Swine Models of Aneurysmal Diseases for Training and Research

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 death^2,3,4. 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 stents^6,7. 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 setting^6,9. 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 animals^{2,6,9,10,11,12}.

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 vessels^2,13.

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetylsalicylic acid	Sanofi	700693	500 mg tablets
Amidotrizoic acid	Bayer Hispania	914614.6	Contrast medium 76%
Anesthesia Machine	Maquet Clinical Care AB	6677200	Maquet Flow-i C20
Bulldog vascular clamp	Dimeda	12.092.07	7.5 cm
Buprenorphine	Richter Pharma Ag	578816	0.3 mg/mL
Clopidogrel	Sandoz	704005	75 mg tablets
Contrast medium	Bayer Hispania	914614	Urografin 36%
Dissector	Dimeda	12.421.01	21 cm
Fentanyl Matrix	Kern Pharma	664823	Transdermic release patch 25 µg/h
Fluoroscopy equipment	Philips Medical Systems		Veradius Unity
Hemostatic gelatin sponge	Takeda Farmaceutica España, SA	324459	Absorbable hemostatic agent. Espongostan
Head hunter catheter	Boston Scientific	RF*YB15110M	5 Fr 100 cm
Heparin	Rovi	641639	Heparin 5%
Hydrophilic guidewire	Terumo	RF*GA35153M	0.035” 150 cm
Introducer sheath	Terumo	RS*B60N10MQ	6 Fr 10 cm
Ketamine	Richter Pharma Ag	580395	100 mg/mL
Ketorolac	Laboratorios Normon, S.A.	603079	30 mg/mL
Micro-forceps	S&T	JFA-5b (1:1)	Forceps for microsugery
Micro-needle holder	S&T	Curved C-14 (Art nº 00088)	Needle holder for microsurgery
Microscissors	S&T	Adventitia SAS-15 R-8 (Art nº 00102)	Straight- scissors for microsurgery
Needle holder	Dimeda	24.114.12	12 cm
Nimodipine	Bayer Hispania, S.L	641969	10 mg/50 mL
Povidone-iodine	CV Medica	193203	Povidone iodine solution (10%)
Propofol	Orion Corporation	588475	10 mg/mL
PTFE prosthesis	Maquet	M00201501086B0	Synthetic prosthesis 6mm
Remifentanil	Laboratorios Normon, S.A.	692295	2 mg
Scalpel handle	Dimeda	06.104.00	13.5 cm
Scissors (Mayo)	Dimeda	07.164.14	14.5 cm
Scissors  (Metzenbaum)	Dimeda	07.287.15	15 cm
Surgical blades	Dimeda	06.122.00	22
Sutures: absorbable suture	Medtronic	GL-123	2/0
Sutures: poplypropylene suture	Aragó	37803	6/0 and 7/0
Swabs	Texpol	1063.01	20 x 20 cm
Tissue forceps	Dimeda	10.102.11 /10.120.11	11.5 cm
Vascular glue	Histoacryl Braun	1050060	Tissue adhesive
Vessel loops	Braun	B1095218	1.5 mm diammeter
Weitlaner	Dimeda	18.670.14	14 cm