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Manganese enhanced MRI can quantify myocardial infarct size earlier than gadolinium enhanced MRI (#81)
Nur H. Jasmin1, 2, May Zaw-Thin1, Mark F. Lythgoe1, Sean Davidson3, Daniel J. Stuckey1
1 University College London, Cantre for Advanced Biomedical Imaging, London, United Kingdom
Late gadolinium enhanced MRI (LGE-MRI) can quantify of infarct size after myocardial infarction (MI) but is non-specific and reflects the increased membrane rupture and extracellular space that develop post MI (1). Mn is a potent T1-contrast agent that enters myocytes through active calcium channels, thus reducing T1 in viable myocardium (2). This active process rapid ceases under ischemia. Hence, we hypothesised that Mn-enhanced MRI (MEMRI) could quantify final infarct size earlier than LGE-MRI and tested this by applying both methods to mice at 1 & 24 hours after myocardial infarction
Myocardial infarction was induced in 7 mice which then underwent MEMRI (n=4, 0.1mmol/kg MnCl2) or LGE-MRI (n=3, 0.5mmol/kg Gd-DTPA) at 1 hour post MI. All animals then underwent both MEMRI and LGE-MRI at ~24 hours post MI with a contrast washout period of at least 5 hours between scans. Imaging was performed using a 9.4T Agilent MRI system and a multi-slice inversion recovery sequence in the short-axis orientation to covered the whole left ventricle (TE/TR = 3.04/1.11ms, TI = ~600ms for MEMRI and ~350ms for LGE-MRI, 90⁰ excitation pulse, slice thickness = 1.0mm, FOV = 25.6 x 25.6 mm, matrix size = 128 x 128) as described (3).
At 1-hour post MI, viable myocardium was enhanced in MEMRI, allowing early delineation of the occlusion zone as 41 ± 8% of the myocardium, whereas only subtle enhancement was seen on LGE-MRI, yielding a significantly lower value of 12 ± 2% (P=0.03. Fig1). At ~24 hours post MI, the MEMRI measure of infarct size remained constant (41 ± 5%) whilst LGE-MRI significantly increased to a level comparable with MEMRI (37 ± 3%). Fig2 shows a direct comparison of MEMRI and LGE-MRI acquired in the same animal at 22 and 27 hours after MRI, respectively, with matching histological TTC staining for infarct size.
Effected myocytes rapidly stop internalising Mn under ischemic conditions, allowing early delineation of the occlusion zone. The membrane rupture that underlies LGE-MRI occurs later, meaning LGE-MRI underestimates the occlusion zone during the first hours post MI
The present study shows MEMRI can quantify final infarct size earlier than LGE-MRI. This provides a sensitive approach which could be used as an early measure of cell death and myocardial viability when assessing the efficacy of new drugs which target acute MI.
(1) Doltra, A., Amundsen, B. H., Gebker, R., Fleck, E., & Kelle, S. (2013). Emerging concepts for myocardial late gadolinium enhancement MRI. Current Cardiology Reviews, 9. 185
(2) Waghorn, B., Schumacher, A., Liu, J., Jacobs, S., Baba, A., Matsuda, T, Hu, T. C.-C. (2011). Indirectly probing Ca(2+) handling alterations following myocardial infarction in a murine model using T(1)-mapping manganese-enhanced magnetic resonance imaging. Magnetic Resonance in Medicine, 65. 239 .
(3) Chow A, Stuckey DJ, Kidher E, Rocco M, Jabbour RJ, Mansfield CA, Darzi A, Harding SE, Stevens MM, Athanasiou T (2017). Human induced pluripotent stem cell-derived cardiomyocyte encapsulating bioactive hydrogels improve rat heart function post myocardial infarction. Stem cell reports 9. 1415
Daniel J Stuckey would like to acknowledge the support of his BHF Fellowship FS/15/33/31608 and MRC Project Grant MR/R026416/1
Fig1: MEMRI and LGE-MRI acquired at 1 and 24 hours post MI
MEMRI or LGE-MRI acquired in the same mouse at 1 & 24h after myocardial infarction. At 1h post MI, Mn uptake in viable myocardium allowed the estimated final infarct area to be distinguished, whilst only subtle enhancement was seen in LGE-MRI resulting in a smaller measure of occlusion zone. At 24h post MI the infarct shows hypoenhancement in MEMRI and hyperenhancement in LGE of similar szes.
Fig2: Direct comparison of MEMRI and LGE-MRI
Fig2: Direct comparison of MEMRI and LGE-MRI acquired in the same animal at 22 and 27 hours after MI, respectively. Arrowhead shows hypoenhancement of the infarct in the MEMRI image and hyperenhancement of the infarct in LGE. In vivo data corresponded with TTC histological staining for infarct.
Keywords: MRI, cardiac, myocardial infarction, contrast, mouse
In vivo imaging of human monocytes reveals a distinct spatial infiltration pattern in the early and late inflammatory phase of mouse myocardial infarction (#254)
Janette Iking1, Lisa Honold2, Michael Kuhlmann2, Holger Reinecke1, Lars Stegger3, Johannes Waltenberger1, Michael Schäfers2, 3, Sven Hermann2, Evangelia Pardali1
1 University Hospital Münster, Dept. of Cardiology I – Coronary and Peripheral Vascular Disease, Heart Failure, Münster, North Rhine-Westphalia, Germany
Distinct infiltration patterns of human monocytes during the inflammatory and the proliferative phase of infarct healing have been shown post-mortem in human hearts. This could have an effect on the remodelling process and might be altered in patients with cardiovascular diseases, as monocytes isolated from these patients show defects in migration, extravasation, and adhesion. So far, no in vivo data is available concerning the accumulation of human monocytes in the infarcted heart. Here, we used an in vivo imaging approach to track human monocytes in mice with myocardial infarction (MI).
Human CD14++ CD16- monocytes were labelled with the 99mTc-HMPAO and the fluorescent dye DiD. Immunodeficient NOD/Scid mice underwent surgery for permanent ligation of the left anterior descending (LAD) coronary artery or a control sham surgery. The area at risk of infarction (AAR) was determined by myocardial perfusion SPECT/CT following surgery. On day 1 or 3 post-MI, 10x106 dual-labelled human monocytes were injected into the tail vein of operated NOD/Scid mice. In vivo SPECT/CT scans were acquired the next day approximately 18h post-injection (2 or 4 days post-MI). Subsequently, organs were explanted and further assessed for biodistribution of human monocytes by ex vivo SPECT/CT imaging, fluorescence reflectance imaging (FRI), autoradiography (AR), gamma counter analysis and histology.
Dual-labelled human monocytes were detected in vivo in the AAR by SPECT/CT imaging on day 2 and day 4 post-MI. In vivo and ex vivo SPECT/CT scans of the heart revealed that human monocytes mainly accumulated in the border zones of the AAR on day 2 post-MI. In contrast, on day 4 post-MI they accumulated more homogeneously within the infarct area. Similar results were obtained with ex vivo FRI of tissue sections. Quantification of the imaging data displayed significantly higher signals in the AAR of MI-operated hearts compared to remote myocardium and sham-operated hearts on both, day 2 and day 4 post-MI. In addition, linear regression analysis revealed that monocyte infiltration increases with infarct size indicating that infarct size is the main determinant for immune cell infiltration.
By using a non-invasive in vivo imaging approach we have shown that human inflammatory monocytes accumulate in distinct regions of the infarcted myocardium in different phases of healing following murine MI. These results are in accordance with observations made in ischaemic human hearts post-mortem indicating that the presented imaging model can be a valuable tool to further characterize human immune cells in vivo in the context of MI.
Pardali, E., Schmitz, T., Borgscheiper, A., Iking, J., Stegger, L., & Waltenberger, J. (2016). Cryopreservation of primary human monocytes does not negatively affect their functionality or their ability to be labelled with radionuclides: basis for molecular imaging and cell therapy. EJNMMI Research, 6(1), 77.
This research was funded by DFG, CRC 656 Münster, project C12 and the DSHF, project F732/13.
SPECT/CT imaging of human CD14++ monocytes in the murine heart.
In vivo (A) & ex vivo (C) SPECT/CT images showing an accumulation of the cells in the border zones of the area at risk of infarction (AAR) on day 2 post-MI (d2). In contrast, on day 4 post-MI (d4), the cells localized homogeneously within the AAR. Quantification of the data (B+D) showing the %ID/ml of the AAR relative to the respective remote myocardium (n=5-9). *p<0.05, **p<0.01 (Kruskal-Wallis).
Keywords: Monocytes, myocardial infarction, in vivo, SPECT, human
Ultrasonographic cardiovascular phenotyping in ApoE-/-Fbn1C1039G +/- murine model of vulnerable atherosclerotic plaque (#265)
Sara Gargiulo1, Sandra Albanese1, Chetan Dhakan1, Matteo Gramanzini1, Adele Ferro1, Flavio Cristofani2, Sharmila Fagoonee1, Lorenzo Silengo2, Fiorella Altruda2, Marcello Mancini1
1 National Council of Research, Institute of Biostructure and Bioimaging , Naples, Italy
Noninvasive cardiovascular phenotyping using state-of-the-art ultrasound technology is an high-sensitivity method for evaluating genes of interest and novel theranostic approaches in murine models of atherosclerosis. The ApoE-/-Fbn1C1039G +/- mouse is an emerging model of vulnerable atherosclerotic plaques, but the in vivo alterations of heart and vessels have not yet been described. To the best of our knowledge, this proof-of-concept study provides for the first time a morphological and functional characterization of cardiovascular system in the ApoE-/-Fbn1C1039G +/- mouse by ultrasonography.
High frequency ultrasound (HFUS) was carried out in ApoE-/-Fbn1C1039G +/- mice (DKO) fed a western-type diet at 16 and 21 weeks of age, and in sex- and age-matched C57Bl/6J control mice (CTRL) fed a standard diet. Vevo 2100-Visualsonics system equipped with a 40 MHz probe and isoflurane anesthesia was used to perform left ventricle (LV) B-mode, M-mode and speckle tracking strain (STS) imaging, as well as B-mode and pulsed wave Doppler scans of aortic arch, abdominal aorta, brachiocephalic and carotid arteries. Cardiac structure, systolic and diastolic function, global and regional myocardial deformation, and arterial elasticity were assessed. Indipendent Student’s test was used to determine statistical significance between DKO and CTRL mice (P <0.05).
Aortic arch pulse wave velocity was significantly higher in DKO than CTRL mice at 16 and 21 weeks of age. The velocity time integral of ascending, descending and abdominal aorta, as of brachiocephalic and carotid arteries were significantly decreased in 21 weeks DKO mice. LV mass, cardiac output and stroke volume, normalized to body weight, were significantly increased in 16 weeks DKO mice. Afterwards, normalized LV diameter and volume in systole and Tei index were significantly decreased in 21 weeks DKO mice. Fractional shortening did not showed a significant difference between DKO and CTRL mice at both 16 and 21 weeks of age. However, LV wall global longitudinal strain decreasing trend was observed in DKO mice, according to age, while global radial strain and strain rate resulted significantly lower at 21 weeks. Regional radial strain and strain rate of LV anterior and posterior wall segments as well the circumferential strain rate were significantly decreased in 21 weeks DKO mice.
These results support that DKO mice develop LV hypertrophy, as a compensatory mechanism related to increased arterial stiffness and afterload. Afterwards, diastolic dysfunction and arteries dilation occurr. STS analysis showed more accuracy than conventional echocardiography to capture subtle changes in DKO mice LV function. Advanced HFUS technology is useful for multiparametric cardiovascular mouse characterization in early atherogenesis stages.
Van der Donckt C, Van Herck JL, Schrijvers DM, Vanhoutte G, Verhoye M, Blockx I, Van Der Linden A, Bauters D, Lijnen HR, Sluimer JC, Roth L, Van Hove CE, Fransen P, Knaapen MW, Hervent AS, De Keulenaer GW, Bult H, Martinet W, Herman AG, De Meyer GR. Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death. Eur Heart J. 2015;36(17):1049-58.
Van Herck JL, De Meyer GR, Martinet W, Van Hove CE, Foubert K, Theunis MH, Apers S, Bult H, Vrints CJ, Herman AG. Impaired fibrillin-1 function promotes features of plaque instability in apolipoprotein E-deficient mice. Circulation. 2009;120(24):2478-87.
Lee L, Cui JZ, Cua M, Esfandiarei M, Sheng X, Chui WA, Xu MH, Sarunic MV, Beg MF, van Breemen C, Sandor GG, Tibbits GF. Aortic and Cardiac Structure and Function Using High-Resolution Echocardiography and Optical Coherence Tomography in a Mouse Model of Marfan Syndrome. PLoS One. 2016;11(11):e0164778.
Schaefer A, Meyer GP, Hilfiker-Kleiner D, Brand B, Drexler H, Klein G. Evaluation of Tissue Doppler Tei index for global left ventricular function in mice after myocardial infarction: comparison with Pulsed Doppler Tei index. Eur J Echocardiogr. 2005;6(5):367-75.
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Figure 1. Representative images on the Vevo 2100 system (Visualsonics, ON, Toronto, Canada)
Evaluation of left ventricular systolic and diastolic function
A) B-mode left ventricle short axis at the level of papillary miscles and tissue doppler imaging trace in corrispondence of posterior wall.
B) B-mode left ventricle long axis and short axis scans with velocity vectrors and corresponding wall Colour M-mode map for speckle tracking-based strain analysis
Keywords: mouse model of vulnerable plaque, cardiovascular phenotyping, High frequency ultrasound (HFUS)
Multimodal in vivo imaging highlights novel features of cardiovascular abnormalities in a new mouse model for Williams syndrome (#448)
Jeroen Essers1, Nicole van Vliet1, Yanto Ridwan1, Laurens Bosman2, Chris de Zeeuw2
1 Erasmus MC, Molecular Genetics, Rotterdam, Netherlands
Williams-Beuren syndrome (WBS) is a developmental disorder that is characterized by distinct facial features, intellectual disability and cardiovascular abnormalities. The underlying etiopathogenesis of this rare disease is due to the de novo microdeletion of a region spanning up to 27 genes, including the elastin gene that is responsible for the elasticity of the arterial wall. Cardiovascular abnormalities encountered in WBS are supravalvular aortic stenosis as well as coronary artery stenosis. Here, we investigated cardiovascular abnormalities in a new WBS mouse model.
We used ultrasound imaging (vevo3100) to determine aortic dimensions, aortic distensibility and left ventricular ejection fraction (LVEF) in a new mouse model with a 18 gene deletion spanning the WBS region. In order to define biomarkers related to cardiac and vascular alterations, we tested the combined use of XCT-FMT using near infrared fluorescent (NIRF) probes. WBS mice and control mice were imaged in vivo using fast and low dose microCT scanning (QuantumFX, Perkin Elmer) and near infrared (NIRF) probes to monitor tissue remodeling activity (MMPsense680). Immediate contrast enhanced CT imaging using EXIA160 during its blood-pool phase allowed registration of changes in ventricular anatomy and of important global parameters, like end-diastolic volume (EDV) and end-systolic volume (ESV).
Extensive ultrasound and microCT analysis showed that aortas of WBS mice exhibited striking tortuosity, an increased length and a smaller internal diameter of the ascending aorta compared to control mice. Functional ultrasound analysis showed that WBS mice had a lower aortic distensibility, and an unexpected but significant increase in LVEF. These anatomical aberrations prompted us to analyze possible differences in remodeling activity in these WBS mice using smart optical probes that report on the activity of matrix metalloproteases; MMPs (MMPsense680, Perkin Elmer). While we detected less MMP activity in the aorta of WBS mice, an increase of MMP activity in the heart region was seen, which appeared to be due to specific activation of the MMPsense probe in the coronaries. Coronary insufﬁciency in WBS puts the patient at high risk of sudden death due to drop of coronary perfusion pressure which may occur during cardiac catheterization or after induction of anesthesia.
Arterial stenosis is a concern for the majority of patients with WBS. Affected patients require careful observation either with echocardiography or other noninvasive imaging. Molecular, non-invasive evaluation of complex cardiovascular abnormalities of WBS including coronary artery disease is feasible with novel probes and could provide accurate information for planning and noninvasive assessment of arterial changes in WBS patients.
Quantitative in and ex vivo imaging of cardiovascular failure in a WBS mouse model.
(A) In vivo microCT visualization of aorta anatomy in a control and WBS mouse highlighting severe tortuosity and lengthening of the aorta (B) Ex-vivo fluorescence analysis of the aorta and heart using a near-infrared MMP activity imaging (MMPsense680). Note the specific labeling of the coronary artery in the WBS mouse (C) Quantification of the MMPsense signal in heart and aorta
Keywords: Williams-Beuren, cardiovascular, microCT, ultrasound, optical imaging
Imaging of hydrogel biomaterials for myocardial regenerative medicine with Magnetization Transfer MRI (#349)
Vitaliy Khlebnikov1, Klaus Neef1, Annette van der Toorn1, Rick Dijkhuizen1, Caroline van Heijningen1, Patricia Dankers2, Carlijn Bouten2, Steven Chamuleau1, Dennis Klomp1, Jeanine J. Prompers1
1 University Medical Center Utrecht, Utrecht, Netherlands
The function of myocardial tissue post-infarction may be restored with the use of tissue engineering. An injectable hydrogel based on poly(ethylene glycol) modified with ureido-pyrimidinone (UPy) moieties (Fig.1a) was proposed as a new delivery system for therapeutic molecules and cells.1 UPy-gel is a pH-switchable hydrogel. At pH≥8.5, it is in a liquid state and can be injected through a catheter, whereas at physiological pH it forms a gel. In this study we developed a non-invasive MRI protocol not only capable of imaging hydrogel biomaterials, but also their pH-switchable sol-gel behavior.
Sample preparation: Liquid (pH≈9) and gelated (pH≈7) were prepared by dissolving UPy-gel in PBS at 3 concentrations (5%, 7.5% and 10% (w/w)).
Ex vivo hearts: UPy10liquid was injected into the myocardial tissue (ventricular wall) of adult rats after isoflurane overdose.
Magnetization transfer (MT) MRI experiments: Z-spectra were acquired by applying a saturation module composed of 100 Gaussian pulses (20ms each) at 137 frequency offsets with varying power levels.
Data analysis: To distinguish two different states of the UPy10 biomaterial, i.e. liquid versus gelated, an artificial neural network (NN) was trained on the Z-spectra from in vitro (Z-spectra for UPy10liquid and UPy10gel) and ex vivo (Z-spectra for the myocardial tissue) experiments.
The UPy-gel generated a detectable MT effect in vitro (Fig.1b). The MT effect was higher in the gelated state when compared with the corresponding liquid biomaterial and scaled with the power of the saturation module. A power level as low as 15dB was sufficient to distinguish two different states of the UPy10 biomaterials. For comparison, we also performed imaging with more traditional MRI contrast, i.e. quantitative T1, T2-weighted, and diffusion-weighted MRI, but none of those was able to differentiate between the liquid and gelated states of the biomaterials. To test the pH-switchable behavior of the UPy-gel, two experiments were performed where UPy10liquid (100μL and 50μL, respectively) was injected in the myocardial tissue of a beating rat heart (Fig.2). A gelated rim and a liquid core (likely a result of hindered diffusion) were identified at the injection site of 100μL of UPy10liquid, whereas at the injection site of 50μL of UPy10liquid, only the gelated state was identified.
We developed and validated a MT-MRI protocol for imaging of hydrogel biomaterials and their pH-switchable behavior.
1. Bastings, M. M. C. et al. A fast pH-switchable and self-healing supramolecular hydrogel carrier for guided, local catheter injection in the infarcted myocardium. Adv Healthc Mater 3, 70–78 (2014).
(a) A schematic of bifunctional UPy-PEG-UPy hydrogelator (Mn(PEG) = 10 kDa). (b) In vitro experiments. MT maps as a function of power (x-axis, in dB) of the MT prepulse for different concentrations of biomaterials: UPy5liquid (5% w/w, liquid), UPy5gel (5% w/w, gelated), UPy7.5liquid (7.5% w/w, liquid), UPy7.5gel (7.5% w/w, gelated), UPy10liquid (10% w/w, liquid), UPy10gel (10% w/w, gelated).
Figure 2. Ex vivo experiments.
MRI images showing the injection site of (a) 100µL and (d) 50µL UPy10liquid (10%, w/w) biomaterial in the rat myocardium. (d) is accompanied with a control-liquid biomaterial (Upy10liquid). (b) and (e) Z spectra at a B1 of 24dB averaged over the hydrogel voxels from the injection site for (a) and (d), respectively. (c) and (f) profiles of the predicted states for (a) and (d), respectively.
Keywords: Myocardial tissue engineering, Hydrogel, Magnetic resonance imaging, Magnetization transfer, Myocardial infarction
Evaluation of [68Ga]Ga-NODAGA-RGD for PET Imaging of Rat Autoimmune Myocarditis (#346)
Arghavan Jahandideh1, 3, Mia Ståhle1, Xiang-Guo Li1, 6, Heidi Liljenbäck1, 2, Juhani Knuuti1, 3, Anne Roivainen1, 2, 3, Antti Saraste1, 3, 4
1 University of Turku, Turku PET Centre, Turku, Finland
Positron emission tomography (PET) imaging of myocarditis has been challenging due to high physiological uptake of 2-deoxy2-[18F]fluoroglucose tracer in the myocardium1. Therefore, new PET tracers are needed. The αvβ3 integrin receptor is expressed in angiogenic endothelial cells, macrophages and myofibroblasts during myocardial injury and inflammatory response 2,3. Arginyl-glycyl-aspartic acid (RGD) motif containing peptides can detect αvβ3 integrin expression. In this study, [68Ga]Ga-NODAGA-RGD PET for the detection of αvβ3 integrin expression in autoimmune myocarditis in rats was evaluated.
Rats (n=6) were immunized twice on day 0 and 7 with subcutaneous injection of porcine cardiac myosin in an equal volume of complete Freund’s adjuvant supplemented with mycobacterium tuberculosis and i.p. pertussis toxin injection. Control rats (n=8) were injected with complete Freund’s adjuvant alone. Twenty min static PET imaging at 60-80 min after i.v. [68Ga]Ga-NODAGA-RGD (50 ± 2.7 MBq) injection was performed on day 21 post-immunization followed by autoradiography and histological analysis of excised heart.
Focal myocardial inflammatory lesions containing high density of macrophages were detected in 5 of immunized rats. In the immunized rats, PET imaging revealed increased target-to-background ratio (maximum standardized uptake value in myocardium/mean standardized uptake value in blood) compared to control rats (2.3 ± 0.47 vs. 1.2 ± 0.15, respectively; P < 0.05).
[68Ga]Ga-NODAGA-RGD shows accumulation in cardiac inflammatory lesions in a rat model of autoimmune myocarditis. Our result indicates that, as a novel application, [68Ga]Ga-NODAGA-RGD is a potential PET tracer for detection and monitoring of active myocarditis.
The authors would like to thank Aake Honkaniemi and Erica Nyman (Histology Unit of Turku Center for Disease Modeling) for their assistance.
Autoimmune myocarditis in rat
Representative short axis histological sections of the left ventricle stained with hematoxylin and eosin (HE) or anti-CD68 antibody detecting macrophages in a rat with autoimmune myocarditis (A) and a control rat (B). In A, focal lesion close to the epicardium (red arrows) shows dense inflammatory cell infiltrate and myocyte damage at high magnification.
Keywords: Positron emission tomography, Myocarditis, Experimental autoimmune myocarditis
In vivo High Frequency Ultrasound detection and analysis of atherosclerotic lesions in ApoE-/-Fbn1C1039G +/- murine model of vulnerable plaque (#281)
Sandra Albanese1, Matteo Gramanzini1, Chetan Dhakan1, Juan C. Cutrin2, Valeria Bitonto2, Flavio Cristofani2, Sharmila Fagoonee1, Lorenzo Silengo2, Fiorella Altruda2, Sara Gargiulo1, Marcello Mancini1
1 National Council of Research, Institute of Biostructure and Bioimaging , Naples, Italy
Noninvasive imaging in mouse models of atherosclerosis has become increasingly important in vascular characterization at early disease stages. Recently, double knockout ApoE-/-Fbn1C1039G +/- mouse (DKO), fed a western type diet, was established as relevant model of human-like vulnerable atherosclerotic plaques, but almost all of the studies focus on ex vivo analysis. To the best of our knowledge, this pilot study is the first describing detection and monitoring of atherosclerotic plaques, and local hemodynamics, in living ApoE-/-Fbn1C1039G +/- mice by means of High Frequency Ultrasound (HFUS).
ApoE-/-Fbn1C1039G +/- mice, feeding 13 weeks of high-fat diet, were examined by HFUS at 16 and 21 weeks of age. Sex- and age-matched C57Bl/6J mice fed a standard diet were used as controls (CTRL). Vevo 2100-Visualsonics system (40 MHz probe) was used to perform aortic arch long-axis view, and lesion-prone sites were examined. Intima-media thickness (IMT) was measured as the distance between the leading edge of the lumen-intima echo and the one of the media-adventitia echo. Lesions were outlined and maximal plaque thickness (MPT) and area (PA) were calculated by leading-to-leading edge approach. Spatio-temporal changes of wall shear stress (WSS) were measured in the ascending and descending aorta. In vivo findings were compared with histology. T-test was used for comparisons (P <0.05).
IMT, an established surrogate marker for human atherosclerosis, was significantly increased for aortic arch lesser curvature in DKO from 16 to 21 weeks of age (p=0.04). Moreover, IMT was significantly increased in 21 weeks DKO at the level of aortic arch greater (p = 0.01) and lesser curvature (p = 0.005). Atherosclerotic plaques were visible in DKO from 16 weeks of age at the level of aortic arch lesser curvature and at the origin of the brachiocephalic artery, as more echolucent and elevated areas than surrounding normal wall and corresponding sites in matched CTRL. MPT and PA showed an increasing trend overtime in DKO from 16 to 21 weeks of age. WSS showed a decreasing trend overtime in DKO, probably related to disease progression, reaching a statistical significance at the level of ascending aorta (t-test p = 0.0002) proximally to detected plaques. Histological analysis confirmed the presence of aortic arch atherosclerotic lesions in DKO. No lesions were observed in CTRL mice.
This study supports the feasibility of noninvasively assessing atherosclerotic plaques and hemodynamic state in ApoE-/-Fbn1C1039G +/- mice in the early atherogenesis using HFUS. By measuring IMT, PMT and PA in the aortic arch, and WSS in the region proximal and distal to lesion sites, HFUS allowed an exploratory monitoring of disease progression in the DKO murine model of vulnerable plaque under investigation.
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Figure 1. Assessment of atherosclerotic plaques in ApoE-/-Fbn1C1039G+/- murine model
B-Mode longitudinal (A) and trasversal (B) scans (Vevo 2100 system, Visualsonics, ON, Toronto, Canada) of aortic arch in DKO mouse, showing an atherosclerotic plaque (arrow) at the level of the lesser curvature, confirmed by Hematoxylin and eosin (C) and Oil Red-O staining (D) histological sections (x100). Corresponding ultrasonographic (E, F) and histological (G, H) evaluation in healthy CTRL.
Keywords: mouse model of vulnerable plaque, plaque characterization, High Frequency Ultrasound (HFUS)
Evaluation of 18F-FTHA as cardiac metabolism tracer for preclinical PET imaging (#574)
Mario González1, Juan Pellico1, Lorena Cussó1, 2, 3, Daniel Calle1, 2, Borja Ibáñez1, Manuel Desco1, 2, 3, Beatriz Salinas1, 2, 3
1 Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
Free fatty acids (FFA) are the primary source of energy in the myocardium. In the development of new radiotracers based on the FFA, 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid (18F-FTHA) has been widely evaluated in pig and clinical studies [1-4]. However, its evaluation in small animals is almost nonexistent, with only a few studies about the use of the tracer for metabolic quantification but without imaging evidences. In this work, we evaluate in vivo pharmacokinetic properties as well as optimal acquisition conditions for the use of this tracer as cardiac imaging agent on mice.
18F-FTHA was synthetized by nucleophilic substitution of the tosyl commercial precursor (90ºC, 8min) and further C-18 sep-pack purification . In vitro stability in PBS (37ºC) of the tracer was measured by HPLC each hour during four hours. Pharmacokinetic evaluation of the radioactive fatty acid was carried out in healthy C57BL/6 mice by ex vivo biodistribution (BD) studies (5min, 20 min and 60 min post injection) and blood half-life analysis by blood extraction during 180 min. Plasma Protein Binding (PPB) was assessed ex vivo on mouse blood sample (0.77 MBq, 0.5 mL, 30 min incubation). Finally, in vivo PET/CT imaging was obtained on C57BL/6 mice (n=4), evaluating different acquisition conditions (uptake times: 5,10,15,20 and 60 min) and fasting/non-fasting conditions.
18F-FTHA was successfully synthetized with a 10% radio-chemical yield, as in previous references . In vitro stability evaluation showed a 7.7% degradation of the tracer after 2h, increasing to 15.2% after 4h. Circulation profile confirmed fast clearance of the tracer, with blood half-life values lower than 5 min. PPB evaluation shown strong affinity between the radioactive fatty acid and plasma proteins, reaching values of 91.92% protein binding. Ex vivo BD studies showed uptake mainly in myocardium and liver, with highest values at 5 min (20.4±3.6% and ID/g31.6±4.8% respectively). Significant decrease in heart uptake is observed at longer time points (60 min= 12.61±7.20%). These results were confirmed by PET imaging, showing in vivo high and specific myocardium uptake as well as hepatic metabolism. Optimal uptake was observed 5 min post injection. In the preliminary evaluation of fasting effect, no significant differences were observed between fasting and non-fasting mice.
This is the first time the radiotracer 18F-FTHA is presented as cardiac metabolism imaging agent for PET on rodents. We successfully optimized acquisition parameter and performed a complete physical-chemical characterization of the radiotracer proving its promising use as PET tracer for cardiac imaging on mice.
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 TAYLOR, Michael, et al. An evaluation of myocardial fatty acid and glucose uptake using PET with [18F] fluoro-6-thia-heptadecanoic acid and [18F] FDG in patients with congestive heart failure. Journal of Nuclear Medicine, 2001, vol. 42, no 1, p. 55-62.
 TAKALA, Teemu, et al. 14 (R, S)-[18 F] Fluoro-6-thia-heptadecanoic acid as a tracer of free fatty acid uptake and oxidation in myocardium and skeletal muscle. European journal of nuclear medicine and molecular imaging, 2002, vol. 29, no 12, p. 1617-1622.
 DEGRADO, Timothy R.; COENEN, Heinz H.; STOCKLIN, G. 14 (R, S)-[18F] fluoro-6-thia-heptadecanoic acid (FTHA): evaluation in mouse of a new probe of myocardial utilization of long chain fatty acids. Journal of nuclear medicine: official publication, Society of Nuclear Medicine, 1991, vol. 32, no 10, p. 1888-1896.
 DEGRADO, Timothy R. Synthesis of 14 (R, S)‐[18F] fluoro‐6‐thia‐heptadecanoic acid (FTHA). Journal of Labelled Compounds and Radiopharmaceuticals, 1991, vol. 29, no 9, p. 989-995.
This work has been supported by the Advanced Imaging Unit (UIA) of Centro Nacional de Investigaciones Cardiovasculares (CNIC), CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505) and Small Animal Image Core at Hospital General Gregorio Marañón (HGGM), Madrid, Spain. This work was partially supported by Comunidad de Madrid (S2017/BMD-3867 RENIM-CM, co-financed by European Structural and Investment Funds), and Ministry of Science, Innovation and Universities (ISCIII-FIS grants PI16/02037, co-financed by ERDF, FEDER, Funds from the European Commission, “A way of making Europe”)
Figure 1. Physicochemical characterization and pharmacokinetic evaluation of 18F-FTHA.
A) Synthesis of 18F-FTHA. B) HPLC chromatogram of purified 18F-FTHA. C) In vitro stability evaluation of the tracer in PBS by HPLC. D) Circulation blood half-life profile of 18F-FTHA in healthy mice. E) Ex vivo biodistribution studies at 5 min, 20 min and 60 min post injection.
Keywords: PET, Fatty acid, Tracer, Cardiac metabolism
Murine Atherosclerosis Characterization Using Lipid-Specific Photoacoustic Imaging and 4D Ultrasound Strain Mapping (#582)
Gurneet S. Sangha1, Katherine Leyba1, Craig J. Goergen1
1 Purdue University, Biomedical Engineering, West Lafayette, Indiana, United States of America
Photoacoustic tomography (PAT) and 4D ultrasound (4DUS) imaging have recently been used to study cardiovascular disease in small animals [1-3]. PAT uses pulsed laser light induced acoustic waves to reconstruct lipid-specific compositional images. 4DUS captures dynamic volumetric information and can be used to estimate 3D Green-Lagrange strain using a direct deformation estimation method . We aim to use a combined PAT/4DUS approach to correlate changes in arterial strain and hemodynamics with lipid localization in mice that have undergone partial carotid ligation induced-atherosclerosis.
A 40 MHz small animal transducer (Vevo2100, VisualSonics) and a Nd:YAG pulsed laser (Surelite EX, Continuum) were used to image five apolipoprotein-E deficient male mice that underwent partial carotid ligation of the left carotid artery while being fed a Western diet . Animals were imaged using ultrasound at days 0, 1, 4, 7, 10, and 14 to obtain long-axis B-mode, M-mode, and pulsed-wave Doppler for morphological and hemodynamic characterization, as well as imaged via 4DUS for strain mapping. At day 14 all animals were euthanized and 3D in situ PAT images of the left carotid artery were acquired using 1210nm light.
Overall the results show that atherosclerotic lesions can be characterized via PAT/4DUS to localize both lipid accumulation and identify regions of low strain. We observed rapid decrease in left carotid artery peak velocity between baseline and day 1 imaging (64±3.8 cm/s to 18.6±7.2 cm/s). Additionally, baseline and day 5 imaging showed a steady decrease in the left carotid artery circumferential cyclic strain (31±6.2% to 11±2.5%) and lumen diameter (0.48±0.03 mm to 0.32±0.05 mm), as well as an increase in right carotid artery diameter (0.48±0.04 mm to 0.56±0.03 mm). Strain mapping from 4DUS images showed heterogenous maximum first principal strain values in both the left (Figure 1) and right carotid artery. The 3D in situ PAT imaging revealed lipid signal from atherosclerotic plaque accumulation in the left carotid artery with no signs of lipid accumulation in the contralateral control right carotid artery (Figure 2).
Dual-modality PAT/4DUS can be used to quantify changes in carotid artery hemodynamics and morphology, as well as lipid localization in a murine model of atherosclerosis. Future work will focus on utilizing this technique for cross-sectional studies to better understand how vessel hemodynamics and mechanics impact changes in atherosclerotic plaque composition.
 Sangha, Gurneet S., Evan H. Phillips, and Craig J. Goergen. "In vivo photoacoustic lipid imaging in mice using the second near-infrared window." Biomedical optics express 8.2 (2017): 736-742.
 Soepriatna, Arvin H., et al. "Cardiac and respiratory-gated volumetric murine ultrasound." The international journal of cardiovascular imaging (2018): 1-12.
 Damen, Frederick W., et al. "High-Frequency 4-Dimensional Ultrasound (4DUS): A Reliable Method for Assessing Murine Cardiac Function." Tomography: a journal for imaging research 3.4 (2017): 180-187.
 Cebull, Hannah., “Strain mapping from 4D ultrasound reveals complex remodeling in dissecting murine abdominal aortic aneurysms.” Journal of Biomechanical Engineering (2018). In Review.
 Nam, Douglas, et al. "Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis." American Journal of Physiology-Heart and Circulatory Physiology 297.4 (2009): H1535.
Funding was provided to Gurneet S. Sangha through the NSF Graduate Research Fellowship (DGE-1333468)
Keywords: photoacoustic, atherosclerosis, murine, ultrasound, imaging
Single chain-antibody variable fragments for the molecular imaging of oxidized LDL in atherosclerosis (#111)
Samata S. Pandey1, 2, Michael Mullin3, Laura Vazquez-Martinez1, Cleo Kontoravdi2, Dorian Haskard1, Ramzi Khamis1
1 National Heart and Lung Institute, Imperial College London, London, United Kingdom
Atherosclerosis is the underlying pathophysiology of the world’s leading cause of death, ischemic heart disease. The high specificity of monoclonal antibodies (mAbs) makes them promising imaging agents for the molecular imaging of atherosclerosis. However, the high molecular weight of full mAbs hinders their ability to be effectively used as biological imaging agents, due to lower tissue penetrance and slower kinetics. Furthermore, many mAbs are hybridoma-derived and thus pose an immunological risk due to their murine origins.
LO1, a monoclonal autoantibody with specificity for malondialdehyde-modified low-density lipoprotein (MDA-LDL) was isolated from a non-immunized LDL receptor deficient mouse (LDLR-/-). Characterization by ELISA, immunohistochemistry on human and mouse atherosclerotic tissue and in vivo imaging using a Near-Infrared Fluorescence (NIRF) fluorophore demonstrated binding to atherosclerotic lesions. In a translational effort, antibody fragments of LO1 were generated. LO1 single-chain variable fragments (scFv) and LO1-humanized Fab (huFab) were expressed in mammalian cells. The humanized Fab was generated using CDR grafting and backmutations. Cys-tags were included on both constructs for labelling of the construct with a NIRF fluorophore using maleimide chemistry.
ScFv and Fabs are a fifth and third of the size of a mAb, respectively, whilst both retaining a full antigen binding site. In vitro characterization of LO1-scFv and LO1-huFab by ELISA and immunohistochemistry demonstrated retention of function and binding of LO1-scFv and LO1-huFab to atherosclerotic lesions and thrombus in mouse tissue sections (Figure 1). Control constructs demonstrated lysines in CDR-H3 to be crucial for binding to MDA-LDL. Humanization of the Fab allows for repeated dosing due to the lower threat of immunogenicity.
In this study, we demonstrated that antibody fragments of LO1 retain function to antigen and thereby have the potential to be used as molecular imaging tools and for future therapeutic targeting of oxidized LDL. Ongoing further work involves in vivo testing of constructs in atherosclerotic LDLR-/- mice for lesional staining.
Keywords: single-chain variable fragment, atherosclerosis, humanization, molecular imaging