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PET and MR assessment of inflammation and mineralization processes in a mouse model of accelerated atherosclerosis. (#162)
G. Rucher1, L. Camelière1, J. Fendri1, K. Dupont1, A. Anfray2, A. Abbas3, 4, L. Berger1, A. Manrique1
1 Université de Caen Normandie, UNICAEN, EA 4650, Caen, France
In this study, we evaluated two imaging procedures targeting inflammation and mineralization processes, both involved in atherosclerosis. Mineralization was assessed using 18F-NaF Positron Emission Tomography (PET) and inflammation using Magnetic Resonance (MR) imaging after the injection of anti-VCAM-1 antibodies conjugated to micro-particles (MPIO-VCAMs). Imaging was performed in a mouse model of accelerated atherosclerosis based on ApoE-/- with chronic kidney failure.
A group of uremic (Ur) and non-uremic (NUr) ApoE-/- were evaluated. Uremia was induced surgically by an electrocautery of the right kidney at 8 week-old (wo), followed by a contralateral nephrectomy at 10 wo. After a low dose CT for attenuation correction, PET was performed using a dedicated preclinical system (Inveon®, Siemens) in12 and 16 wo mice. Telediastolic T2* weighted MR sequences (TR/TE: 100/4.25 ms, 12 slices, voxel: 0.1x0.1x0.15 mm3) encompassing the thoracic aorta were performed using a 7T magnet (Pharmascan®, Bruker) in 16 wo mice, after intravenous injection of MPIO-VCAMs. Then, urea and calcium serum levels were measured and aortas harvested to assess intra-aortic calcium content with quantitative von Kossa staining or photoelectric flame photometry (PFP).
Renal failure, confirmed by urea serum level increase (NUr: 8.6 mM±0.2 vs. Ur: 22.9±1.3, p<0.0001) was associated with an increased calcium serum level (NUr: 2.24 mM±0.01 vs. Ur: 2.54±0.09, p=0.003) and aortic calcium content with PFP (NUr: 0.32±0.05 vs. 0.49±0.07, p<0.05). We observed an active aortic metabolism using 18F-NaF PET in 5 NUr (n=9, 55%) and 8 Ur (n=12, 66%, NS) in 12 wo mice. In 16 wo mice, only 4 Ur (n=9, 45%) had aortic 18F-NaF uptake. Although von Kossa staining found no difference between Ur and NUr, there was an inverse relationship between PET and von Kossa (PET-: 0.89%±0.09 vs. PET+: 0.42%±0.07, p=0.028). MR showed a T2* signal void after MPIO-VCAM injection in 4 Ur (n=5, 80%) and 1 NUr (n=4, 25%) mice, predominantly in the aortic arch. Finally, aortic arch and brachiocephalic trunk were enlarged in Ur compared to NUr (68.1±8.2 µm/g vs. 61.9±7.4, p=0.03 and 35.5±2.9 vs. 32.2±2.2, p=0.003).
In this study, uremic ApoE-/- mice showed an early and sustained increased vascular mineralization process. Active mineralization was associated with persistent vascular inflammation as demonstrated using T2* MR after MPIO-VCAMs.
Keywords: Atherosclerosis, mineralization, inflammation, aorta, PET, MRI
Cardiac Fibrosis: virtual tissue biopsy by high resolution computed tomography (#127)
A. Khan1, A. M. Markus2, C. Dullin1, F. Ramos-Gomes2, M. Silter3, E. Zeisberg3, F. Alves1, 2, 4
1 University Medical Center Göttingen, Institute for Diagnostic and Interventional Radiology, Göttingen, Lower Saxony, Germany
Chronic heart diseases are associated with fibrosis, a pathological process of scarring that has profound consequences on myocardial function. Therefore, the detection, prevention, and regression of myocardial fibrosis have emerged as important targets for improving heart failure therapy. Our aim was to evaluate the feasibility to detect and characterize the pattern of interstitial fibrosis in a mouse model with Angiotensin II (Ang II) infusion, by using multiscale imaging techniques including X-ray based three-dimensional histology.
A mouse model of cardiac fibrosis was established via Ang II infusion. Ex-vivo virtual histology of whole hearts was performed using lab-based microCT and synchrotron radiation-microCT (SR-microCT) of phosphotungstic acid (PTA) stained hearts. Data from Quantum microCT and SR-microCT was aligned to cut heart sections at a region of interest (ROI) for Masson’s Trichrome staining (MTS) and two-photon laser-scanning microscopy (2P-LSM).
The slower rate and pattern of PTA staining indicated potential structural alterations in the tissue of fibrotic heart obtained from Ang II infused mice. After scanning the hearts with SR-microCT possible fibrotic lesions were identified. Tissue sections stained with MTS showed the highest level of collagen deposition at the ROI. Furthermore, second harmonic generation (SHG) visualized by 2P-LSM revealed a significant increase in collagen content within heart sections obtained from the fibrotic heart as compared to the healthy controls. Histological analysis confirmed the presence of fibrosis mainly within the walls of the ventricles of the heart.
We show that areas of fibrosis within hearts obtained from a mouse model of Ang II induced cardiac fibrosis could be visualized in great detail by microCT virtual histology and 2P-LSM.
1). Dullin C., et al. (2017). μCT of ex -vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology, and immunochemistry. PLoS ONE. 12(2), e0170597.
2). Sopel M. J., et al. (2011). Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells. Laboratory Investigation. 91(4), 565–578.
We thank Bettina Jeep, Sabine Wolfgramm and Baerbel Heidrich for excellent technical assistance. SR-microCT images were acquired at the high-resolution SYRMEP beamline of the ELETTRA synchrotron radiation facility (Trieste, Italy).
The reconstruction of SR-microCT acquired images of paraffin embedded PTA stained hearts.
(A) The reconstruction of ventricular walls of the fibrotic heart and (B) the healthy heart. The brighter regions display the fibrotic pericardium present in both hearts. The fibrotic lesions appear as brighter regions within the myocardium of the fibrotic heart which are absent in the healthy heart.
Keywords: Cardiac fibrosis, Angiotensin II, microCT, Two-photon laser scanning microscopy.
Experiences using a hind limb ischemia mouse model to evaluate the process of angiogenesis by non-invasive High Resolution Laser Doppler Imaging. (#86)
1 Instituto Aragonés de Ciencias de la Salud (IACS), Medical Imaging & Phenotyping, Zaragoza, Spain
The hind limb ischemia model is a well-established and popular "gold standard" tool to test and quantify the effect of novel therapies on the angiogenesis. Different strains of mice are often used as animal models. Once the femoral ischemia has developed, changes in blood flow are evaluated for 3-5 weeks. We want to check what the response in control animals of two strains of mice, athymic nude and c57s. Both strains are frequently used in angiogenesis studies in our laboratory.
Hind Limb Ischemia Model
Laser Doppler Perfusion Imaging
Immediately after unilateral femoral artery excision, marked reduction in blood flow of one hindlimb. In C57 mice, perfusion remained impaired for 7 days, increased to 68% of nonischemic limb by day 10, and ultimately returned to 73% by day 35. Perfusion in athymic nude remained impaired for 7 days, increased to 58% of nonischemic limb by day 10, and ultimately returned to 66% by day 35.
Approximately 6% of c57 mice developed signs of toe necrosis at distal portion of ischemic hindlimb during the first postoperative week but then healed rapidly during the following weeks. 26% of athymic mice developed signs of toe necrosis during the first postoperative week and 55% and none of them healed during the following weeks.
Variability was observed in the response to femoral ischemia between the two mouse strains evaluated. The athymic nude strain recovered with more difficulty with respect to the C57BL/6JRj mice (Day 10, foot perfusion ratio 68% vs 58% and day 35, 66% vs 73%) and presented a higher rate of associated necrotic events (6% vs 26%) during the first week.
Vascular flow evaluation, using High Resolution Laser Doppler Imaging in this type of model, should be performed for a máximum of 2-3 weeks as for no significant change will be expected after this time.
Keywords: Laser, Doppler, angiogenesis, Hind limb ischemia, mouse
Protocols for Fetal Mouse Echocardiography Imaging (#133)
L. Flores1, A. V. Alonso1, L. Cussó1, 2, 3, L. J. Jiménez-Borreguero4, M. Desco Menéndez1, 2, 3
1 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Advanced Imaging Unit, Madrid, Spain
Fetal echocardiography (FE) is a very valuable technique to diagnose the causes of fetal death when there is suspicion of cardiac pathology. FE is extremely useful working in cardiac transgenic mouse models. As in human medicine, intrauterine echocardiography has gained relevance in recent years for preclinical research [1-7]. Its main drawback is the reduced echocardiographic window which restrict the number of parameters obtain for each fetus . We present two new FE methods that enable more complete echocardiography assessments as compared with conventional intrauterine techniques.
Ultrasonography was carried out with a VEVO 2100 system and a 40 MHz probe on anesthetized pregnant mouse females. We compared the following acquisition methods: A) conventional intrauterine FE (gold standard ); B) uterine horns extraction ; C) fetus individualization. Briefly, each method consists on: A) conventional study ; B) a ventromedial laparotomy was done to externalize uterine horns, FE is obtained by placing the probe on top of the uterine horns wall; and C) once the uterine horns are exhibit as in protocol B, the fetuses are extracted one by one to acquire their individual FE. We analyzed a total of 449 fetuses from 72 pregnant mouse females: (N=24 protocol A (10 animals), N=252 protocol B (37 animals), and N=172 protocol C (25 animals).
Table I summarizes advantages and shortcomings identified, and data that can be obtained with each protocol.
Conventional intrauterine FE is the most common method for cardiac fetal evaluation since it is a non-invasive procedure allowing longitudinal studies. However, the cardiac planes that can be visualized and the functional data that can be obtained is very limited. This study demonstrates that the extraction of the uterine horns or the fetus individualization are efficient and complementary alternatives to the intrauterine image, to obtain a broader assessment of cardiac parameters related to fetal death, although these methods may imply animal the sacrifice, preventing longitudinal studies.
 MacGrogan D. et al. Circ Res. 2016;  Golden HB. et al. Methods Mol Biol. 2012;  Liu X. et al. Circ Cardiovasc Imaging. 2014;  Yu Q. et al. Ultrasound Med Biol. 2008;  Menendez-Montes I. et al. Dev Cell. 2016.
We thank María Villalba Orero for her advice, and Miguel Torres Sanchez, Silvia Martín Puig and Jose Luis de la Pompa Mínguez for kindly providing the echocardiographic images. The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).
Keywords: Fetal echocardiography, cardiac pathology
Rapid non-invasive functional assessment of the human carotid artery with handheld volumetric multi-spectral optoacoustic tomography (#505)
I. Ivankovic1, 2, E. Mercep3, X. L. Dean-Ben2, D. Razansky1, 2
1 Technical University Munich, Faculty of Medicine, Munich, Bavaria, Germany
In carotid artery disease (CAD), unambiguous detection of plaque vulnerability as well as core biological processes involved in its formation on a cellular and molecular level is limited with the existing clinical imaging modalities. Optoacoustic imaging has recently demonstrated promise in specific detection of activity markers typically associated with CAD [1,2]. However, non-invasive visualization and rapid functional and molecular assessment of the deeply embedded carotid artery remained an unmet clinical need.
We devised a custom handheld volumetric multi-spectral optoacoustic tomography (vMSOT) scanner capable of real-time 3D imaging of human carotid artery non-invasively. Carotid arteries of healthy volunteers were imaged non-invasively at depths ranging between 6-15 mm. Rapid volumetric images were acquired at varying excitation wavelengths with the entire carotid bifurcation visualised with 200µm spatial resolution.
Volumetric anatomical views of the bifurcation were analysed in different planes and compared with pulse-echo ultrasonography images. Real-time volumetric image acquisition allowed dynamic characterisation of the arterial pulse and physiological carotid waveform extraction. The spectrally enriched data has further enabled revealing the levels of oxygenated and deoxygenated haemoglobin in the artery.
The handheld vMSOT method was found capable of clinical real-time 3D imaging of the human carotid artery, making it less prone to motion artefacts and inter-user variability then the conventional imaging methods used in clinical practice. Combined with the proven capacity of spectroscopic optoacoustics for label-free characterization of blood oxygenation and chemical tissue composition, vMSOT holds promise for non-invasive functional assessment of cardiovascular disease.
 Deán-Ben, X. L., Gottschalk, S., Mc Larney, B., Shoham, S., & Razansky, D. (2017). Advanced optoacoustic methods for multiscale imaging of in vivo dynamics. Chemical Society Reviews, 46(8), 2158-2198.
 Jansen, K., van Soest, G., & van der Steen, A. F. (2014). Intravascular photoacoustic imaging: a new tool for vulnerable plaque identification. Ultrasound in medicine & biology, 40(6), 1037-1048.
Figure 1: Non-invasive 3D optoacoustic imaging of the human carotid artery in vivo.
(A) Depth characterisation of the carotid artery, where the top and lateral maximum intensity projections (MIP) of the 3D images are colour coded for depth (mm) (H: Hair, CW: Carotid Wall). (B) Top MIP of the 3D image of the carotid bifurcation after surface removal (EC: External carotid, IC: Internal Carotid, CC: Common carotid).
Endothelial integrity during atherosclerotic plaque development and therapeutic interventions (#414)
T. Beldman1, T. Malinova1, E. Desclos3, A. Grootemaat3, N. van der Wel3, M. de Winther1, E. Lutgens1, W. J. M. Mulder1, 2, S. Huveneers1, E. Kluza1
1 Academic Medical Center, Medical Biochemistry, Amsterdam, Netherlands
Endothelial dysfunction presents during atherogenesis as morphological, molecular and metabolic abnormalities of vascular endothelium. The goal of this study was to determine the structural and functional integrity of endothelial lining on atherosclerotic plaques in comparison to the healthy vessel wall. Furthermore, we hypothesized that inhibition of monocyte migration or modulation of endothelial metabolism could improve endothelial integrity in atherosclerosis.
6 ApoE-/- mice were 6 weeks on a high-fat diet and received intravenous injection of fluorescently labeled hyaluronan nanoparticles (HA-NPs). 2h post-injection, the mouse aortas were excised. Endothelial junctions and macrophages were stained with vascular endothelial-cadherin (VEC) and MAC-3 antibody, respectively. The aortas were imaged using confocal, two-color super-resolution ground state depletion (GSD) and correlative light electron microscopy. In the therapeutic study, ApoE-/- mice received either an inhibitor of monocyte recruitment (TRAF6 inhibitor) or glycolysis inhibitor (3PO) (10 mice/group). After 6 weeks of therapy and high-fat diet, the mice were injected with HA-NPs and fluorescent deoxyglucose (2-NBDG). The excised aortas were investigated with IVIS imaging system.
The endothelium covering plaque surface displayed activated phenotype i.e., fibroblastoid cell morphology and focal adherens junctions. The quantitative analysis of junctional structure revealed circa 30% loss of VEC continuity in atherosclerotic endothelium (Figure 1A).
HA-NP injection resulted in high NP levels in atherosclerotic plaques. At the plaque surface, the HA-NP signal colocalized with endothelial junctions. By using two-color GSD, we were able to resolve single HA-NPs confined between VEC foci. CLEM experiments confirmed the junctional pathway of NP accumulation (Figure 1B).
In the therapeutic study, the aortic permeability probed by HA-NPs was 40% (anti-inflammatory treatment) and 30% (metabolic treatment) lower compared to the control group. Interestingly, the glucose uptake was significantly enhanced by both treatments by circa 30%, which might by a consequence of therapy-induced oxygen deprivation (Figure 1C).
We demonstrate an active endothelial cell phenotype and remodeling junctions in the endothelium of mouse atherosclerotic plaques. By using fluorescent nanoparticles and advanced microscopy methods, we provide direct evidence on the loss of functional endothelial integrity. Furthermore, our initial results suggest modulation of endothelial integrity in atherosclerosis by anti-inflammatory and metabolic therapy.
Keywords: atherosclerosis, endothelial dysfunction, nanoparticles, super-resolution microscopy
Noninvasive Imaging of the Coronary Vasculature Using 3D Ultrafast Ultrasound (#320)
M. Correia1, D. Maresca1, M. Tanter1, B. Ghaleh2, M. Pernot1
1 INSERM, ESPCI, Paris, France
Dysfunctions on the coronary circulation can lead to adverse and severe clinical outcomes, e.g., ischemic heart failure (IHF) or coronary artery disease (CAD). However, the imaging of intramyocardial coronary vasculature remains challenging. Recently, we have shown that ultrafast ultrasound Doppler Imaging  can assess the coronary vasculature without contrast agent. Yet, given the complex architecture of the coronary vasculature, 3D imaging is of high importance. In this study, we propose 3D Ultrafast Ultrasound Doppler Imaging  to map the intramyocardial coronary circulation.
Acquisitions were conducted in open-chest swine hearts (N=5) using a customized, programmable, 1024-channel 3-D ultrafast ultrasound scanner and a 2-D matrix-array probe (9-MHz, 0.3mm pitch, 1024 elmts, Vermon). The myocardial signal was filtered using an adaptive temporal-weighted spatial-temporal clutter filter to separate the blood signal throughout the cardiac-cycle. Power Doppler images were computed and overlapped on B-mode to explore anatomical and blood flow functional information. Quantification of the flow change was performed during hyperemia after a 30-s left anterior descending (LAD) artery occlusion followed by reperfusion and was compared to gold standard measurements provided by a flowmeter probe placed at a proximal location on the LAD.
We successfully imaged epicardial and intramyocardial coronary vessels in two and three-dimensions between early and late diastole. The epicardial coronary veins and arteries were assessed exhibiting diameters of a few millimeters. The intramural coronary veins and arteries (i.e. prearterioles) of diameters ranging from 500 μm down to 300 μm were also imaged. Ultrahigh-sensitive cine loops of venous and arterial intramyocardial blood flows were obtained within 1 cardiac cycle. Quantification of the coronary flow changes during hyperemia was in good agreement with gold standard measurements (r2 = 0.89), as well as the assessment of coronary flow reserve (2.35 ± 0.65 vs. 2.28 ± 0.84; p = NS).
We successfully imaged epicardial and intramyocardial coronary vessels in three-dimensions between early and late diastole. Assessing the full anatomy of the coronary vasculature at high volume rates given by ultrafast imaging could open new insights in cardiovascular imaging. 3-D Ultrafast Doppler imaging could become an important non-invasive imaging tool to evaluate CAD and IHF.
 Maresca D, Correia M, Villemain O, Bizé A, Sambin L, Tanter M, Ghaleh B, Pernot M. Noninvasive Imaging of the Coronary Vasculature Using Ultrafast Ultrasound. JACC Cardiovasc Imaging. 2017
 J Provost, C Papadacci, JL Gennisson, J Arrango, M. Imbault, M. Fink, M. Tanter, M. Pernot, « 3D Ultrafast Ultrasound imaging in vivo », Physics in Medicine and Biology, 59, 19, L1-L13, 2014
This work was funded by the European Research Council
Keywords: ultrasound imaging, ultrafast, doppler, coronary, blood flow
Molecular Imaging of Tropoelastin in Aortic Aneurysm (#46)
B. Lavin1, S. Lacerda2, M. E. Andia3, R. M. Botnar1, A. Phinikaridou1
1 King's College London, Biomedical Engineering, London, United Kingdom
Elastin degradation and impaired synthesis favour the accumulation of tropoelastin, rather than cross-linked elastin, that may promote the growth of abdominal aortic aneurysms (AAAs) . We have developed a tropoelastin-binding MR contrast agent (Gd-TESMA) to test whether molecular imaging of tropoelastin can be used as a biomarker for AAA development and the risk of rupture.
ApoE-/- mice (n=50) were infused with angiotensin-II (Angio-II)  for up to 4 weeks to induce formation of AAA. Gd-TESMA was synthesized and fully characterized prior to in vivo use . Mice were imaged at 3 Tesla cross-sectionally before and 7, 14 and 21 days after infusion of Ang-II. Additionally, ApoE-/- infused with Angio-II (n=10) were scanned longitudinally for up to 4 weeks. Mice were imaged 30min post-injection of Gd-TESMA. 3D contrast-enhanced angiography and 3D gradient-echo late gadolinium enhancement (LGE) images were acquired with resolution=0.1×0.1x0.5mm. Elastin and tropoelastin histological stainings were used for validation.
Example of MR angiograms and LGE images, after administration of Gd-TESMA, in an animal infused with Angio-II for 3 weeks are shown in Figure 1. MRA images show the dilation of the aorta, with or without, formation of a false channel and LGE images show the enhancement of the vessel wall. Corresponding histology validates the remodeling of the aneurysmal tissue and the deposition of tropoelastin molecules. Tropoelastin accumulation occurs only within the remodelled aneurysmal wall but not in non-diseased aortic segments as seen by in vivo MRI using the it is validated histologically. Longitudinal MRA and LGE images show the growth of the AAA and the deposition of tropoelastin over time. Remodeling of tropoelastin occurred early in the development of the AAA (week 1), persisted and increased over time (Fig. 2). The rate of tropoelastin deposition correlated with the rate of AAA growth.
Molecular imaging of tropoelastin maybe a surrogate marker of AAA expansion.
1. Krettek, A., et al. ATVB, 2003.
2. Botnar M., et al., Circulation; Cardiov.Img. 2014.
3. Phinikaridou, A., et al., ISMRM proceedings, 2016.
British Heart Foundation
Figure 1: In vivo MRI of tropoelastin remodeling in AAA.
A: The reconstructed MRA shows the formation of AAAs. MRI after injection of Gd-TESMA and ex vivo histology show:
1) Low enhancement and lack of tropoelastin in normal aortic wall (B-G);
2) Dilated aortas, with or without false channels (FC), vascular enhancement and deposition of tropoelastin in the two AAAs (H-S).
3) Flow MRI showed retrograde blood flow in the FC (T-V).
Longitudinal remodeling of tropoelastin during AAA evolution by MRI
A: The rate of AAA growth correlates with the deposition of tropoelastin. B: When tropoelastin accumulated at a rate > 3mm3/wk, the growth rate of the AAA was higher than 10mm3/ wk and was significantly higher than the growth rate of the AAA when tropoelastin accumulated at a rate < 3mm3/week C: Vascular enhancement was higher in AAAs that expand over time compared with AAA that did not increase.
Keywords: molecular imaging, tropoelastin, elastin, MRI