A Reproducible Porcine ePTFE Arterial Bypass Model for Neointimal Hyperplasia

A Reproducible Porcine ePTFE Arterial Bypass Model for Neointimal Hyperplasia
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  A Reproducible Porcine ePTFE Arterial Bypass Model forNeointimal Hyperplasia 1 Muneera R. Kapadia, M.D., Oliver O. Aalami, M.D., Samer F. Najjar, M.D., Qun Jiang, M.D.,Jozef Murar, B.A., Brian Lyle, B.A., Jason W. Eng, Bonnie Kane, B.S., Timothy Carroll, Ph.D.,Patricia M. Cahill, D.V.M., and Melina R. Kibbe, M.D. 2  Division of Vascular Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois Submitted for publication May 29, 2007  Background. Late failure of prosthetic vascular by-pass grafting using expanded polytetrafluoroethylene(ePTFE) is secondary to the development of neointi-mal hyperplasia, most commonly at the distal anasto-mosis. To develop therapies that can improve uponcurrent prosthetic vascular bypass grafting, a largeanimal model of prosthetic bypass grafting that re-sults in reproducible neointimal hyperplasia is nec-essary.  Methods. We performed bilateral end-to-side carotidartery bypasses with 6 mm ePTFE in a porcine model( n   11). We studied graft patency using magneticresonance angiography (MRA, 3 wk), duplex ultra-sonography (4 wk), and digital-subtraction contrastangiography (4 wk). Animals were sacrificed at 4 wkand morphometric analysis was performed.  Results. Of the 11 animals that underwent surgery,one pig died from respiratory compromise; of the re-maining 10, graft patency was 90% at 4 wk. Peak sys-tolic and end diastolic velocities were established forthis model using ultrasonography. MRA, ultrasonogra-phy, and angiography confirmed graft patency andwere complimentary tools to evaluate the grafts. De-velopment of neointimal hyperplasia was reproduc-ible at 4 wk in both the proximal and distal anastomo-ses (2.5 to 3 mm 2 ) of the ePTFE bypass grafts. Conclusion. We developed a reproducible porcineePTFE carotid artery bypass model for studying neo-intimal hyperplasia. Not only does this model allow forthe manipulation and evaluation of potential thera-pies, but patency and neointimal hyperplasia can beeasily evaluated by traditional means, such as MRA,ultrasonography, and angiography. This preclinicalmodel is ideal for evaluation of novel therapies in vivo designed to inhibit neointimal hyperplasia follow-ing arterial reconstruction with prosthetic bypassgrafting.  © 2008 Elsevier Inc. All rights reserved.  Key Words: restenosis; neointimal hyperplasia; por-cine; ePTFE; peripheral arterial disease; bypass. INTRODUCTION Failure of arterial revascularization secondary toneointimal hyperplasia remains a clinical problem as-sociated with significant morbidity and mortality. All vascular procedures result in endothelial cell damage,platelet and leukocyte adherence to the site of injury,and vascular smooth muscle cell proliferation and mi-gration, which result in neointimal hyperplasia andarterial restenosis [1– 4]. In patients requiring vascu- lar bypass grafting with prosthetic material, neointi-mal hyperplasia develops aggressively, especially atthe distal anastomosis. In fact, according to Veith  et al. ,only 54% of below-knee bypass grafts with expandedpolytetrafluoroethylene (ePTFE) are patent at 4 y [5].More recently, Stonebridge  et al. reported 2-y patencyrates for uncuffed femoral to below-knee ePTFE bypassgrafts to be only 29% [6]. The exaggerated neointimal hyperplasia forms secondary to graft thrombogenicitydue to lack of endothelium, low flow states when usedin diameters less then 6 mm, and compliance mis-match between the prosthetic material and the native vessel [7, 8].Because of these significant problems with pros-thetic grafts, researchers have devoted significant timeand resources to developing alternate materials for vascular bypass grafting. Large animal models that 1 Muneera R. Kapadia and Oliver A. Aalami share co-first author-ship. 2 To whom correspondence and reprint requests should be ad-dressed at Division of Vascular Surgery, Northwestern UniversityFeinberg School of Medicine, Galter 10-105, 201 E. Huron Street,Chicago, IL 60611. E-mail: .Journal of Surgical Research 148,  230–237 (2008)doi:10.1016/j.jss.2007.08.003230 0022-4804/08 $34.00 © 2008 Elsevier Inc. All rights reserved.  tend to have similar anatomy and physiology to hu-mans are instrumental, especially when evaluating new potential therapies [9]. For the study of neointimal hyperplasia, rabbit, canine, primate, and porcine mod-els have been described. While each of these modelshas advantages, each also has significant limitations.Therefore, we sought to develop a reproducible largeanimal model of prosthetic arterial bypass grafting toreliably evaluate therapies designed to reduce the de- velopment of neointimal hyperplasia following pros-thetic bypass grafting. We designed and evaluated aporcine carotid artery ePTFE bypass graft model. Thebenefits of our model include reproducibility, simpleexposurewithouttheneedtoenteramajorbodycavity,and the ability for accurate noninvasive monitoring using duplex ultrasonography or magnetic resonanceangiography (MRA). METHODSPresurgical Care and Anesthesia   All animal procedures were performed by use of aseptic techniquein accordance with the Northwestern University Animal Care andUse Committee. Domestic juvenile castrated male Yorkshire-Landrace pigs (Oak Hill Genetics, Ewing, IL) weighing 25 to 30 kg were used. Animals received antibiotic prophylaxis with one dose of cefazolin (25 mg/kg intramuscular [i.m.]) preoperatively and postop-eratively. Aspirin (325 mg oral [p.o.]) was administered daily start-ing 5 d prior to surgery and continued throughout the entire post-operative course. On the day of surgery, the animals also received anaspirin suppository (325 mg).Preop analgesia and sedation included buprenorphine (0.01 mg/kg i.m.), acepromazine (0.15 mg/kg i.m.), ketamine (20 mg/kg i.m.), andatropine (0.05 mg/kg i.m.). After intubation, anesthesia was main-tained with inhaled isoflurane (0.5% to 2.0%) delivered with 100%oxygen. Temperature, heart rate, respiratory rate, and oxygen sat-uration were monitored continuously and recorded every 15 minthroughout the procedure. Surgical Procedure  Animals were placed in the supine position, and their necks wereshaved, then prepped with betadine and alcohol (70%). Pigs receivedbilateral ePTFE bypass grafts ( n    11) (Fig. 1). Through a midline neck incision, both right and left common carotid arteries (CCA)wereexposed.Followingheparin(150U/kgintravenous[i.v.])admin-istration, the right CCA was occluded proximally and in the mid-section with noncrushing vascular clamps. A longitudinal arteriot-omy was made, and a 6 cm length of ePTFE graft (6 mm thin wallstretch ePTFE; Gore, Flagstaff, AZ) was anastomosed in an end-to-side fashion with running 6-0 polypropylene suture. Care was takento ensure that the ePTFE graft was anastomosed at a 45° angle withrespect to the native artery, thereby dictating a standard arteriot-omy length. Prior to completion of the proximal anastomosis, thenative vessel was flushed and irrigated with heparinized saline(2000 U heparin per 1 L normal saline). After completion of the prox-imal anastomosis, flow was restored in the CCA for 5 min while thegraft was clamped near the anastomosis. Next, the right CCA wasoccluded distally and in the mid-section with noncrushing vascularclamps. The distal anastomosis was created in a manner similar tothe proximal anastomosis. Just prior to its completion, the graft andnative artery were vigorously flushed with heparinized saline. Oncethe distal anastomosis was completed, the distal arterial clamp wasremoved, restoring blood flow to the CCA through the ePTFE graft.Sub-adventitial papaverine injections (30 mg/mL) were used to pre- vent arterial spasm, especially in the distal CCA. The mid-section vascular clamp was replaced with double ligation using 2-0 silksuture to simulate an occlusion. After completion of the right bypassgraft, a second dose of heparin (75 U/kg i.v.) was administered, andthe left bypass graft was created in a similar manner. After bothbypass grafts were completed, meticulous hemostasis was achieved,and the incision was closed in multiple layers using absorbablesuture. Animals were monitored until awake, alert, and sternal.Postoperative analgesia consisted of buprenex (0.01 mg/kg i.m.)given every 12 h for the first 48 h postoperatively. Magnetic Resonance Angiography  Magnetic resonance angiography was performed to evaluate graftpatency 3 wk postoperatively. After sedation with acepromazine(0.15 mg/kg), atropine (0.05 mg/kg), and ketamine (20 mg/kg), MRA was performed using a time-resolved T1-weighted gradient echopulse sequence. A time-series of 3D contrast-enhanced images wereacquired with a gadolinium-based contrast agent (0.1 mmol/kg i.v.;Magnevist, Berlex, Princeton, NJ). Noncontrast angiograms werealso performed using a time-of-flight imaging protocol. Duplex Ultrasonography and Angiography  Four weeks postoperatively, just prior to sacrifice, both ultra-sonography and contrast angiography were conducted. Following exposure of both the right and left CCA and bypass grafts, intraop- FIG.1.  Intraoperative photographs of (A) the right ePTFE ca-rotid artery bypass graft and (B) bilateral ePTFE carotid arterybypass grafts. Arrows indicate ePTFE grafts. (Color version of figureis available online.) 231 KAPADIA ET AL.: PORCINE ePTFE BYPASS MODEL FOR NEOINTIMAL HYPERPLASIA   erative duplex ultrasonography was performed, obtaining B-modeimages and Doppler velocity measurements including peak systolic velocity (PSV) and end diastolic velocity (EDV). For each side, mea-surements were obtained at the proximal CCA, proximal anastomo-sis, proximal graft, mid-graft, distal graft, distal anastomosis, anddistal CCA. A significant stenosis was defined as PSV greater thantwo times the normal inflow artery velocity.Next, digital subtraction contrast angiography was performedpercutaneously by accessing the common femoral artery and placing a 5 F introducer sheath. Using a 0.035-in. J-wire, a 5F multisided-hole catheter was advanced into the aortic arch under fluoroscopy. An arch angiogram was obtained with contrast injection (20 mL;Omnipaque, Amersham, Piscataway, NJ) through the catheter. EachCCA was selected using an angled guide catheter and the J-wire, andangiograms were obtained of both CCA and ePTFE grafts using 10ml of contrast media. Following angiogram completion, the animalswere euthanized with pentobarbital (72 mg/kg). Tissue Processing  The ePTFE bypass grafts and native artery extending 2 cm fromeach anastomosis were harvested  enbloc  and underwent  exvivo perfusion-fixation in formalin overnight. Next, the samples weredehydrated with a graded ethanol series, cut into 1 cm sections, andplaced in plastic tissue cassettes (Tissue-Tek, Hatfield, PA). Thesamples were then embedded in paraffin, and blocks were cut into 5  m sections. Tissue Staining and Morphometric Analysis Sections were examined histologically for evidence of neointimalhyperplasia using routine hematoxylin and eosin staining. Digitalimages were collected with light microscopy using an Olympus BHTmicroscope (Melville, NY). Five equally-spaced sections throughouteach proximal and distal anastomoses were analyzed for each bypassgraft (Fig. 2). Neointimal hyperplasia (area in millimeters) under the ePTFE graft was assessed using ImageJ software (National Insti-tutes of Health, Bethesda, MD). To accurately compare the degree of neointimal hyperplasia from each anastomosis, the angle of embed-ding and cutting the block with respect to the angle of the ePTFE tothe native artery was accounted for and controlled so that each blockwas similar, as changes to the block orientation can artificiallyunderestimate or overestimate the degree of neointimal hyperplasia. Statistical Analysis Results are expressed as mean    standard error of the mean.Differences between multiple groups were analyzed by use of one-way analysis of variance with the Student-Newman-Keuls post hoctest for all pair wise comparisons. Differences between two groupswere analyzed with the Student’s t -test (SigmaStat; SPSS, Chicago,IL). Statistical significance was assumed when  P  0.05. RESULTSSurgery and Outcomes Juvenile male Yorkshire-Landrace pigs underwentcarotid artery bypass withePTFEgrafts( n  11)(Fig.1). Early on, the intraoperative acute thrombosis rate was50%. These occurred in the beginning of the series andcaused us to modify our intraoperative anticoagulationregimen. Initially, we were administering only onedose of heparin (100 U/kg i.v.). After experiencing in-traoperative graft thromboses, we began administer-ing two heparin doses; 150 U/kg was administeredbefore the first graft and an additional 75 U/kg prior tothe second graft. This dramatically reduced the occur-rence of intraoperative graft thrombosis for all subse-quent operations. All grafts were patent at the comple-tion of the procedure. One animal developed malignanthyperthermia at the completion of the procedure, how-ever recovered without sequelae. One animal diedimmediately postoperatively secondary to respiratorycompromise.The postoperative course for each animal was un-complicated. No late neurological complications wereobserved. At sacrifice, 10% of the grafts were throm-bosed (2 out of 20). Several animals had minor woundinfections; however, there were no septic complica-tions. FIG.2.  Diagram depicting the five equally-spaced sectionsthroughout the ePTFE-arterial anastomosis that were used for mor-phometric analysis. (Color version of figure is available online.) FIG.3.  Magnetic resonance angiography of the bilateral ePTFEcarotid artery bypass grafts from the (A) anterior-posterior view and(B) right anterior oblique view. Arrows indicate proximal anastomo-ses, and arrowheads indicate distal anastomoses. 232  JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 2, AUGUST 2008  Magnetic Resonance Angiography  MRA was performed 3 wk following the initial pro-cedure to assess graft patency (Fig. 3). Reconstructedimages demonstrated that the ePTFE grafts werepatent. Minimal stenosis was present at either theproximal or distal anastomosis. Ultrasonography  On the day of sacrifice, intraoperative duplex ultra-sonography was performed (Fig. 4). The PSV and EDV  for each segment are shown in Table 1. Note that while there is a more significant variation in the PSV com-pared with the EDV, there is no statistically significantdifference within the PSV or EDV for the differentareas of the graft analyzed. Thus, we have establishedbaseline PSV and EDV in this porcine ePTFE carotidartery bypass model. Contrast Angiography  Following ultrasonography and just prior to sacri-fice, digital subtraction contrast angiography was per-formed (Fig. 5). Angiography is the gold standard when evaluating vessel and graft patency. Results were con-current with the MRA and ultrasonography data. Neointimal Hyperplasia  Both the proximal and distal anastomoses of eachePTFE graft were analyzed and quantified for neointi-mal hyperplasia (Fig. 6 A). The neointimal hyperplasia(Fig. 6B) at the proximal anastomosis was 2.93  0.24mm 2 and the distal anastomosis was 2.64  0.26 mm 2 ;there was no statistically significant difference be-tween these measurements (  P  NS). Importantly, thestandard error for each of these is 10% or less than themean value for either the proximal or distal anastomo-sis, indicating that our model is reproducible. DISCUSSION Neointimal hyperplasia and restenosis following ar-terial bypass grafting with prosthetic material oftenresults in treatment failure and significant morbidityand mortality. Therefore, it is critical to study this FIG.4.  Characteristic ultrasound waveform and B-mode imagesof the (A) proximal anastomosis (PA), (B) mid-graft (MG), and (C)distal anastomosis (DA). (D) Mean peak systolic velocity (PSV) andmean end diastolic velocity (EDV) measurements throughout thegrafts. PArt    proximal artery; PG    proximal graft; DG    distalgraft; DArt    distal artery. (Color version of figure is availableonline.) FIG.5.  Contrast angiography of the right ePTFE carotid arterybypass graft from the right anterior oblique (RAO) view. Arrowindicates proximal anastomosis, and arrowhead indicates distalanastomosis. TABLE1DuplexUltrasonographyVelocityMeasurements SectionPeak systolic velocity (m/s)mean  SEEnd diastolic velocity (m/s)mean  SEProximal artery (PArt) 0.583  0.178 0.128  0.064Proximal anastomosis (PA) 0.677  0.285 0.135  0.047Proximal graft (PG) 0.548  0.207 0.103  0.044Mid graft (MG) 0.444  0.158 0.078  0.032Distal graft (DG) 0.515  0.192 0.107  0.053Distal anastomosis (DA) 0.590  0.200 0.095  0.028Distal artery (DArt) 0.732  0.232 0.114  0.049  Note.P  NS for all sections within each group. 233 KAPADIA ET AL.: PORCINE ePTFE BYPASS MODEL FOR NEOINTIMAL HYPERPLASIA   problem and important to have animal models thatallow us to do so. In this manuscript, we present aporcine carotid artery ePTFE bypass graft model forthe study of neointimal hyperplasia. This model simu-lates conditions seen in human arterial bypass proce-dures with prosthetic material. It is reproducible asevidenced by the consistent development of neointimalhyperplasia seen in all of our pigs. The surgical expo-sure is simple and avoids violation of any major bodycavities. The carotid arteries and bypass grafts areeasily evaluated with MRA, duplex ultrasonography,and digital subtraction contrast angiography. Thismodel is ideally suited to study novel therapies aimedat reducing neointimal hyperplasia following arterialbypass grafting procedures with prosthetic materials. Animal models have been instrumental in the studyof neointimal hyperplasia. Small animal (e.g., mouseand rat) and rabbit models are inexpensive and providea good initial invivo  environment to examine potentialtherapies. However, to determine whether a therapywill be effective in patients, it should be evaluated in alarge animal model. Large animal models more closelyresemble human physiology and anatomy, and aremost predictive of results in humans [9]. This point has been illustrated recently with the E2F decoy trials.Initially, the E2F decoy therapy, an oligodeoxynucle-otide competitive inhibitor of the E2F transcriptionfactor that results in cell-cycle blockade was shown tobe quite effective in rat and rabbit models of arterialinjury and vein bypass grafting  [10, 11]. Before addi- tional large animal data were collected, this therapywas initiated in patients with the PREVENT trial,demonstrating safety and feasibility [12]. Unfortu- nately, in the PREVENT III and IV trials, no signifi-cant improvement in outcome was observed in patientsundergoing lower extremity arterial reconstruction orcoronary artery bypass grafting, respectively [13, 14].This example emphasizes the importance of evaluating potential therapies in more clinically relevant animalmodels prior to administration to patients.Many investigators have used nonhuman primate,canine, and porcine models to study therapies for theinhibition of neointimal hyperplasia following arterialinterventions; however, each of these species has itsown unique advantages and disadvantages. Nonhu-man primates are most anatomically and physiologi-cally similar to humans, but are often cost prohibitive. Additionally, they may not offer sufficiently better re-sults than other large animal models to justify theiruse. Dogs are less costly and easier to work with thannonhuman primates, but their unpredictable hyperco-agulability and potent fibrinolytic system can be prob-lematic in vascular research [15]. Porcine models, while hypercoagulable, are less expensive and do notpresent the ethical dilemmas associated with non-human primates [15]. Furthermore, when compared with canine models, porcine models of arterial injuryhave been shown to be more predictive of results inhumans [9, 16]. Taking these considerations into ac- count, we chose to focus on porcine models.Currently described porcine restenosis models withprosthetic materials include variations of arterio- venous and arterio-arterial bypass grafts [17–23]. The arterio-venous bypass graft represents a differentpathophysiologic process that results in neointimal hy-perplasia when compared to arterial reconstruction[24]. Hemodynamic forces are thought to play an im-portant role in this differential response. Arterial by-pass grafts are pulsatile; combined with compliancemismatch, particularly with ePTFE grafts, neointimalhyperplasia typically forms at the heel, toe, and a shortsegment of the floor of the distal anastomosis [24]. Incontrast, arterio-venous fistulas have blood flows 5 to10 times higher than arterial bypass grafts and, as a FIG.6.  (A) Representative hematoxylin and eosin-stained cross-sections from the proximal artery, proximal anastomosis (PA), distalanastomosis (DA), and distal artery. (B) Morphometric analysis of PA and DA conducted on five equally-spaced sections throughouteach anastomosis ( n  10 pigs). NS  not significant. (Color versionof figure is available online.) 234  JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 2, AUGUST 2008
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