQI Clinical Practice Guideline for Vascular Access: 2019 update.</a> American Journal of Kidney Diseases. 75, 1 (2020).</li><li>Schmidli, 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=Editor's+choice+-+Vascular+access:+2018+Clinical+Practice+Guidelines+of+the+European+Society+for+Vascular+Surgery+(ESVS).">Editor's choice - Vascular access: 2018 Clinical Practice Guidelines of the European Society for Vascular Surgery (ESVS).</a> European Journal of Vascular and Endovascular Surgery. 55 (6), 757-818 (2018).</li><li>Grogan, 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=Frequency+of+critical+stenosis+in+primary+arteriovenous+fistulae+before+hemodialysis+access:+Should+duplex+ultrasound+surveillance+be+the+standard+of+care.">Frequency of critical stenosis in primary arteriovenous fistulae before hemodialysis access: Should duplex ultrasound surveillance be the standard of care.</a> Journal of Vascular Surgery. 41 (6), 1000-1006 (2005).</li><li>Swinnen, J., Lean, T. K., Allen, R., Burgess, D., Mohan, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxta-anastomotic+stenting+with+aggressive+angioplasty+will+salvage+the+native+radiocephalic+fistula+for+dialysis.">Juxta-anastomotic stenting with aggressive angioplasty will salvage the native radiocephalic fistula for dialysis.</a> Journal of Vascular Surgery. 61 (2), 436-442 (2015).</li><li>Bharat, A., Jaenicke, M., Shenoy, S. <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+technique+of+vascular+anastomosis+to+prevent+juxta-anastomotic+stenosis+following+arteriovenous+fistula+creation.">A novel technique of vascular anastomosis to prevent juxta-anastomotic stenosis following arteriovenous fistula creation.</a> Journal of Vascular Surgery. 55 (1), 274-280 (2012).</li><li>H&#246;rer, T. 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=No-touch+technique+for+radiocephalic+arteriovenous+fistula--Surgical+technique+and+preliminary+results.">No-touch technique for radiocephalic arteriovenous fistula--Surgical technique and preliminary results.</a> The Journal of Vascular Access. 17 (1), 6-12 (2016).</li><li>Sadaghianloo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salvage+of+early-failing+radiocephalic+fistulae+with+techniques+that+minimize+venous+dissection.">Salvage of early-failing radiocephalic fistulae with techniques that minimize venous dissection.</a> Annals of Vascular Surgery. 29 (7), 1475-1479 (2015).</li><li>Sadaghianloo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+artery+deviation+and+reimplantation+inhibits+venous+juxta-anastomotic+stenosis+and+increases+primary+patency+of+radial-cephalic+fistulas+for+hemodialysis.">Radial artery deviation and reimplantation inhibits venous juxta-anastomotic stenosis and increases primary patency of radial-cephalic fistulas for hemodialysis.</a> Journal of Vascular Surgery. 64 (3), 698-706 (2016).</li><li>Bai, 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=Artery+to+vein+configuration+of+arteriovenous+fistula+improves+hemodynamics+to+increase+maturation+and+patency.">Artery to vein configuration of arteriovenous fistula improves hemodynamics to increase maturation and patency.</a> Science Translational Medicine. 12 (557), (2020).</li><li>Zhang, Y. Y., Wang, X. H., Liu, Z., Hou, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+radio-cephalic+arteriovenous+fistula+in+the+forearm+with+a+modified+no-touch+technique.">Creating radio-cephalic arteriovenous fistula in the forearm with a modified no-touch technique.</a> Journal of Visualized Experiments. (182), e62784 (2022).</li><li>Hou, G. 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=Modified+no-touch+technique+for+radio-cephalic+arteriovenous+fistula+increases+primary+patency+and+decreases+juxta-anastomotic+stenosis.">Modified no-touch technique for radio-cephalic arteriovenous fistula increases primary patency and decreases juxta-anastomotic stenosis.</a> The Journal of Vascular Access. , (2022).</li><li>Kunlin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+vein+transplantation+in+treatment+of+ischemia+caused+by+arteritis.">Long vein transplantation in treatment of ischemia caused by arteritis.</a> Revue de Chirurgie. 70 (7-8), 206-235 (1951).</li><li>Wang, 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=Venous+stenosis+in+a+pig+arteriovenous+fistula+model--Anatomy,+mechanisms+and+cellular+phenotypes.">Venous stenosis in a pig arteriovenous fistula model--Anatomy, mechanisms and cellular phenotypes.</a> Nephrology, Dialysis, Transplantation. 23 (2), 525-533 (2008).</li><li>Marius, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sheep+model+of+hemodialysis+arteriovenous+fistula+using+superficial+veins.">Sheep model of hemodialysis arteriovenous fistula using superficial veins.</a> Seminars in Dialysis. 28 (6), 687-691 (2015).</li><li>Ramacciotti, E., 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=Fistula+size+and+hemodynamics:+An+experimental+model+in+canine+femoral+arteriovenous+fistulas.">Fistula size and hemodynamics: An experimental model in canine femoral arteriovenous fistulas.</a> The Journal of Vascular Access. 8 (1), 33-43 (2008).</li><li>Eiketsu, 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=Arterial+enlargement,+tortuosity,+and+intimal+thickening+in+response+to+sequential+exposure+to+high+and+low+wall+shear+stress.">Arterial enlargement, tortuosity, and intimal thickening in response to sequential exposure to high and low wall shear stress.</a> Journal of Vascular Surgery. 39 (3), 601-612 (2004).</li><li>Eddie, 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=A+new+arteriovenous+fistula+model+to+study+the+development+of+neointimal+hyperplasia.">A new arteriovenous fistula model to study the development of neointimal hyperplasia.</a> Journal of Vascular Research. 49 (2), 123-131 (2012).</li><li>Karl, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+dialysis+fistula+model+exhibits+a+senescence+phenotype:+pathobiological+mechanisms+and+therapeutic+potential.">The murine dialysis fistula model exhibits a senescence phenotype: pathobiological mechanisms and therapeutic potential.</a> American Journal of Physiology. Renal Physiology. 315 (5), 1493-1499 (2018).</li><li>Hong, S. 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=Clinical+analysis+of+radiocephalic+fistula+using+side-to-side+anastomosis+with+distal+cephalic+vein+ligation.">Clinical analysis of radiocephalic fistula using side-to-side anastomosis with distal cephalic vein ligation.</a> The Korean Journal of Thoracic and Cardiovascular Surgery. 46 (6), 439-443 (2013).</li><li>Tang, W. 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=A+meta-analysis+of+traditional+and+functional+end-to-side+anastomosis+in+radiocephalic+fistula+for+dialysis+access.">A meta-analysis of traditional and functional end-to-side anastomosis in radiocephalic fistula for dialysis access.</a> International Urology and Nephrology. 53 (7), 1373-1382 (2021).</li><li>Marie, 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=Patterns+of+blood+flow+as+a+predictor+of+maturation+of+arteriovenous+fistula+for+haemodialysis.">Patterns of blood flow as a predictor of maturation of arteriovenous fistula for haemodialysis.</a> The Journal of Vascular Access. 15 (3), 169-174 (2014).</li><li>Srivastava, 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=Spiral+laminar+flow,+the+earliest+predictor+for+maturation+of+arteriovenous+fistula+for+hemodialysis+access.">Spiral laminar flow, the earliest predictor for maturation of arteriovenous fistula for hemodialysis access.</a> Indian Journal of Urology. 31 (3), 240-244 (2015).</li><li>Loveland-Jones, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+arteriovenous+fistula+to+study+hemodialysis+access+complications.">A new model of arteriovenous fistula to study hemodialysis access complications.</a> The Journal of Vascular Access. 15 (5), 351-357 (2014).</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> Journal of Vascular Surgery. 59 (1), 192-201 (2014).</li></ol>

Currently, several animal models are available for AVF. Among them, pigs, sheep, and dogs are mostly used as large animal models13,14,15. The small animal models used include rabbits, rats, and mice16,17,18. New Zealand rabbits were used in this study. New Zealand rabbits have abundant perivenous tissues around the EJV, which makes them...

The construction of an arteriovenous fistula (AVF) is widely used in clinical practice for patients undergoing maintenance hemodialysis (MHD), and it has higher patency and fewer complications than an arteriovenous graft (AVG) or tunneled cuffed catheter (TCC)1,2. Although AVF is the preferred mode of vascular access, it is not perfect and has inherent limitations. The 1 year primary AVF patency rates are only 60%-65%, with many failures occurring in the near anastomotic region3,4,5.
Vessels undergo different degrees of damage during the traditional surgical approach, which ultimately affects the maturation of the AVF. New surgical modalities, such as the no-touch technique (NTT)&#160;(Supplementary Figure 1) proposed by H&#246;rer et al.6 and radial artery excursion and reimplantation (RADAR) proposed by Sadaghianloo et al.7,8 and Bai et al.9, were designed to decrease the rate of juxta-anastomotic stenosis and to improve the fistula patency by modifying the surgical technique. Although the effect of RADAR was better than that of NTT, inflow arterial stenosis was observed to be more prominent with RADAR. To further reduce injury to the veins and arteries during the operation, in 2021, a new modified no-touch technique (MNTT) was proposed to create a radio-cephalic AVF by preserving the perivenous tissue around the cephalic vein without cutting the radial artery (Supplementary Figure 1 and Supplementary Figure 2). The preliminary results showed increased primary patency, decreased juxta-anastomotic stenosis, and no arterial stenosis10,11.
Considering the current lack of animal models of AVF using MNTT, and to further explore the mechanism of MNTT in AVF surgery, this study introduces a common carotid artery (CCA)-external jugular vein (EJV) AVF procedure using MNTT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal Depilatory</td>
			<td>Fuzhou Feijing Biotechnology Co., Ltd.</td>
			<td>PH1877</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved hemostatic forceps</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZH131R/RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZDO25R/RN</td>
			<td></td>
		</tr>
		<tr>
			<td>electrical razor</td>
			<td>Shenbao Technology Co., Ltd</td>
			<td>PGC-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed Table</td>
			<td>Zhenhua Biomedical Instrument Co., Ltd</td>
			<td>ZH-DSB019</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needleholder</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZM208R/RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Dodium Injection</td>
			<td>Jiangsu Wanbang Biochemical Pharmaceutical Group Co., Ltd.</td>
			<td>H32020612</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical gauze dressing</td>
			<td>Nanchang Kangjie medical hygiene products Co., Ltd</td>
			<td>20172640135</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceops</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZD275RN/T</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder forceps</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZF2618RB/T</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZF022T</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-silk sutures 4-0</td>
			<td>Kollsut Medical Instrument Co., Ltd.</td>
			<td>NMB020RRCN26C075-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-absorbable sutures 8-0 (double needle)</td>
			<td>Yangzhou Yuankang Medical Instrument Co., Ltd.</td>
			<td>10299023602</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodine solution</td>
			<td>Shanghai Likang Disinfection High-tech Co., Ltd.</td>
			<td>310512</td>
			<td></td>
		</tr>
		<tr>
			<td>Rinse needle</td>
			<td>Jiangsu Tonghui Medical Instrument Co., Ltd</td>
			<td>20180039</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel handle</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd. Surgical Instruments Factory</td>
			<td>J11030</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp blade</td>
			<td>Suzhou Medical Products Factory Co., Ltd.</td>
			<td>TY21232001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride Injection&#160; (100 mL)</td>
			<td>Guangdong Otsuka Pharmaceutical Co., Ltd.</td>
			<td>B21K0904</td>
			<td></td>
		</tr>
		<tr>
			<td>Sugical Scissors</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZC120R/RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Sumianxin II</td>
			<td>Jilin Dunhua Shengda Animal Pharmaceutical Co., Ltd.</td>
			<td>20180801</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe with needle&#65288;5 mL)</td>
			<td>BD medical devices (Shanghai) Co., Ltd</td>
			<td>2006116</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine Hydrochloride and Zolazepam Hydrochloride for Injection</td>
			<td>Virbac Pet Health, France</td>
			<td>83888204</td>
			<td></td>
		</tr>
		<tr>
			<td>Triangle needle</td>
			<td>Hangzhou Huawei medical supplies Co., Ltd</td>
			<td>7X17</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamp</td>
			<td>Xinhua Surgical Instrument Co., Ltd.</td>
			<td>ZF220RN</td>
			<td></td>
		</tr>
		<tr>
			<td>New Zealand rabbits</td>
			<td>Suzhou Huqiao Biological Co., Ltd.</td>
			<td>SCXK2020-0001</td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified technique for arteriovenous fistula construction in rabbits

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury to the veins and arteries during the operation and hemodynamic changes can lead to intimal hyperplasia, leading to juxta-anastomotic stenosis. To reduce injury to the veins and arteries during the operation, this study proposes a new modified no-touch technique (MNTT) for AVF construction that can decrease the rate of juxta-anastomotic stenosis and improve the AVF patency. To unravel the hemodynamic changes and mechanisms of the MNTT, this study presented an AVF procedure using this technique. Although this procedure is technically challenging, 94.4% procedural success was achieved after adequate training. Ultimately, 13 out of 34 rabbits had a functional AVF 4 weeks after the surgery, leading to a 38.2% AVF patency rate. However, at 4 weeks, the survival rate was 86.1%. Ultrasonography showed active blood flow through AVF anastomosis. Furthermore, the spiral laminar flow was observed in the vein and artery near the anastomosis, suggesting that this technique may improve the hemodynamics of the AVF. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was observed at the proximal external jugular vein (EJV) of the anastomosis. This technique will improve the understanding of the mechanisms underlying the use of MNTT for AVF construction and provide technical support for the further optimization of the surgical approach in AVF construction.

The outcome of the successful application of this technique is a patent AVF in the rabbit neck. This study used the following criteria to evaluate the success: (1) when the vascular anastomosis is completed, the venous tremor of the AVF can be touched, and the vascular murmur can be heard; (2) 4 weeks after the AVF is established, the active blood flow through the internal fistula anastomosis can be measured by color Doppler ultrasound; (3) 4 weeks after the AVF is established, hematoxylin-eosin (H&#38;E) staining shows ...

Watch this Scientific Journal Video about A Modified Technique for Arteriovenous Fistula Construction in Rabbits at JoVE.com

A Modified Technique for Arteriovenous Fistula Construction in Rabbits

The present protocol proposes the creation of an arteriovenous fistula in rabbits using a modified no-touch technique. The technique involves the side-to-side anastomosis of the common carotid artery and external jugular vein without the dissection of the perivenous tissues or cutting off the artery.

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury ...

Clinically, radiocephalic AVF created with a modified no-touch technique can increase primary patency and decrease juxta-anastomotic stenosis. To explore the mechanism of MNTT, this animal model needs to be established. The main advantage of the modified no-touch technique is to reduce injury to the external jugular vein when establishing AVF.When dealing with the external jugular vein care should be taken to preserve the paravenous tissue as much as possible and reduce the injury to the vein. To begin, place an anesthetized rabbit in a supine position on a fixed table. Bind its limbs and incisors.Using an electric razor, shave the rabbit's neck and upper chest, and remove the hair with animal depilatory cream. Next, using a scissor, make a three centimeter long incision between the mandible and the sternoclavicular joint. Identify the right extra jugular vein, or EJV, and make a tunnel perpendicular to it.Place a vascular clamp along the tunnel and similarly make another tunnel at the distal EJV. Control the blood flow by applying a 4-O suture and a clamp along the tunnel. To dissect the common carotid artery, begin by exploring the CCA lateral to the trachea and medial to the sternocleidomastoid muscle.Now, dissect two centimeters of the CCA. Using a 4-0 suture thread, control the blood flow when needed. Place vascular clamps at the distal and proximal lens of the CCA.For phlebotomy and anastomosis, use micro scissors to dissect four millimeters of the internal part of the EJV. Then, make a four millimeter long incision in the middle of the vein. Rinse the vein with heparin solution to prevent thrombosis.Make a similar incision in the anterior wall of the artery and rinse as demonstrated. Pull the EJV and the CCA as close together as possible. Apply Connell's technique using 8-0 non-absorbable sutures to perform side-by-side anastomosis of the vessels.Suture the posterior wall of the vessels first, followed by the anterior vessel wall. To observe active blood flow through the anastomosis, remove all vascular clamps except for the one at the distal EJV. Using the 4-0 suture placed earlier, ligate the distal end of the EJV, then remove the distal clamp.Remove the suture thread around the CCA. Of the 34 rabbits that had immediately successful arterial venous fistula using the modified no-touch technique, only 31 rabbits survived, resulting in a survival rate of 86.1%13 out of 34 rabbits had a functional AVF four weeks after the surgery leading to a 38.2%AVF patency rate. The patent arterial venous fistula was confirmed after four weeks.Spiral laminar flow was observed in both vessels near the anastomosis. Upon HE staining, significant venous intimal hyperplasia was observed at the site of the arteriovenous fistula anastomosis, but not at the proximal EJV of the anastomosis. AVF animal model established using MNTT can provide technical support for further exploration of the molecular biological mechanism of juxta-anastomotic stenosis.

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Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

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.

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 ...

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.

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 ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Creating Radio-cephalic Arteriovenous Fistula in the Forearm with a Modified No-Touch Technique

Autologous arteriovenous fistula (AVF) is the primary and best option to obtain vascular access for hemodialysis treatment; other options are arteriovenous graft (AVG) and central venous catheterization (CVC). The implementation of radio-cephalic autologous arteriovenous fistula (RC-AVF) in the forearm was preferred among patients with superior vascular conditions. However, there is a high rate of early fistula failure. The chosen surgical method is understood to have an effect on the maturation of the fistula. New surgical procedures such as radial artery deviation and reimplantation (RADAR) have been significantly improved for juxta-anastomotic stenosis. Nevertheless, new problems such as stenosis of arteries and narrowing of surgical indication were also found. In this report, we presented a modified no-touch technique (MNTT) to create an RC-AVF, in which the venous and arterial wall avoid devascularization and the radial artery does not sever.

We present a modified no-touch technique (MNTT) to create a radio-cephalic arteriovenous fistula (RC-AVF) in which the venous and arterial wall avoid devascularization and the radial artery does not sever.

Autologous arteriovenous fistula (AVF) is the primary and best option to obtain vascular access for hemodialysis treatment; other options are ...

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.