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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Description of two different aneurysmal swine models for neuroradiology training courses and research studies. This study provides evidence of the feasibility of these aneurysm porcine model creations and the reproducible methods that are close to the clinical setting.

Abstract

Large animal models, specifically swine, are widely used to research cardiovascular diseases and therapies, as well as for training purposes. This paper describes two different aneurysmal swine models that may help researchers to study new therapies for aneurysmal diseases. These aneurysmal models are created by surgically adding a pouch of tissue to carotid arteries in swine. When the model is used for research, the pouch must be autologous; for training purposes, a synthetic pouch suffices.

First, the right external jugular vein (EJV) and right common carotid artery (CCA) must be surgically exposed. The EJV is ligated and a vein pouch fashioned from a short segment. This pouch is then sutured to an elliptical arteriotomy performed in the CCA. Animals must be kept heparinized during model creation, and local vasodilators may be used to decrease vasospasms. Once the suture is completed, correct blood flow should be inspected, checking for bleeding from the suture line and vessel patency. Finally, the surgical incision is closed by layers and an angiography performed to image the aneurysmal model.

A simplification of this aneurysmal carotid model that decreases invasiveness and surgical time is the use of a synthetic, rather than venous, pouch. For this purpose, a pouch is tailored in advance with a segment of a polytetrafluoroethylene (PTFE) prosthesis, one end of which is sutured close using polypropylene vascular suture and sterilized prior to surgery. This "sac" is then attached to an arteriotomy performed in the CCA as described.

Although these models do not reproduce many of the physiopathological events related to aneurysm formation, they are hemodynamically similar to the situation found in the clinical setting. Therefore, they can be used for research or training purposes, allowing physicians to learn and practice different endovascular techniques in animal models that are close to the human system.

Introduction

Intracranial aneurysm (IA) is a severe cerebrovascular disease associated with up to a 50% mortality rate when ruptured. It is a relatively common and potentially lethal condition, with a reported prevalence between 3.6% and 6% in angiographic studies1. The intracranial vessels are abnormally dilated and suffer distension due to multifactorial risk factors, including, but not limited to, smoking, hypertension, excessive alcohol intake, or increasing age. When left untreated, IA can spontaneously rupture, resulting in subarachnoid hemorrhage (SAH) that is responsible for significant morbidity and death2,3,4. Additionally, one third of patients require hospitalization or nursing care, and only 30% of patients with SAH can return to independent living, thus representing a serious disease burden in humans that actually justifies the need for animal experiments5.

Nowadays, patients with high risk of IA rupture and hemorrhage are treated with occlusion mainly by endovascular coiling, microsurgical clipping, or flow diverting stents6,7. The endovascular procedure has been evaluated by the International Subarachnoid Aneurysm Trial (ISAT), demonstrating that coiling is safer, less invasive, and therefore has less significant adverse effects than microsurgical therapy3. For these reasons, the endovascular procedures are the most common techniques used for IA treatment3. Specialized training is required for physicians to perform these minimally invasive procedures correctly8.

Moreover, the development of new devices or therapies for IA treatment needs to be well-established and tested in preclinical studies before their translation to the clinical setting6,9. There are different IA experimental animal models according to the main objective of the research or training purposes. These models have been performed in numerous species, with their limitations and advantages. However, all of them entail artificial induction or surgical creation due to the absence of natural IA in animals2,6,9,10,11,12.

Although no animal model perfectly reproduces the human pathophysiology, small animals, such as rodents, are the most frequently used in IA research studies6. Large species are usually employed for the development of new endovascular devices or training in therapeutic interventions2. Among large animal models, it is common to use swine to research IA disorders and therapies, as well as for training courses. This is because of their ability to tolerate the surgical procedure and their similar vascular diameter and blood flow when compared to human cerebral vessels2,13.

The method of choice for IA animal model creation varies depending on the main objective of each individual research project, such as whether angiographic or histologic endpoints will be evaluated. In this sense, models created by surgical ligation or by adding an autologous pouch of tissue to the CCA are used for IA growth research. Surgical models must be combined with hypertension induction if the primary endpoint of the study is IA rupture. When the model is used for training purposes, the technique can be simplified by using a synthetic pouch sutured onto the CCA without the need for hypertension6.

This paper describes two different aneurysmal swine models that may help researchers to study new therapies or training in endovascular interventions for IA diseases. These aneurysmal models are created by surgically adding a pouch of tissue to the CCA in swine. When the model is used for research, the pouch is autologous, thus providing the ability to study healing of the aneurysm after exclusion without the interference of any exogenous material. For training purposes, a synthetic pouch that recapitulates the endovascular anatomy to reproduce the procedure suffices.

Protocol

The experiment was approved by the ethical committee of the Jesús Usón Minimally Invasive Surgery Centre, and all procedures were performed according to Spanish Royal Decree 53/2013 and the European regulation (2010/63/EC).

1. Presurgical preparation and anesthesia

  1. House large white swine weighing 35-40 kg individually, with free access to water and feed once a day. Acclimate for 2 weeks before the date of the intervention, to perform clinical examination and allow time for detecting silent diseases.
  2. Administer the following oral drugs to prevent thrombotic events during the study: Acetylsalicylic acid (1 g/animal/24 h) and Clopidogrel (75 mg/animal/24 h) from 7 days before model induction until IA therapy.
  3. Inject ketamine (10 mg/kg) intramuscularly after a 24 h fasting period. Ten min later, induce anesthesia by 1% propofol intravenously (3 mg/kg).
  4. After endotracheal intubation, use inhaled sevoflurane to maintain anesthesia (3%-4.5% inspiratory fraction). Connect endotracheal tubes to a semiclosed, circular anesthetic circuit attached to a ventilator with a fresh gas flow rate of 0.5-1 L/min.
  5. To obtain normocapnia (35-45 mmHg CO2), control ventilation by a tidal volume of 8-10 mL/kg. Ensure an adequate intraoperative analgesia by an initial intravenous dose of ketorolac (1 mg/kg) and tramadol (1 mg/kg) combination and a continuous remifentanil infusion (15-18 µg/kg/h).
  6. Confirm proper animal plane of anesthesia considering unconsciousness (hypnosis), insensitivity to pain, muscle relaxation, and the absence of reflex responses14.
  7. To prevent dryness while under anesthesia, use a vet ointment on eyes during the surgery procedure.
  8. Fix the anesthetized animals at the operating table in supine position. Shave the animals´ necks, scrub with povidone-iodine, and drape under sterile conditions.
  9. Perform all procedures under sterile conditions, using sterile gloves and material.

2. Surgery

  1. Surgical approach
    1. Perform a 10 cm long longitudinal skin incision 2-3 cm to the right of the neck´s midline.
  2. Pouch tailoring
    1. Autologous pouch
      1. To expose the EJV, dissect the subcutaneous and fat tissue and perform hemostasis.
      2. Separate the right sternocephalicus muscle from the connective tissue and retract it with a Weitlaner retractor to facilitate EJV exposure.
      3. Expose and identify the EJV, which is lateral and deeper than the CCA (Figure 1A).
      4. Use two bulldog vascular clamps to stop blood flow inside the vessel during the venous segment extraction.
      5. Isolate a 15-20 mm segment of the EJV to obtain the autologous pouch.
      6. Ligate the proximal and distal ends of the EJV.
      7. Flush the extracted vein segment with heparinized saline (5,000 IU/L).
      8. Check the inside of the excised EJV and select a 7-8 mm long segment where no venous valves are present.
      9. Fashion this segment into a pouch by closing one end with a 7/0 polypropylene running suture (Figure 1B).
      10. Keep the pouch immersed in heparinized saline until use.
      11. Confirm no bleeding from the ligated EJV.
    2. Synthetic pouch
      1. Cut a 1 cm long segment from a PTFE prosthesis (6-8 mm in diameter, depending on the aneurysmal size that is going to be created).
      2. Suture one end close using a 6/0 polypropylene vascular suture. Seal this closed part of the prosthesis with vascular glue to avoid bleeding from the suture line.
      3. Sterilize this synthetic graft prior to surgical aneurysm creation.
  3. Aneurysm creation surgery
    1. To expose the CCA, dissect the subcutaneous and fat tissue and perform hemostasis.
    2. Separate the right sternocephalicus muscle from the surrounding connective tissue and retract it with a Weitlaner retractor to facilitate CCA exposure.
    3. Identify the CCA. Place 2 silicon vessel loops at the cranial and distal end of the exposed CCA (Figure 2A). Dissect 5 cm of this vessel, removing its adventitia with a dissector (Figure 2B). During this surgical access, take care to avoid injuring the vagus nerve that can lead to Horner´s syndrome.
    4. Administer vasodilators locally (such as 1-2 mL of nimodipine 10 mg/50 mL) to prevent vasospasms.
    5. Administer heparin intravenously (150 IU/kg) 5 min before CCA crossclamping.
    6. Place one bulldog vascular clamp at the caudal dissected part of the CCA and another bulldog vascular clamp 4-5 cm apart at the cranial part of the vessel (Figure 2C).
    7. Use microscissors to perform an 8 mm elliptical arteriotomy in the CCA between the two bulldog vascular clamps (Figure 2D).
    8. Use heparinized saline solution (5,000 IU/L) to flush the segment of the CCA intraluminally.
    9. Suture the autologous or synthetic pouch to the elliptical arteriotomy using a 6/0 polypropylene running suture (Figure 3A,B). Flush the distal and proximal clamps before finishing the pouch suture.
    10. Protect the vessel and nearby structures with warm, heparinized saline solution during the microsurgical procedure.
    11. Once the prosthesis or autologous pouch is sutured to the CCA, check there is no bleeding (Figure 3C,D). First, remove the cranial bulldog vascular clamp, and then the caudal one.
    12. Perform hemostasis by applying pressure with wet swabs if there is some bleeding from the suture line. If required, apply traction to the vessel loops, or replace the bulldog vascular clamps and perform hemostatic stitches at the bleeding site. If necessary, place a piece of hemostatic gelatin sponge around the prosthesis.
    13. Inspect the CCA pulse cranial to the aneurysmal sac to ensure that correct carotid patency has been recovered.
    14. Close the surgical incision by layers using 2/0 absorbable sutures and the skin with single stitches with 0 nonabsorbable sutures.
    15. Administer buprenorphine (10 µg/kg/12 h) intramuscularly during the first 24 h and place a fentanyl transdermic release patch (25 µg/h) after the surgical procedures to achieve postoperative analgesia.
    16. Decrease the inhaled sevoflurane and increase fresh gas flow rate (20 L/min) to obtain recovery conditions. Remove the endotracheal tube when the animals breathe spontaneously and physiological parameters have been recovered, such as oxygen saturation and heart rate.
      NOTE: The fresh gas is a mixture of pure O2 (100%) and medical air (21% O2). Finally, the fraction of inspired oxygen is 50% to 45% (FiO2 = 0.5-0.45).
    17. Do not leave the animals unattended until they have regained sufficient consciousness to maintain sternal recumbence.
    18. Keep the animals that have undergone surgery isolated from other animals until fully recovered.

3. Angiography test and postoperative phase

  1. Wait for 24-48 h to avoid damaging the suture line.
  2. Anesthetize the animal again as described above and shave the groin area. Prepare the zone with povidone-iodine and apply sterile draping.
  3. Access a femoral artery using the modified Seldinger technique with a 6Fr introducer sheath.
  4. Insert a 5Fr headhunter catheter through the femoral sheath over a 0.035 in hydrophilic guidewire. Under fluoroscopic guidance, advance this catheter to the origin of the CCA and remove the guidewire.
  5. Inject contrast medium (amidotrizoic acid diluted to 50% with a saline solution) through the headhunter catheter to image the CCA with aneurysm model.
  6. Once the angiography confirms correct aneurysm model creation, use animal models for research or for training endovascular treatment procedures.
  7. Euthanize the animals when the studies or training courses finish with a lethal intravenous administration of potassium chloride (2 mmol/kg) while under deep anesthesia.

Results

The presented technique has been used for different purposes, namely research into postcoiling aneurysm healing and training in embolization techniques. Venous pouches have been used for testing differential healing using both platinum and bioactive coils. The pouches were sutured as described above and, 24 h after model creation, an angiogram was obtained to document the dimensions and appearance of the aneurysms. Endovascular coil embolization was performed successfully in all the pigs. In each case, the left-side aneu...

Discussion

There are different techniques to create aneurysm animal models based on the objective of the study. Some aneurysm model protocols include surgical procedures combined with hypertension or hemodynamic stress induction by angiotensin II administration, nephrectomies, or high-salt diet, among others, because the main objective of these studies is aneurysm rupture research. However, in the present study, these conditions are not induced since these animal models are used for neuroradiology training or nonrupture aneurysm re...

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

The study was performed by the ICTS 'NANBIOSIS', more specifically by U-21 (Experimental Operating Rooms), U-22 (Animal Housing), and U-24 (Medical imaging) of the Jesús Usón Minimally Invasive Surgery Centre (JUMISC). This work was funded by the Instituto de Salud Carlos III (CB16/11/00494) and the Consejeria de Economía, Ciencia y Agenda Digital, Junta de Extremadura (GR21201), cofunded by the European Regional Development Fund "A way to make Europe". The authors acknowledge all the work performed by the animal housing, experimental technicians, and Joaquín González for taking photos of the surgical procedure.

Materials

NameCompanyCatalog NumberComments
Acetylsalicylic acidSanofi700693500 mg tablets
Amidotrizoic acidBayer Hispania914614.6Contrast medium 76%
Anesthesia MachineMaquet Clinical Care AB6677200Maquet Flow-i C20
Bulldog vascular clampDimeda12.092.077.5 cm
BuprenorphineRichter Pharma Ag5788160.3 mg/mL
ClopidogrelSandoz70400575 mg tablets
Contrast mediumBayer Hispania914614Urografin 36%
DissectorDimeda12.421.0121 cm
Fentanyl MatrixKern Pharma664823Transdermic release patch 25 µg/h
Fluoroscopy equipmentPhilips Medical SystemsVeradius Unity
Hemostatic gelatin spongeTakeda Farmaceutica España, SA324459Absorbable hemostatic agent. Espongostan
Head hunter catheterBoston ScientificRF*YB15110M5 Fr 100 cm
HeparinRovi641639Heparin 5%
Hydrophilic guidewireTerumoRF*GA35153M0.035” 150 cm
Introducer sheathTerumoRS*B60N10MQ6 Fr 10 cm
KetamineRichter Pharma Ag580395100 mg/mL
KetorolacLaboratorios Normon, S.A.60307930 mg/mL
Micro-forcepsS&TJFA-5b (1:1)Forceps for microsugery
Micro-needle holderS&TCurved C-14 (Art nº 00088)Needle holder for microsurgery
MicroscissorsS&TAdventitia SAS-15 R-8 (Art nº 00102)Straight- scissors for microsurgery
Needle holderDimeda24.114.1212 cm
NimodipineBayer Hispania, S.L64196910 mg/50 mL
Povidone-iodineCV Medica193203Povidone iodine solution (10%)
PropofolOrion Corporation58847510 mg/mL
PTFE prosthesisMaquetM00201501086B0Synthetic prosthesis 6mm
RemifentanilLaboratorios Normon, S.A.6922952 mg
Scalpel handleDimeda06.104.0013.5 cm
Scissors (Mayo)Dimeda07.164.1414.5 cm
Scissors  (Metzenbaum)Dimeda07.287.1515 cm
Surgical bladesDimeda06.122.0022
Sutures: absorbable sutureMedtronicGL-1232/0
Sutures: poplypropylene sutureAragó378036/0 and 7/0
SwabsTexpol1063.0120 x 20 cm
Tissue forcepsDimeda10.102.11 /10.120.1111.5 cm
Vascular glueHistoacryl Braun1050060Tissue adhesive
Vessel loopsBraunB10952181.5 mm diammeter
WeitlanerDimeda18.670.1414 cm

References

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