Animal procedures were approved by our institution’s Animal Care Committee in accordance with Canadian Council on Animal Care guidelines. The method for the creation of occlusive and sclerosing gels and their safety/efficacy to treat endoleak was previously reported including six animals of the current study [8]. The current publication focus on the potential of SWI for characterising aneurysm healing after endoleak embolisation. The first draft of this article is available on a university repository website (https://papyrus.bib.umontreal.ca/xmlui/bitstream/handle/1866/16033/Bertrand-Grenier_Antony_2015_these.pdf?isAllowed=y&sequence=6).
Creation of bilateral iliac aneurysms
Eight mongrel dogs (25–50 kg of bodyweight) underwent surgical construction of bilateral aneurysms in the common iliac arteries (16 aneurysms in total) using a venous patch taken from the external jugular vein. A collateral vessel (branch division of the sacral artery) was re-implanted in the aneurysm as previously reported [11]. Procedures were performed by a vascular surgeon with 20 years of experience in experimental surgery (I.S.).
EVAR with type I endoleak creation
After eight weeks of recovery, EVAR was performed by an interventional radiologist with a 22 years of experience (G.S.) using a 59-mm-long balloon-expandable stent-graft (iCAST, Atrium, Hudson, NH, USA) deployed to a diameter of 7 mm or 8 mm. A type I endoleak was then created in all aneurysms by inflating a 3-mm diameter balloon catheter alongside the proximal landing zone of the stent-graft after deployment, creating a misfit between the stent-graft and the vessel wall [12].
Endoleak embolisation
The injectable chitosan hydrogel (Chi) was prepared by mixing an acidic solution of chitosan containing a radiopaque agent with beta-glycerophosphate [13], with only occlusive property. A second occlusive and sclerosing gel formulation was obtained combining Chi with sodium tetradecyl sulfate (Chi-STS) [13]. Both embolisation agents were slowly injected in the same animal, with random side attribution, in a blind fashion for the type of gel (Chi or Chi-STS), under fluoroscopy through a 4-French catheter positioned alongside the stent-graft (Glidecath, Terumo, Tokyo, Japan). The operator was asked to occlude the entire aneurysm sac while avoiding gel migration in the stent-graft lumen.
Angiography
Percutaneous transfemoral angiography (Koordinat 3D II, Siemens Healthineers, Erlangen, Germany) was performed after stent-graft implantation, endoleak embolisation and before sacrifice at three months (n = 5 dogs) or six months (n = 3 dogs). Type I endoleak was defined as residual opacification of the aneurysm through an antegrade flow coming from the proximal neck and type II endoleak as a retrograde flow coming from the collateral vessel [14]. CT was the reference standard for endoleak detection while angiography was only used to classify endoleaks.
Computed tomography
CT was performed before sacrifice and reviewed by the same interventional radiologist. Arterial and venous phases were acquired, with a retrospective electrocardiographic gating reconstructed at 70% of the RR interval (60 mL at 4 mL/s, Omnipaque 300 mg I/mL, General Electric Healthcare Canada, Mississauga, ON, Canada) with acquisition parameters set at 120 kVp and 724 mAs (Somatom Sensation 64, Siemens Healthineers, Forcheim, Germany). Endoleaks were characterised as areas of contrast enhancement in the aneurysm sac visible in the arterial or venous phase.
Ultrasound
A research technician with 20 years of experience performed independently DUS and SWI examinations at one week, one month, three months and six months (only three dogs at the last time point). Post-processing of SWI, segmentation and registration of region of interests (ROIs) on imaging acquisition and pathology examination were performed by a PhD student in medical physics (A.B.G.).
A 256-element linear probe (SuperLinear™ SL15–4, 7.5 MHz) was used for all B-mode, DUS and SWI acquisitions (Aixplorer, Aix-en-Provence, France). Three axial acquisition planes were taken on the aneurysm (proximal, middle and distal). The diameters and areas of the three axial planes were measured and averaged. Aneurysm growth or shrinkage was estimated as the variation in percentage of the mean aneurysm cross-sectional surface area between baseline and follow-up.
Standard parameters for DUS examinations to detect endoleak were set to a scale of 10 cm/s, smoothing to 0, wall filter to low, and high-definition frame rate to middle. The steer angle was first set to 0° then to 60° right anterior oblique and left anterior oblique.
Dynamic elastography parameters were selected as a smoothing of 5, opacity of 50% and low acoustic power. For elasticity measurement, the colour code scale displayed on SWI images was converted to grayscale after calibration. SWI was used to observe the evolution of the mechanical property of the thrombus and embolisation agent over time. Endoleak on SWI was defined as the absence of elasticity values within the aneurysm sac outside the stent-graft with the presence of signal on the posterior wall [10]. Areas without signal on the posterior wall were deemed as non-diagnostic.
Pathology
Each dog was sacrificed with a barbiturate overdose (108 mg/kg, euthanyl forte, Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada). Aneurysms were collected and fixed in buffered formalin. Consecutive 3–5 mm axial macroscopic sections were prepared with a cutting-grinding system to keep the implant/tissue interface intact (EXAKT Advanced Technologies GmbH, Norderstedt, Germany). In some samples, the SG was removed and tissues were sent to histology. Macroscopic cuts and histology analyses were processed independently by a PhD student (F.Z.) supervised by a biomedical engineer with 15 years of experience in endovascular biomaterials (S.L.) to identify and segment the different ROIs. Endoleaks were identified as defect zones, organised thrombus as a dense tissue with a yellowish colouration, fresh thrombus as areas with dark bluish loose tissue and embolisation agents as yellow/pink areas without appearance of tissue organisation. Macroscopic cuts were used as the reference standard for ROI segmentation and to characterise thrombus organisation and agent degradation.
Histopathologic examination was performed to confirm the content of the different ROIs assessed on macroscopic results.
ROI segmentation and co-registration
At sacrifice, axial images acquired on B-mode ultrasound, DUS, SWI, CT and histopathology were co-registered based on the level of acquisition, aneurysm area and stent-graft localisation in the sac. TeraRecon (AquariusNET iNtuition, version 4.4.7, TeraRecon Headquarters, Foster City, CA, USA) and Image J (Rasband, W.S., version 1.47b, National Institutes of Health, Bethesda, MD, USA) were used to segment ROIs of the aneurysms.
Presence of contrast enhancement on CT scans combined with the presence of a defect in the aneurysm sac on the macroscopic examination was used as the reference standard for endoleak diagnosis [10].
For ultrasound examinations acquired before sacrifice at one week, one month and three months (for the three dogs with six-month follow-up), on each acquisition plane, the aneurysm was divided into three different ROIs: endoleak; total thrombus; and embolisation agents. ROI segmentation and positioning were based on the fusion of B-mode, DUS and elasticity images which were acquired at the same level.
Endoleak ROIs were traced based either on colour DUS or SWI examinations. Total thrombus was defined as ROIs without endoleak or embolising agent in the aneurysm sac. The ROI segmentation of embolising gel was based on the echogenicity of the gel on B-mode ultrasound which was hyperechoic compared to native thrombus. In the case of discordance or uncertainty for embolising gel contouring, the operator relied on the gel localisation on pathologic examinations.
To determine stiffness values corresponding to different levels of thrombus organisation, the thrombus was sub-classified in fresh or organised thrombus on the pre-sacrifice examination. To evaluate fresh thrombus maturation over time, ROIs with elasticity values compatible with the presence of a fresh thrombus (in the range of 3–19 kPa as previously reported [10]) on the one-week SWI examination were segmented and registered on the three-month SWI examination.
The different ROIs elasticity measurements were averaged for the three axial acquisition planes. The percentage of growth or shrinkage of aneurysms between the one-week time point and sacrifice was correlated with the averaged surface area of endoleak and/or fresh thrombus found at sacrifice on pathology (Pearson coefficient). Thresholds for significance of p values were adjusted with Bonferroni correction by testing each individual hypothesis at a significance level of α/m, where α is the desired overall alpha level and m is the number of hypotheses. The overall significant level was α = 5% and the significant level of comparison for multiple testing was also 5%. Statistical analyses were performed using R software (version 3.2.1, R Foundation for Statistical Computing, Vienna, Austria).
