Creation of Two Saccular Elastase-Digested Aneurysms with Different Hemodynamics in One Rabbit

Summary

This protocol describes the steps for the creation of a rabbit model with two elastase-digested aneurysms with different hemodynamics (stump and bifurcation constellation). This allows the testing of novel endovascular devices in aneurysms with different angioarchitecture and hemodynamic conditions within a single animal.

Abstract

Preclinical animal models with hemodynamic, morphologic, and histologic characteristics close to human intracranial aneurysms play a key role in the understanding of the pathophysiological processes and the development and testing of new therapeutic strategies. This study aims to describe a new rabbit aneurysm model that allows the creation of two elastase-digested saccular aneurysms with different hemodynamic conditions within the same animal.

Five female New Zealand white rabbits with a mean weight of 4.0 (± 0.3) kg and mean age of 25 (±5) weeks underwent microsurgical stump and bifurcation aneurysm creation. One aneurysm (stump) was created by right common carotid artery (CCA) exposure at its origin at the brachiocephalic trunk. A temporary clip was applied at the CCA origin and another, 2 cm above. This segment was treated with a local injection of 100 U of elastase for 20 min. A second aneurysm (bifurcation) was created by suturing an elastase-treated arterial pouch into the end-to-side anastomosis of the right CCA to left CCA. Patency was controlled by fluorescence angiography immediately after creation.

The average duration of surgery was 221 min. The creation of two aneurysms in the same animal was successful in all rabbits without complication. All aneurysms were patent immediately after surgery except for one bifurcation aneurysm, which showed an extreme tissue reaction due to elastase incubation and an immediate intraluminal thrombosis. No mortality was observed during surgery and up to one-month follow-up. Morbidity was limited to a transient vestibular syndrome (one rabbit), which recovered spontaneously within one day.

Demonstrated here for the first time is the feasibility of creating a two-aneurysm rabbit model with stump and bifurcation hemodynamic characteristics and highly degenerated wall conditions. This model allows the study of the natural course and potential treatment strategies on the basis of aneurysm biology under different flow conditions.

Introduction

Intracranial aneurysm is a severe condition with a mortality rate after rupture reaching 50% and long-term disability in 10-20% of patients¹. The last decade has seen a rapid development of endovascular treatment options but, at the same time, also an increasing rate of recurrence with up to 33% of aneurysm recanalization after coiling^2,3. To better understand the pathophysiology underlying aneurysm occlusion and recanalization, as well as for the development and testing of new endovascular devices, there is currently a need for reliable preclinical models whose hemodynamic, morphological, and histologic characteristics mimic those of human intracranial aneurysms^4,5,6. As of today, there is no defined model as a standard for preclinical trial, and a large range of species and techniques are available to researchers^7,8.

However, the rabbit is a species of particular interest due to the size and hemodynamic similarities between its neck arteries and the human cerebral vessels, as well as its similar coagulation and thrombolysis profiles. Several models with elastase-digested saccular aneurysms on the common carotid arteries (CCAs) have shown qualitative and quantitative similarities with human intracranial aneurysms in terms of flow conditions, geometric features, and wall characteristics^9,10,11,12. This study aims to describe a technique to create a new rabbit aneurysm model with both stump and bifurcation elastase-digested aneurysms in the same animal. The surgical techniques are inspired by those of Hoh et al.¹³ and Wanderer et al.¹⁴ with slight modifications to provide a good standardization and reproducibility and to ensure low mortality and morbidity.

Protocol

NOTE: The experiment was approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (Application Number BE108/16), and all animal care and procedures were performed in accordance with institutional guidelines and 3R principles^15,16. Data are reported according the ARRIVE guidelines. Peri-operative management was conducted by a board-certified veterinarian anesthesiologist. For the study, female New Zealand white rabbits, with a mean weight of 4.0 (± 0.3) kg and mean age of 25 (±5) weeks, were housed at a room temperature of 22-24 °C with a 12-h light/dark cycle with free access to water, pellets, and hay.

1. Pre-surgical phase and anesthesia

1. Perform a clinical examination as recommended by the Association of veterinary Anesthetists and the European and American College of Veterinary Anesthesia and analgesia to confirm that the rabbits are healthy by weighing each animal, evaluating the mucous membrane, documenting the capillary refill time and pulse quality, and performing a pulmonary and cardiac auscultation as well as an abdominal palpation.
2. Based on the clinical finding, attribute an American Society of Anesthesiologists (ASA) classification to each rabbit¹⁷. Perform surgery only on animals with an ASA I score.
3. Shave both outer ears, and apply prilocaine-lidocaine cream on auricular arteries and veins. Achieve deep sedation with a combination of ketamine 20 mg/kg, dexmedetomidine 0.1 mg/kg, and methadone 0.3 mg/kg injected subcutaneously (SC). Leave the animals undisturbed for 15 min. Give supplementary oxygenation (3 L/min) through a loosened face mask, and monitor with a pulse oximeter.
4. Place a 22 G cannula in the left auricular central artery as well as in an auricular vein. Induce general anesthesia with propofol 1-2 mg/kg intravenous (IV) until effect (loss of swallowing reflex). Proceed with endo-tracheal intubation via a silicone tube (3 mm internal diameter).
5. Shave the forehead to place the pediatric electroencephalographic (EEG) sensors. Shave the surgical field, and inject ropivacaine hydrochloride 0.75 % intradermally.
6. Place the rabbit on the operation table in dorsal recumbency, install full monitoring, and connect the endo-tracheal tube to a low-resistance pediatric circle system. Maintain anesthesia with administration of isoflurane in oxygen, targeting a maximal end tidal (Et) concentration of 1.3 %.
7. Provide a continuous infusion of ringer lactate 5 mL/kg/h through the venous access. Ensure clinical and instrumental monitoring until extubation by means of pulse oximetry, doppler and invasive blood pressure, 3-lead electrocardiogram, EEG, rectal temperature, and inhaled and exhaled gases.
8. Disinfect the surgical field with povidone iodine from the manubrium sterni to jaw angles, and apply the sterile draping. During surgery, provide analgesia with lidocaine (constant rate infusion (CRI) of 50 μg/kg/min) and fentanyl (CRI of 3-10 μg/kg/h). Perform spontaneous or assisted ventilation. Allow permissive hypercapnia.
9. Perform at least one arterial blood gas analysis during surgery. In case of hypotension (mean arterial pressure below 60 mmHg), treat it with noradrenaline, titrated until effect. Use a heating pad or a heating forced-air warming system to prevent hypothermia (aim: rectal temperature 37.5-38.5 °C).
   ​NOTE: As the invasive arterial blood pressure is measured at the left ear artery, the clipping of the left CCA will stop the blood flow and suppress the curve. The blood pressure has then to be measured with Doppler technique until reopening of the vessel.

2. Surgery

1. Approach
   1. Make a median skin incision from the hyoid bone until a point 1.5 cm caudal to the manubrium sterni with a scalpel. Prepare the subcutaneous and fat tissue from the medial incision while performing meticulous hemostasis.
   2. Free the sternocephalicus muscle from the adherent connective tissue, and apply lidocaine topically (2-4 mg/kg, prefer lidocaine 1%) to avoid myoclonus. Expose the right CCA medially of the sternocephalicus muscle and keep it wet with wet swabs.
   3. Now prepare the lateral and proximal parts of the sternocephalicus muscle and retract it medially with a vessel loop to expose the CCA. Identify the external jugular vein and protect it with a wet micro swab.
   4. Dissect the connective tissue carefully along the proximal CCA until the bifurcation of the brachiocephalic trunk to expose the artery. In the presence of small branches coming from the artery, coagulate them with the cauterizer.
      NOTE: Take care to avoid any nerve damage.
2. Stump aneurysm creation and tissue harvesting for the bifurcation aneurysm
   1. Before clipping the right CCA, measure the anti-clotting time (ACT), and give natrium heparin (80 EI/kg) systemically via the ear vein (performed by the anesthesia team) to avoid thromboembolic events.
   2. Now apply 2 temporary clips: the first one at the origin of the CCA and the second one 2 cm distal from it (Figure 1A). Place a rubber pad under the vessel and rinse with papaverine HCL (40 mg/ mL; 1:1 dissolved in 0.9% saline) for vasodilatation.
   3. Remove the adventitia carefully using microscissors. Perform an arteriotomy below the distal clip with a 22 G IV-catheter, and insert the catheter caudally up to the proximal clip (Figure 1A,B).
   4. Flush the segment intraluminally with heparinized NaCl (500 U/100 mL in 0.9% saline) until there is no blood visible, and finally fix the catheter with a ligature (4-0). Now, through the catheter, inject 0.1-0.2 mL of elastase (100 IU previously dissolved in 5 mL of Tris-Buffer) into the artery segment and incubate for 20 min (Figure 1B).
   5. Start with the dissection on the left side to expose the left CCA (see section 2.3). After 20 min of incubation time with elastase, clear the elastase solution, and change the syringe to rinse the artery segment about 10 times with 0.9% NaCl.
   6. Apply 2 ligatures (6-0): the first one 5 mm distal of proximal clip and the second just proximally, under the arteriotomy (Figure 1C). Cut the vessel ~3 mm above the first ligature and one more time between the second ligature and the distal clip. Keep this autologous graft in a heparinized solution (500 U/100 mL in 0.9% saline) until the creation of the bifurcation aneurysm (Figure 1D). Finally, carefully open the first proximal clip, and measure the aneurysm (length, width, and depth).
3. Bifurcation aneurysm creation
   1. Prepare the left side by dissecting the sternocephalicus muscle medially to expose ~2 cm of the left CCA. Apply lidocaine topically on the muscle to avoid myoclonus.
   2. Underlay the carotid artery with a gauze ball and a small swab with a piece of glove. Apply some papaverine.HCl topically (40 mg/mL; 1:1 dissolved in 0.9% saline). Continue to work under microscopic view: prepare the aneurysm pouch and remove the adventitia. Measure the aneurysm pouch (length, width, depth).
   3. Flush the open part of the right CCA with heparinized NaCl and if needed, replace the clip to have ~1 cm to allow free manipulations for the suture. Remove the adventitia carefully, and make a ~2 mm longitudinal incision laterally in the stump of the right CCA.
   4. Now apply two temporary clips on the left CCA to delimit a segment of ~1 cm and remove the adventitia in between. Perform an arteriotomy with a 23 G needle. Flush the segment with heparinized NaCl (500 U/100 mL in 0.9% saline). Enlarge the arteriotomy using microscissors to ~4-5 mm to allow the suturing of the right CCA and the aneurysm pouch (Figure 1E). Irrigate the vessels during the whole suturing procedure and protect them with wet micro swaps.
   5. Perform the anastomosis with 9-0 non resorbable suture.
      1. Suture the proximal back wall of the right carotid blunt with 5 stitches, starting at the proximal edge of the arteriotomy on the left CCA. Then, suture the backside of the aneurysm pouch with 4-5 stitches, starting at the distal edge of the arteriotomy on the left CCA.
      2. Continue with the distal backside at the level of the fish mouth incision to suture with the vertical backside of the aneurysm graft with 3 stitches. Suture the front side of the fish mouth incision with 3 stitches, starting upwards and moving downwards.
      3. Finish with the front suture between the left CCA and front side of the aneurysm graft and right CCA with ~6 stitches. Before finishing the anastomosis, rinse the vessels with heparinized 0.9% saline solution intraluminally.
   6. Before removing the clamp, measure the anti-clotting time (ACT) one more time, and administer an adapted dose of heparin systemically (target: 2-3 times baseline ACT).
   7. Remove the clip on the right CCA while putting some pressure on the anastomosis with micro swabs for hemostasis. Then, continue by removing the distal clip from the left CCA. If there is no major bleeding, continue with taking out the proximal clip on the left CCA, to allow blood flow. If there is some bleeding from the anastomose, apply some pressure with the gauze ball and swab; wait for a couple of minutes. If it persists, replace the clips and perform re-stitches.
      NOTE: A blood loss of more than 20-30 mL can endanger the recovery phase.
4. Patency control and documentation
   1. After opening all the vessels, document the results photographically and measure them (Figure 1F and Figure 2A,B).
   2. Confirm the restoration of the flow in the distal CCA through the invasive arterial blood pressure curve (measured at the ear artery, a direct branch of the external carotid), which should also return to normal.
   3. Perform fluorescence angiography by administering 1 mL of fluorescein IV, using 2 bandpass filters, a video camera, and a bicycle spotlight. See previous publications for the description of the whole procedure^18,19.
5. Closure
   1. Readapt the fat pad on the anastomosis and suture it with a 4-0 resorbable suture. Finally suture subcutis and skin with single stitches using 4-0 resorbable suture.

3. Postsurgical phase

1. At the end of the surgery, discontinue isoflurane and systemic analgesia without reversion in order to maintain the analgesic effect. Ensure that control of the swallowing reflex has returned before performing tracheal extubation.
2. Administer meloxicam 0.5 mg/kg IV to ensure analgesia, aspirin (ASS) 10 mg/kg IV to prevent immediate thrombotic events, vitamin B12 100 μg SC and clamoxyl 20 mg/kg IV as antibiotic prophylaxis.
3. Provide supplementary oxygenation and warming until the rabbit spontaneously regains the sternal recumbency. Perform rescue analgesia with methadone if any sign of pain is observed. Perform postoperative follow-up and care 4 times a day for the first 3 preoperative days, in accordance with the guidelines for the assessment and management of pain in rodents and rabbits^23,24.
4. Ensure postoperative analgesia with fentanyl patch (12 μg/h) applied on the outer ear, meloxicam 1x/ SC for 3 days, and methadone as rescue therapy, along with a score sheet for pain evaluation (Supplementary File).

Results

The creation of a stump and a bifurcation aneurysm was successful in all 5 New Zealand white rabbits without intraoperative complications. No mortality was observed during surgery or during the follow-up period of 24 ± 2 days. One rabbit experienced postoperative complications with a vestibular syndrome and a blindness of the right side. The animal recovered completely and spontaneously after 24 h. This complication did not interfere with its normal activities (free movements, water and food intake, interactions with oth...

Discussion

The most common technique for aneurysm creation involves the creation of a stump aneurysm at the origin of the right CCA, either through an open or an endovascular method. The model has been validated to be a stable non-growing aneurysm that remains open with time^20,21. The second possible technique involves the microsurgical creation of an arterial bifurcation aneurysm by anastomosing the right CCA on the left one and suturing an aneurysm pouch on the bifurcatio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-0 resorbable suture	Ethicon Inc., USA	VCP292ZH
4-0 resorbable suture	Ethicon Inc., USA	VCP304H
6-0 non absorbable suture	B. Braun, Germany	C0766070
9-0 non absorbable suture	B. Braun, Germany	G1111140
Adrenaline	Amino AG	1445419	any generic
Amiodarone	Helvepharm AG	5078567	any generic
Anesthesia machine	Dräger		any other
Aspirin	Sanofi-Aventis (Suisse) SA	622693	any generic
Atropine	Labatec Pharma SA	6577083	any generic
Bandpass filter blue	Thorlabs	FD1B	any other
Bandpass filter green	Thorlabs	FGV9	any other
Biemer vessel clip (2x)	B. Braun Medical AG, Aesculap, Switzerland	FD560R	any other
Bipolar forceps			any other
Bispectral index (neonatal)			any other
Blood pressure cuff (neonatal)		any other
Bycilces spotlight			any other
Clamoxyl	GlaxoSmithKline AG	758808	any generic
Dexmedetomidine	Ever Pharma	136740-1	any generic
Elastase	Sigma Aldrich	E7885
Electrocardiogram electrodes
Ephedrine	Amino AG	1435734
Esmolol	OrPha Swiss GmbH	3284044
Fentanyl (intravenous use)	Janssen-Cilag AG	98683
Fentanyl (transdermal)	Mepha Pharma AG	4008286
Fluoresceine	Curatis AG	5030376
Fragmin	Pfizer PFE Switzerland GmbH	1906725
Heating pad or heating forced-air warming system
Isotonic sodium chloride solution (0.9%)	Fresenius KABI	336769
Ketamine	Pfizer PFE Switzerland GmbH	342261
lid retractor			Approach
Lidocaine	Streuli Pharma AG	747466
Longuettes
Metacam	Boehringer Ingelheim	P7626406	Medication
Methadone	Streuli Pharma AG	1084546	Sedaton
Micro-forceps  curved	Ulrich Swiss, Switzerland	U52-015-15
Micro-forceps  straight 2x	Ulrich Swiss, Switzerland	U52-010-15
Microscissors	Ulrich Swiss , Switzerland	U52-327-15
Midazolam	Accord Healthcare AG	7752484
Needle 23 G			arteriotomy
Needle holder
O2-Face mask
Operation microscope	Wild Heerbrugg
Papaverin	Bichsel		topical application
Povidone iodine	Mundipharma Medical Company		any generic
Prilocaine-lidocaine creme	Emla
Propofol	B. Braun Medical AG, Switzerland		General anesthesia
Pulse oxymeter
Rectal temperature probe (neonatal)
Ringer Lactate	Bioren Sintetica SA		Infusion
Ropivacain	Aspen Pharma Schweiz GmbH	1882249	Local anesthesia
Scalpell	Swann-Morton	210
Small animal shaver
Soft tissue forceps
Soft tissue spreader
Stainless steel sponge bowls
Sterile micro swabs
Stethoscope
Surgery drape
Surgical scissors
Syringes 1 mL, 2 mL, and 5 mL
Tris-Buffer	Sigma Aldrich	93302	Elastase solution
Vascular clip applicator	B. Braun, Germany	FT495T
Vein and arterial catheter 22 G
vessel loop			Approach
video camera or smartphone
Vitarubin	Streuli Pharma AG	6847559
Yasargil titan standard clip (2x)	B. Braun Medical AG, Aesculap, Switzerland	FT242T