Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit

Summary

Developing and testing endovascular devices for intracranial aneurysm treatment is still of great importance. Most aneurysm models used today miss either the important characteristics of an arterial degenerated wall or the hemodynamics of a true bifurcation. Therefore, we aimed to design a novel arterial pouch bifurcation model in rabbits.

Abstract

Endovascular treatment for intracranial aneurysms gained importance over the past decades, consequently there is an increased need of testing endovascular devices. Animal models respecting rheological, hemodynamic and aneurysm wall conditions are highly warranted. Therefore, the aim of the present study was to design a novel standardized and reproducible surgical technique to create autologous arterial pouch bifurcation aneurysms with non-modified and modified wall conditions in rabbits.

Bifurcation aneurysms were created by end-to-side anastomosis of the right on the left common carotid artery, both serving as parent arteries for the arterial pouch, which was microsurgically sewn on. Grafts were taken from the proximal right common carotid artery, either for the control (n = 7, immediate autologous re-implantation) or modified (n = 7, incubated with 100 international units elastase for 20 minutes before autologous re-implantation) group. Pouch and parent artery patency were controlled by fluorescence angiography immediately after creation. At follow-up (28 days), all rabbits underwent contrast enhanced magnetic resonance angiography and fluorescence angiography followed by aneurysm harvesting, macroscopic and histological evaluation.

A total of 16 female New Zealand White rabbits were operated upon. Two animals died prematurely. At follow-up, 85.72% of all aneurysms remained patent. Both groups revealed an increase in aneurysm size over time; this was more pronounced in the control group (6.48 ± 1.81 mm³ at time of creation vs. 19.85 ± 6.40 mm³ at follow-up, p = 0.037) than in the modified group (8.03 ± 1.08 mm³ at time of creation vs. 20.29 ± 6.16 mm³ at follow-up, p = 0.054).

Our findings demonstrate the adequacy of this new rabbit model which allows for the creation of bifurcation aneurysms with different wall conditions in a microsurgical approach. Given the excellent long-term patency and the property of aneurysm growth over time, this model may serve as an important tool for preclinical evaluation of novel endovascular therapies.

Introduction

Subarachnoid hemorrhage resulting from intracranial aneurysm (IA) rupture can effectively be controlled by either endovascular or microsurgical occlusion techniques^1,2,3,4. Different endovascular therapies, to overcome the main limitation of IA recurrence after coiling, gained importance over the past decades generating an increased need of testing endovascular devices. To test these novel treatment approaches, appropriate animal models that respect rheological properties, hemodynamics and aneurysm wall conditions are highly warranted^5,6,7. In this context, clinical as well as preclinical studies have already revealed the important role of aneurysm wall conditions regarding aneurysm rupture and recurrence after occlusion, especially focusing on the loss of mural cells^7,8,9.

So far, experimental aneurysms in rabbits have most often been created either by elastase incubated common carotid artery (CCA) stumps or venous pouches sutured into an artificial CCA bifurcation.^{10,11,12,13,14,15,16} Thus, a true arterial pouch bifurcation model has never been described.

The aim of this study was to design a safe, fast, and standardized technique for microsurgical creation of bifurcation aneurysms with different wall conditions in a rabbit model (Figure 1). This was achieved by suturing non-modified and modified arterial pouches into an artificial created bifurcation of both CCAs.

Protocol

All veterinary care was performed in accordance with the institutional guidelines (all experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16)) and conducted under supervision of a board-certified veterinarian anesthesiologist. The ARRIVE guidelines and the 3R principles were strictly followed^17,18.

NOTE: House all animals at a room temperature of 22‒24 Celsius (°C) and maintain a 12 hours (h) light/dark cycle. Provide free access to water, pellet and ad libitum hay diet every time. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value (p) of ≤ 0.05 was considered significant.

1. Presurgical phase

1. Perform a detailed preoperative clinical examination of all rabbits planned for surgery immediately next to a quiet, aseptic operating room maintaining a temperature of 23 ± 3 °C.
   1. Record the weight of each animal, macroscopically evaluate the mucous membranes, capillary refill time and pulse quality.
   2. Further on perform cardiac auscultation with a stethoscope and abdominal palpation.
   3. Based on the clinical findings, attribute an American Society of Anesthesiologists (ASA) classification to each rabbit¹⁹. Include only animals with an ASA I score in the study.
   4. Shave both outer ears with an electric shaver and apply prilocaine-lidocaine cream on both auricular arteries and veins.
2. Sedate the rabbit with a combination of 20 milligram (mg)/killogram (kg) of ketamine, 100 mg/kg of dexmedetomidine and 0.3 mg/kg of methadone injected subcutaneously (SC) via a syringe.
3. Leave each animal undisturbed for at least 15 min.
4. Thereafter, under supplementary oxygenation with 3 liter (l) /minute (min) through a loose face mask and steady monitoring through a pulse oximeter, place a 22 G cannula in the left auricular central artery and another 22 G cannula in the auricular vein of the contralateral ear.
5. Shave the surgical field (neck) and inject 0.75% peri-incisional ropivacaine intradermally. Next shave the forehead and prepare to place pediatric electroencephalographic (EEG) sensors.
6. Induce general anesthesia with propofol 1-2 mg/kg intravenously (IV) to effect. Then immediately intubate the trachea of all rabbits with a silicone tube (3 millimeter (mm) internal diameter) under capnographic control. Afterwards, transport all rabbits to the operating room, place them in dorsal recumbency and connect the tube to a pediatric circle system.
7. Achieve anesthesia deepening and maintenance through isoflurane in oxygen, targeting a maximal end tidal isoflurane concentration of 1.3%.
8. Ensure clinical and instrumental monitoring (pulse oximetry, doppler and invasive blood pressure, 3-lead electrocardiogram, EEG, rectal temperature monitoring and inhaled and exhaled gases) until tracheal extubation.
9. To maintain hydration, provide Ringer’s lactate at a continuous rate infusion (CRI) of 5 ml/kg/h through the venous access. Always confirm proper anesthesia using toe pinches at an interval of 10 min.
10. Disinfect the surgical field using povidone iodine from the manubrium sterni to both jaw angles. Now, perform sterile draping of the surgical field.
11. During surgery, provide analgesia with lidocaine at a CRI of 50 microgram (µg)/kg/min and fentanyl at 3‒10 µg/kg/h. Apply spontaneous or assisted ventilation as well as permissive hypercapnia. Perform arterial blood gas analysis at least one time during surgery.
12. Treat relevant hypotension (mean arterial pressure < 60 mmHg) with noradrenaline. Prevent hypothermia (rectal temperature ≤ 38 °C) using a heating pad or a heating forced-air warming system.

2. Surgical phase – Step I

1. Start the surgery with a median skin incision from the manubrium sterni to the level of the jaw angles/larynx. Sharply dissect the skin and soft tissue with a scalpel, surgical scissors and forceps. Separate the subcutis and the fat pad medially by blunt dissection.
2. Enter the anterior upper ridge of the sternocleidomastoid muscle medially on the left side by blunt dissection, using micro forceps and surgical scissors.
3. Macroscopically, perform blunt preparation and carefully separate the left CCA from the vagal nerve distally to avoid laryngeal paresis by further using micro forceps and surgical scissors (Figure 2). Note that the bifurcation of the left CCA serves as intraoperative landmark (Figure 3 and Figure 4A). For all the following steps, use a soft tissue spreader to improve surgical visualization.
4. After successful preparation and liberation of the left distal CCA from the vagal nerve, administer papaverine (40 mg/ml, 1:1 diluted in 0.9% isotonic sodium chloride solution) locally. Continuously protect all vessel segments with micro swabs followed by further papaverine administration externally. Place the papaverine-soaked left CCA below the autologous muscle tissue to protect the vessel from drying under the light of the operation microscope.
5. Switch sides while maximizing the surgeon’s comfort during the operative procedure. Repeat the same surgical procedure on the right side. Dissect the CCA distally and proximally up to the predefined landmarks (carotid bifurcation at the level of the jaw angles/larynx and internal jugular vein; Figure 4A,B). Reinsert a spreader and administer micro swabs and papaverine as described previously.
6. Before the ligation of the right proximal CCA, inject heparin (500 international units (IU)/kg) systemically via a venous ear catheter.
7. Use a surgical microscope from now on. First, ligate the right proximal CCA with a 4-0 non-absorbable suture directly at the end of the macroscopically visible proximal landmark to avoid any tension on the arterial vessel.
   1. Secondly, apply a 6-0 non-absorbable ligature exactly 4‒5 mm distally by using a vessel clip for measurement, considering that after cutting distally from the first 4-0 ligature, the resulting arterial pouch will be of standardized length of about 3‒4 mm in every animal (Figure 5A,C).
8. After tightening the 6-0 ligature, clamp the right CCA as far distally as possible with a temporary vessel clip (as normally used in cerebral aneurysm surgery) to avoid any endothelial damage and to create a long vessel segment for irrigation in order to prevent thrombogenesis (Figure 5B).
9. Now perform a cut distally to the 4-0 non-absorbable ligature. To harvest the arterial pouch (Figure 5C), perform a second cut distally to the 6-0 non-absorbable ligature.
10. Clean the arterial pouch meticulously from all soft tissue and measure its length, width and depth (Figure 5C) with a vessel clip. If no further modification is needed, keep the autologous arterial graft in a heparinized solution (500 IU/100 ml in 0.9% isotonic sodium chloride) at room temperature until further use.

3. Arterial pouch degradation

1. If an arterial pouch degradation is needed, clean it meticulously of soft tissue and preincubate it with 100 IU of porcine elastase dissolved in 5 ml of Tris-buffer at room temperature on the day of experiment for 20 min. Do not use a brush technique. Incubate the arterial pouch intra- and extra-luminally by using a shaker.
2. Before putting the pouch in a heparinized solution of 0.9% isotonic sodium chloride, gently swipe it three times for 3 min with anatomical forceps in 0.9% isotonic sodium chloride solution to wash out the remaining porcine elastase.
3. If needed, keep the lumen of the arterial pouch opened up with a microtube made of silicone; meticulously protect the left and right CCA during the whole surgical procedure with wet micro paddings.

4. Surgical phase – Step II

1. For further preparation of the CCA, place two round micro swabs directly beneath it to move the artery more superficially. Now, put one micro swab with a purple padding under the left CCA at the distal third for better visualization of the artery.
2. Flush the right proximal CCA with a solution of 0.9% isotonic sodium chloride combined with 500 IU of heparin dissolved in 100 ml of 0.9% isotonic sodium chloride. In order to create a tension free anastomosis, place the right CCA under the fat pad/peritracheal musculature by using surgical scissors for tunneling it to the left side. Remove the soft tissue of the artery.
   1. Now perform a 2 mm fish mouth incision on the proximal side of the right CCA using a micro scissor and forceps.
3. Change the side on the operating table. Clip the left distal CCA with another temporary vessel clip followed by the proximal left CCA with two temporary vessel clips. Protect all exposed vessel segments from drying out under the surgical light using wet micro swabs.
4. Liberate the distal third of the left CCA completely from soft tissue and perform an arteriotomy. Use surgical micro forceps and gently grab some soft tissue. Now elevate the artery and incise the left distal CCA slowly with a surgical micro scissor. Flush the vessel segments with heparin (500 IU dissolved in 100 ml of 0.9% isotonic sodium chloride solution).
5. After performing the arteriotomy with curved micro forceps and micro scissors, enlarge the arteriotomy located at the distal third of the left CCA distally, measuring about 2-fold of the diameter of the right blunt of the carotid artery and the autologous graft. This allows sufficient blood flow into the arterial pouch.
6. Take the arterial pouch out of the heparinized saline solution. Place the pouch in the surgical field, where the bifurcation is planned. Start suturing the rear of the right carotid blunt caudally-located with a non-absorbable 9-0 suture, followed by a suture on the cranially-located rear side at the level of the fish mouth incision. Finish sewing the rear from distal to proximal by single stitches.
7. While suturing, keep all elastase preincubated pouches moist with continuous irrigation. While suturing the vessel wall of the pouch, use curved surgical micro forceps to gently open up the lumen with its tip. Whenever suturing parts of the left or proximal right CCA, use straight surgical micro forceps. Afterwards, suture the horizontal back side.
8. Next suture the horizontal front side, starting at the dome of the aneurysm moving to its base. Afterwards, start with single stitches distally on the front side moving caudally.
   1. For all steps 4.5‒4.8 while suturing the anastomosis pay attention just to grab the part of the vessel close to the arteriotomy to avoid iatrogenic stenosis. Also, continuously moisten all vessel segments during the whole surgical procedure extraluminally with a syringe filled with heparinized sodium chloride solution (500 IU dissolved in 100 ml of 0.9% isotonic sodium chloride) and protect them with wet micro swabs.
   2. Before finishing the anastomosis, irrigate the whole complex with heparinized 0.9% isotonic sodium chloride solution intraluminal (500 IU dissolved in 100 ml of 0.9% isotonic sodium chloride). Beware that elastase modified arterial pouches have to be sewn on as quickly as possible because of their strong tendency to dry out and to thrombose. Because of the aggressive behavior of the residual elastase concentration in the pouch regarding digesting circumferential vessels, proceed fast with the surgery to re-perfuse the vessel complex quickly.
9. Remove all temporary vascular clamps stepwise.
   1. Remove the distal clamp from the left CCA. Accept minor bleeding and staunch it by gently imprinting micro swabs on the anastomosis. Afterwards, remove the clamp of the right CCA, press gently with micro swab and forceps to avoid thrombus formation.
   2. If needed, replace the temporary vascular clips to provide enough coagulation. Afterwards, relieve both vessel clips from the left side proximally. If needed in any step, replace clips to allow coagulation or to perform re-stitching.
10. At this stage perform fluorescence angiography of the vessel complex (Figure 6 and Figure 7).
    NOTE: Fluorescence angiography is performed by administering 1 ml of fluorescein IV, using 2 bandpass filters, a smartphone with video camera, and a bicycle spotlight. This procedure has been already described elsewhere^20,21,22.
11. Lastly, close the operative situs. Readapt and gently suture the fat pad with a 3-0 resorbable suture with single nodes to protect the anastomosis. Close subcutis and skin in the same fashion.

5. Postsurgical phase

1. Discontinue the isoflurane and systemic analgesia administration at the end of the surgery and provide tracheal extubation as soon as the swallowing reflex has returned.
2. Administer 0.5 mg/kg of meloxicam IV, 10 mg/kg aspirin (ASS) IV, 100 µg of vitamin B₁₂ SC and 20 mg/kg of clamoxyl IV.
3. Provide supplementary oxygenation and active warming until the rabbits have spontaneously regained sternal recumbency.
4. Perform postoperative follow-up and animal care four times a day for the first three days, in accordance to the guidelines for the assessment and management of pain in rodents and rabbits^23,24.
5. Administer post-operative analgesia via a fentanyl patch (12 µg/h) applied on the outer ear, meloxicam once a day SC for three days and methadone as rescue therapy SC, according with the score sheet for pain evaluation. Administer 250 IU/kg low-molecular heparin (LMH) subcutaneously for three days in all rabbits.

Results

Following a pilot series of seven animals, totally 16 animals were included in the experimental protocol. Two animals died prematurely and were therefore excluded from the final analysis (12.5% mortality). Calculated on 14 animals, immediate aneurysm patency rate during fluorescence angiography was 71.43% in both, the control and modified group. Four aneurysms had to be reopened with consecutive thrombus evacuation and after a repeated fluorescence angiography there was a documented patency in all cases (100%). Aneurysm ...

Discussion

Our study demonstrates the feasibility of creating a true bifurcation aneurysm model with different wall conditions in rabbits. Overall, 14 female New Zealand White rabbits with a mean weight of 3.7 ± 0.09 kg and mean age of 112 ± 3 days were included in the study. 85.72% of all aneurysms remained patent during a follow-up at 28 days. Two animals died prematurely (12.5% mortality).

Previous studies suggested a variety of extracranial aneurysm models to analyze the management of endov...

Materials

Name	Company	Catalog Number	Comments
3-0 resorbable suture	Ethicon Inc., USA	VCP428G
4-0 non-absorbable suture	B. Braun, Germany	G0762563
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
Bipolar forceps			any other
Bicycle spotlight			any other
Biemer vessel clip (2 x)	B. Braun Medical AG, Aesculap, Switzerland	FD560R	temporary
Bispectral index (neonatal)			any other
Blood pressure cuff (neonatal)			any other
Clamoxyl	GlaxoSmithKline AG	758808	any generic
Dexmedetomidine	Ever Pharma	136740-1	any generic
Electrocardiogram electrodes			any other
Elastase	Sigma Aldrich	45125	any generic
Ephedrine	Amino AG	1435734	any generic
Esmolol	OrPha Swiss GmbH	3284044	any generic
Fentanyl (intravenous use)	Janssen-Cilag AG	98683	any generic
Fentanyl (transdermal)	Mepha Pharma AG	4008286	any generic
Fluoresceine	Curatis AG	5030376	any generic
Fragmin	Pfizer PFE Switzerland GmbH	1906725	any generic
Glyco			any generic
Heating pad			any other
Isotonic sodium chloride solution (0.9%)	Fresenius KABI	336769	any generic
Ketamine	Pfizer	342261	any generic
Laboratory shaker	Stuart	SRT6	any other
Lidocaine	Streuli Pharma AG	747466	any generic
Longuettes			any other
Metacam	Boehringer Ingelheim	P7626406	any generic
Methadone	Streuli Pharma AG	1084546	any generic
Microtubes			any other
Micro needle holder			any other
Midazolam	Accord Healthcare AG	7752484	any generic
Needle holder			any other
O2-Face mask			any other
Operation microscope	Wild Heerbrugg		any other
Papaverine	Bichsel		any generic
Prilocaine-lidocaine creme	Emla		any generic
Propofol	B. Braun Medical AG, Switzerland		any generic
Pulse oxymeter			any generic
Rectal temperature probe (neonatal)			any other
Ropivacaine	Aspen Pharma Schweiz GmbH	1882249	any generic
Scalpell	Swann-Morton	210	any other
Small animal shaver			any other
Smartphone			any other
Soft tissue forceps			any other
Soft tissue spreader			any other
Stainless steel sponge bowls			any other
Sterile micro swabs			any other
Stethoscope			any other
Straight and curved micro-forceps			any other
Straight and curved micro-scissors			any other
Straight and curved forceps			any other
Surgery drape			any other
Surgical scissors			any other
Syringes 1 ml, 2ml and 5 ml			any other
Tris-Buffer	Sigma Aldrich	93302	any generic
Vascular clip applicator	B. Braun, Germany	FT495T
Vein and arterial catheter 22 G			any generic
Vitarubin	Streuli Pharma AG	6847559	any generic
Yasargil titan standard clip (2 x)	B. Braun Medical AG, Aesculap, Switzerland	FT242T	temporary