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An Arteriovenous Fistula Model of Intimal Hyperplasia for Evaluation of a Nitinol U-Clip Anastomosis

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An Arteriovenous Fistula Model of Intimal Hyperplasia for Evaluation of a Nitinol U-Clip Anastomosis
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy An Arteriovenous Fistula Model of Intimal Hyperplasia for Evaluation of a NitinolU-Clip Anastomosis R.L. Varcoe a , b , c , * , A.B.P. Teo a , b , M.H. Pelletier a , b , c , Y. Yu a , b , c , J.-L. Yang a , b , c , P.J. Crowe a , b , c ,W.R. Walsh a , b , c a Department of Surgery, Prince of Wales Hospital, Sydney, Australia b Surgical and Orthopaedic Research Laboratories, Prince of Wales Clinical School, Faculty of Medicine, Australia c The University of New South Wales, Sydney, Australia a r t i c l e i n f o  Article history: Received 17 May 2011Accepted 1 November 2011Available online 21 November 2011 Keywords: Arteriovenous  fi stulaHemodialysis vascular accessIntimal hyperplasiaOvine modelVascular anastomotic clip a b s t r a c t Objectives:  The aim of this study was to create an ovine arteriovenous  fi stula (AVF) model which wouldclosely replicate a human forearm  fi stula and use this to quantify the degree of intimal hyperplasia inthose created with the U-Clip compared to a conventional sutured anastomosis. Materials and methods:  Twenty AVFs were created in 10 Border Leicester e Merino sheep between thesuper fi cial femoral artery and vein of each hind limb. On one side the U-Clip and on the othera continuous polypropylene suture was used to perform the anastomosis. The animals were sacri fi ced at2 ( n  ¼  3), 4 ( n  ¼  4), 6 ( n  ¼  3) weeks and histological slices were taken of each AVF in cross section todetermine the intimal media area per unit length (IMA/L). Results:  Intimal hyperplasia (IH) was observed at all time points with one AVF found occluded withthrombus at the time of harvest. The IMA/L was signi fi cantly lower in the U-Clip groups by 24% at 2weeks, 32% at 4 weeks and 23% at 6 weeks (Two-wayANOVA,  p ¼ 0.019, observed power ¼ 0.825, time orside  p  0.766, type  p ¼ 0.001; Paired  t  -test,  p < 0.001 between matched anastomotic types). Time takento perform the anastomosis was similar between the two anastomotic techniques (Polypropylene 14(8 e 18) vs. U-Clip 15.3(11 e 23) min;  p  ¼  0.47). Conclusion:  This ovine AVF model results in IH similar to that seen in a human AVF. The IH that occurswith the U-Clip is less than that of continuous polypropylene suture.   2011 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. Introduction Intimal hyperplasia (IH) is ubiquitous after the vascular injurythat occurs during surgery. It affects all types of vascular anasto-mosis but is most pronounced in the arteriovenous  fi stula (AVF)created forhemodialysis access. This IH results in restenosis, whichthreatens AVF patencyand causes signi fi cant morbidity. The failureor dysfunction of arteriovenous  fi stulae has an annual cost of morethan $1 billion dollars in the United States 1 e 3 where there werealmost 328,000 patients receiving hemodialysis therapy at the endof 2006. 4 The primary patency of an AVF is less than 50% after 3 years andmay be as low as 20% in patients who are elderly, diabetic orfemale. 5,6 Despite such poor durability alternatives to AVF such ascuffed central venous catheters and synthetic arteriovenous graftssuffer higher rates of infectious complications, central venousstenoses and even lower patency rates. 6,7 The AVF remains there-fore the best option for patients with end stage renal disease whorequire hemodialysis.The nitinol U-Clip device (Medtronic, Minneapolis, MN, USA) isdesigned to reduce the use of sutures thus eliminating knot tyingwhilst facilitating the creation of a circumferentially interruptedvascular anastomosis 8 (Fig. 1). In support of this device ’ s ef  fi cacya single prospective human study has shown superior patency andmaturation rates of forearm AVFs created with U-Clips whencompared to continuous polypropylene suture. 9 The aim of ourstudy was to create an AVF model with vessel size, handlingproperties,con fi gurationandhemodynamicstressesakintohumanforearm AVFs to quantify the degree of intimal hyperplasia andmake comparison between continuous polypropylene suture andthe Nitinol U-Clip anastomosis. *  Corresponding author. R.L. Varcoe, Suite 8, Level 7, Prince of Wales PrivateHospital, Barker St, Randwick, NSW 2031, Australia. Tel.: þ 61 2 96504981; fax: þ 612 96504910. E-mail address:  r.varcoe@unsw.edu.au (R.L. Varcoe). Contents lists available at SciVerse ScienceDirect European Journal of Vascular and Endovascular Surgery journal homepage: www.ejves.com 1078-5884/$  e  see front matter    2011 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.ejvs.2011.11.002 European Journal of Vascular and Endovascular Surgery 43 (2012) 224 e 231  Author's personal copy Materials and Methods  Animals Ten 2-year old crossbred Border Leicester e Merino wetherswere used with the approval of our local Animal Care and EthicsCommittee (ACEC 08/113A). They were kept in the research facilityat the St. George Hospital Campus, University of New South Walesin accordance with the research guidelines of the National Healthand Medical Research Council of Australia. Surgery Sheep were  fi rst sedated with Intramuscular Zolazopam(8 e 12mg/kg),analgesedwithsubcutaneousCarprofen(2 e 4mg/kg)andintramuscularBurprenorphine(0.005mg/kg)thenadministeredinhaled Iso fl uorane (2 e 3%) and 100% oxygen (3 L/min) throughoutthesurgery. Laryngoscopy wasperformedandanendotracheal tubeplaced above the carina for ventilation. Antibiotics (IntravenousCephalothin 0.016 e 0.024 mg/kg; Benacillin 0.079 e 0.122 ml/kg IM)wereadministeredandcrystalloid fl uids(Hartmann ’ ssolution)weregivenintravenouslyat4 e 10ml/kg/hpriortoandduringthesurgery.Bothhindlimbgroincreaseswerecleanedwithpovidone-iodineand draped in accordance with conventional surgical practice. Skincreaseincisionsweremadejustbelowtheinguinalligamentandtheincisiondeepenedtoexposeboththesuper fi cialfemoralarteryandvein. 5000 IU of Sodium Heparin was administered intravenouslyand the vessels were double slung both proximally and distally toachieve hemostasis for AVF creation. A 2 cm length longitudinalarteriotomy and corresponding venotomy was performed in thesuper fi cial femoral vessels. This length was standardized for allanastomoses and all animals. A conventional Brescia-Cimino side-to-side anastomosis was created using continuous 6 e 0 poly-propylene(Prolene,Ethicon,Johnson&Johnson,Warren,NJ,USA)inone hind limb and interrupted U-Clips in the contralateral one 10 (Fig. 2). In each anastomosis care was taken to evert the vesseledges and avoid adventitia within the suture line. Flushing withheparinised saline and hemostasis were given careful attention atthe time of sling release. A marking suture was placed at the ceph-alad and caudad ends of the anastomosis to aid and orient thecadaveric dissection. Patency of   fi stula was con fi rmed with pulseandthrillattheendoftheanastomosis.Layeredclosureoffasciaandskin with 3 e 0 polypropylene suture (Prolene, Ethicon, Johnson & Johnson, Warren, NJ, USA) was performed. The side of hind limbchosen for suture/U-Clip was alternated in consecutive animals.The nitinol U-Clip is applied with a standard needle andsuturing action. It is connected to the surgical needle bya  fi ne wireandreleasedbygraspingatriggerpointwiththeteethoftheneedleholder, at which point it resumes its circular shape holding theblood vessel walls in apposition (Fig. 1A,B). The retention knob at Figure 1.  The nitinol U-Clip with its delivery apparatus intact (A), demonstrating the release trigger point (B, Arrow) and resuming its circular shape (C). (B and C Images takenwithenvironmental scanning electron microscopy (TM-1000, Hitachi, Japan)). R.L. Varcoe et al. / European Journal of Vascular and Endovascular Surgery 43 (2012) 224 e  231  225  Author's personal copy theendoftheclipsnugsagainstthevesselwalltopreventitpullingthrough (Fig. 1C). U-Clips are placed every 1 e 2 mm as one wouldwith an interrupted suture. Study design Ten Sheep were utilized to create 20 AVFs. They were sacri fi cedat 2 weeks (3 sheep, 6 AVF), 4 weeks (4 sheep, 8 AVF) and 6 weeks(3 sheep, 6 AVF). Specimen explantation and preparation Each sheep was administered 20,000 IU of intravenous SodiumHeparin prior to euthanasia with a lethal injection of SodiumPentothal. Hind limb dissection was carried out to identify andexcise each AVF en-bloc with its associated few centimeters of feeding artery and vein. The AVF was gently  fl ushed with 10%buffered formalin so as notto remove any thrombus or IH and thenallowed to fi x in formalin (40%) for 48h. High resolution X-Raywasused to con fi rm the presence of U-Clips and aid with orientation inthe 20 specimens. The specimens were then divided through thelongitudinalmidpointof the anastomosis ina crosssectionalplane.Half of the specimen was processed for paraf  fi n embedding afterremoval of the U-Clips, the other half for Polymethyl-Methacrylate(PMMA) embedding. The paraf  fi n embedded blocks were cut  fi vemicrons thick on slides using a Leica RM 2165 microtome (LeicaMicrosystems, Germany), stained with Harris ’ s Hematoxylin andEosin (H&E) and Van Gieson for histological and morphometric Figure 2.  A diagram showing the formation of side-to-side arteriovenous  fi stulae in the super fi cial femoral artery and vein (A). Photography of the ovine surgery showing  fi stulaecreation with continuous polypropylene suture (B and C) and nitinol U-Clip (D and E). R.L. Varcoe et al. / European Journal of Vascular and Endovascular Surgery 43 (2012) 224 e  231 226  Author's personal copy analysis.ThePMMAembeddedspecimenswerecutintotenmicronthick sections on a Leica SP 1600 saw microtome, acid etched, andstained withbasic fuschinand methyleneblue. The PMMAsamplesallowed sectioning directly through the metallic U-Clips. Histology and morphometric analysis Sections from each block were examined to determine repre-sentative samples of cross section through the midsection of eachanastomosis. These cross sections were then processed to deter-mine intima media area per unit length. Digital images of H&E andVan Gieson stained slides were taken at 1.25, 2, 4, 10 and 20  objective magni fi cations on a BX51 Olympus microscope witha DP2-BSW camera and software (Olympus Corporation, Japan).Olympus DP2-BSW software was used to manually trace a linewhich followed the outer border of the media and a second line totrace the lumen so as to encapsulate both the intima and media(Fig. 3). The intima and media area were calculated together ratherthan attempting to trace the internal elastic lamina that separatesthem as the neo-intima and media were histologically indistin-guishable. The areawithin the fi rst circle was calculated (B), as wastheareawithintheinnercircle(A).TheIntimaMediaArea(IMA)wasthen calculated as B e A, and divided by the luminal circumference(IMA/L) to correct for artefactual exaggeration from obliquesectioningofthespecimenandalsovesseldilatation.Thiscalculatedarea per unit length measurement was used to precisely detect anytrueincrease inneo-intimalmassand discountanydifferencesthatmight occur in the alignment of the blood vessel wall as a result of variation in surgical technique. For each anastomosis a mean valuewas calculated using all representative sections. Statistics All quantitative data were expressed as mean    SD. Two-wayanalysis of variance (ANOVA) was performed to identify thedifference between the two types of suture technique among allsamples,with side and time beingthe variables.A paired  t  -testwasalso used to compare the differences between individual pairIMA/L. To look at time dependency a multiple regression analysiswas performed to identify the differences of individual types of techniques. A sample size of 20 was selected to provide a statisticalpower of 80% ( a  ¼  0.05, two-tail) to detect an estimated 30%difference in the primary endpoint of IMA/L. Data processing andanalysis were accomplished using a computer program (PASWStatistics18,IBMSPSS,Chicago,IL,USA).Avalueof  P  (2sided) < 0.05was considered statistically signi fi cant. Results AllAVFspecimensdemonstratedsubstantialintimalhyperplasiaat the suture line both on macroscopic and histological evaluation.One U-Clip AVF in the 4 week group was occluded with thrombusand therefore excluded from the analysis. Surgical details The mean time taken to complete the anastomosis in thePolypropylene group was 14 min (Range 8 e 18 min). In the U-Clipgroup this was 15.3 min (Range 11 e 23 min). This time taken wasnot signi fi cantly different (  p  ¼  0.47) despite an absence of assis-tant to pre-load the next U-Clip. No technical dif  fi culties wereencountered with either anastomotic technique. The meannumber of U-Clips used in each animal was 20.5 (Range 15 e 26),Table 1. Macroscopic appearance Atthe timeof specimenharvestthe AVFswere encasedindense fi brotic scar tissue. The blood vessels proximal and distal to theanastomosis had preserved tissue planes which facilitatedcompleteremovaloftheAVF.Oncesectioned,theluminalsurfaceatthe anastomotic suture line could be inspected. At each time point Figure 3.  Polypropylene AVF with demarcation of the luminal surface and borderof the media to calculate intima media area. The artery is seen on the right, the vein on the left andthe IH mass at the junction between the two. R.L. Varcoe et al. / European Journal of Vascular and Endovascular Surgery 43 (2012) 224 e  231  227
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