3D quantitative MRI of aerosolized Gd_based AGuIX nanoparticles in isolated ventilated pig lungs (#535)
Yannick Crémillieux1, Yoann Montigaud2, Clémence Bal3, Noël Pinaud1, Vi Pham1, François Lux4, Olivier Tillement4, Bei Zhang5, Jérémie Pourchez2
1 Université de Bordeaux, CNRS, Institut des Sciences Moléculaires, Bordeaux, France
2 Mines Saint-Etienne, Univ Lyon, Univ Jean Monnet, Inserm, Sainbiose, Centre CIS, Saint-Etienne, France
3 Université de Bordeaux, Bordeaux, France
4 Université de Lyon, Institut Matière Lumière, Villeurbanne, France
5 Canon Medical Systems Europe, Zoetermeer, Netherlands
Aerosol therapy represents an attractive and efficient administration route for delivering therapeutic compounds either locally or systemically (1). Indeed, the lungs present a large surface area for drug absorption and an extensive vasculature with a weak anatomical barrier that does not limit access to the body (2). MR imaging of nebulized theranostic nanoparticles (NP) in the lung of small animal was demonstrated previously (3). Here we report MR imaging of the distribution of aerosolized Gd_based theranostic AGuIX NP, already used in patients, in a model of isolated ventilated pig lungs.
Aerosol was administered to ex vivo porcine lung connected to a 3D-printed human head replica and respiratory tract (4). The lungs were ventilated by passive expansion, simulating pleural depressions and was synchonized to aerosol administration generated with a medical jet nebulizer (Fig.1). Solutions (6 ml) of AGuIX (NHTheAguix, France) nanoparticles were aerosolized during 15 minutes. AGuIX NP is composed of a polysiloxane core with 10 DOTA cyclic ligands chelating Gd ion (5). Its diameter is 3 ± 0.1 nm, its mass 8.5 ± 1 kDa and its relaxivity r1 at 3 Tesla is equal to 8.9 mM-1.s-1 per Gd ion. MRI acquisitions were performed at 3 Tesla (Vantage Galan 3T ZGO, Canon Medical Systems Corporation, Japan) using a 3D UTE sequence with a total acquisition time of 3.5 min (TR=3.7ms, TE=96μs).
In this lung model, large signal enhancement (SE) of the lung was observed after aerosolization of 6 ml solution containing 1.8 mmol of Gd3+. As a point of comparison, the dose of i.v. administered Gd3+ with clinical contrast agent in patient is in the order of 8 mmol. The SE is conspicuous for the whole lung up to the distal regions of the lung (distribution similar to that previously reported with SPECT techniques in ref. 4) with a more pronounced SE in the airways. The large SE can be appraised in Fig. 2 where the left lung was clamped during aerosolization. The concentration of Gd3+, assessed from 4 consecutive 3D UTE acquisitions at different flip angles, was evaluated, in regions excluding large airways, to be 130 μM. Considering a total lung tissue volume of 1 liter, it can be estimated that 7 % of Gd3+ ion have diffused into the lung tissue and contribute to the SE in our experimental set-up, the missing fraction being located in the upper respiratory tract and large airways.
We demonstrate In this study that the distribution of aerosolized Gd_based nanoparticles can be vizualized and quantified using MRI in large animal ventilated lung model. This protocol can be used for assessing aerosol deposition with high spatial resolution (1 mm 3D isotropic) without ionizing radiation. Moreover, the AGuIX NPs have well known radiosentitizing properties (6, 7) that can be applied for enhancing radiotherapy to treat lung cancer.
- Dolovich MB et al. Device selection and outcomes of aerosol therapy: Evidence-based guidelines: American College of Chest Physicians/American College of Asthma, Allergy, and Immunology. Chest. 2005 Jan;127(1):335-71.
- Choi HS, Ashitate Y, Lee JH, et al. Rapid translocation of nanoparticles from the lung airspaces to the body. Nat Biotech 2010;28:1300–1303
- Bianchi A, Lux F, Tillement O, Crémillieux Y. Contrast enhanced lung MRI in mice using ultra-short echo time radial imaging and intratracheally administrated Gd-DOTA-based nanoparticles. Magnetic Resonance in Medicine. 2013;70:1419-26
- Perinel S, Pourchez et al. Development of an ex vivo human-porcine respiratory model for preclinica studies.Sci Rep. 2017 Feb 24;7:43121.
- Lux F et al. Ultrasmall rigid particles as multimodal probes for medical applications. Angew Chem Int Ed Engl. 2011 Dec 16;50(51):12299-303.
- A. Bianchi, S. Dufort, F. Lux F, P.Y. Fortin, N. Tassali, O. Tillement, J.L. Coll, Y. Crémillieux. Targeting and in vivo imaging of non-small-cell lung cancer using nebulized multimodal contrast agents. Proc Natl Acad Sci U S A. 111 9247-9252 (2014).
- Lux F. et al. AGuIX(®) from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine. Br J Radiol. 18:20180365 (2018).
The authors are grateful to the company NH TherAguix for providing AGuIX nanoparticles. This study was carried out within the IBIO (Institut de Bio-Imagerie de Bordeaux).
Overview of the experimental set-up with the ENT (ear, nose, throat) replica and the pig lung in the sealed enclosure. The pump for lung ventilation and aerosol device are not shown in the picture.
Coronal slice from a UTE 3D data set acquired after aerosolization of 6 ml solution of AGuIX nanoparticles. Left bronchus was clamped during the aerosolization.
Keywords: Gd_based nanoparticle, aerosol, MRI, Lung, theranostic nanoparticle
Magnetic Resonance Imaging-observable Phenotypic and Functional Changes associated with Steatosis Development in the Mouse Liver (#15)
Frauke Conny Waschkies1, 2, Lukas Frick1, Bostjan Humar1, Udo Ungethuem1, Rolf Graf1, Pierre-Alain Clavien1
1 University Hospital Zurich, Visceral- and Transplantation Surgery, Zurich, Switzerland
2 ETH and University of Zurich, Institute for Biomedical Engineering, Zurich, Switzerland
Capacity of the liver for regeneration is adversely affected by preconditions such as steatosis or fibrosis – a risk factor for postoperative liver failure. For this purpose it is important to monitor hepatic functional reserve and liver regeneration. BOLD MRI may assess early changes pertaining to microenvironmental changes in tissue oxygenation, perfusion & metabolism, yet studies in liver are scarce [1,2]. Here we report on response of the liver to vasodilatory challenges in three animal groups, under control diet (CD), high-fat diet (HFD) and HFD substituted with Ω3 (HFD+Ω3).
MR imaging was performed on a 4.7T Bruker Pharmascan unit. Mice were intubated, mechanically ventilated and maintained under 2% isoflurane anesthesia. 4-5 male C57/B6 mice were studied in each group. T1w, ip/op FLASH images with and w/o Magnetization Transfer (MT) prepulse and multi-echo gradient-echo images for T2* maps (qT2*) were recorded and BOLD response to hypercapnic and pharmacological (acetazolamide, Diamox™ parenteral, Goldschield Pharm.) stimulus quantified. Signal fat fraction, MT ratio, basal qT2* and BOLD response were computed; graphs & statics were generated with R v3.5.0.
Abdominal MR images were obtained from all three animal groups (Fig. 1), depicting different contrasts pertaining to changes in water-fat content (signal fat fraction), altered protein-water interaction (MT ratio), alterations in basal oxygenation/perfusion and/or iron load (qT2*) and the ability to respond to a functional challenge (BOLD). ROI analysis was performed for quantitative assessment of these changes, shown here for the left lateral liver lobe (Fig. 2). HFD group displayed the highest fat fraction and lowest MT ratio, however, MTR may be affected by presence of fat. Interestingly, basal qT2* was lowest in this group, and is suggestive for less efficient perfusion and/or lower oxygenation status, but may also relate to iron accumulation, as implicated for NAFLD . Increased (rather than the expected decreased) BOLD response might relate to the drop in baseline. HFD+Ω3 group was similarly affected, though to a much lesser extent.
BOLD MRI in response to vasodilatory challenge assesses microenvironmental changes in tissue oxygenation, perfusion and metabolism and combination with structural MRI markers allows a comprehensive characterization of pathological changes during steatosis development in mouse liver. However, our preliminary findings require substantiation in larger animal cohorts and by corroborative histology.
 Ganesh et al. J Magn Reson Imaging 2016. 44(2): 305-16.
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 Britton et al. World J Gastroenterol. 2016 22(36): 8112-8122
Fig. 1: MRI-based characterization of steatosis-associated changes in the mouse liver.
Sample liver images of each a mouse under control diet (CD, top), HFD (HFD, middle) and Omega-3-based HFD for two weeks (HFD+Ω3, bottom row). Standard anatomical T1w images are shown with corresponding color-coded quantitative maps of the signal fat-fraction, magnetization transfer ratio (MTR), basal qT2* and BOLD signal change (∆T2*) in response to hypercapnia and acetazolamide, respectively. Map colour scales indicated under each map. Scalebar 500 um.
Fig. 2: Quantitative analysis of MRI markers in the left lateral lobe of the mouse liver.
Comparison between CD (blue), HFD (yellow) and HFD+Ω3 groups (gray) as characterized by signal-fat-fraction, MTR as a marker for macromolecular content, basal qT2* and BOLD signal in response to the hypercapnic and pharmacological stimulus acetazolamide as a marker for liver vascular function.
Keywords: murine liver steatosis, vascular dysfunction, Blood Oxygenation Level Dependent (BOLD) MRI
MRI Phenotypization, Vasoreactivity and Hypoxia in MC-38 colon and A549 lung adeno-carcinoma cell grafts grown on the Chorioallantoic Membrane of the Chick Embryo in ovo (#29)
Frauke Conny Waschkies1, 2, Fatma Kivrak Pfiffner3, Dorothea M. Heuberger4, Petra Wolint5, Marcel Andre Schneider1, Yinghua Tian1, Maurizio Calcagni6, Pietro Giovanoli6, Johanna Buschmann5, 6
1 University Hospital Zurich, Visceral- and Transplantation Surgery, Zurich, Switzerland
2 ETH and University of Zurich, Institute for Biomedical Engineering, Zurich, Switzerland
3 University of Zurich, Institute of Medical Molecular Genetics, Schlieren, Switzerland
4 University Hospital Zurich, Clinic of Intensive Care, Zurich, Switzerland
5 University Hospital Zurich, Surgical Research Division, Zurich, Switzerland
6 University Hospital Zurich, Plastic Surgery and Hand Surgery, Zurich, Switzerland
Recently, a tumor model based on the chorioallantoic membrane (CAM) was characterized structurally with MRI . Yet, vascular functional reserve and oxygenation-sensitive MRI measures [2-3] remain largely unexplored in this model. In our preliminary experiments we compare MC-38 colon and A549 adeno-carcinoma cell grafts with regards to their vascular and oxygenation phenotypes. We demonstrate that a functional gas challenge with carbogen is feasible through gas exchange on the CAM, allowing to access vascular function and oxygenation status of the tumor graft in this experimental model.
Fertilized Lohman white LSL chick eggs were opened on incubation day (ID) 3.5 and on ID7 5 x 105 matrigel embedded MC-38 colon or A549 lung adenocarcinoma cells were planted onto the CAM. MRI was performed in ovo on ID 14 in 5 samples for each graft type on a 4.7T Bruker PharmaScan system, with chicken embryos sedated (0.3 mg/kg medetomidine). T1w and T2w anatomical reference images obtained and quantitative T1 and T2* maps were compared between periods of air and carbogen (95% O2, 5% CO2). Gases were delivered through a plastic tubing to the CAM. T1w scans were repeated in selected samples after i.v. injection Gd-DOTA (Dotarem®, Guerbet S.A., Switzerland) to study enhancement in the tumor graft. Corroborative histology was obtained from H&E and Ki-67 staining.
MC-38 colon and A549 lung adenocarcinoma cell grafts were compared using quantitative T1 and T2* MRI, readouts associated with vascular responsivity and oxygenation status, when compared between periods of air and carbogen. Since the CAM serves as a breathing organ during chick embryo development, these markers might be also applicable for the grafts on the CAM (Fig. 1). Our preliminary data show that in A549 lung adenocarcinoma cell grafts T2* values increased upon carbogen exposure, while MC-38 grafts displayed a decreasing trend in T1 (Fig. 2). Qualitative assessment of Gd-enhancement, suggests that A549 grafts display a more prominent enhancement compared to MC-38 grafts (not shown). Furthermore, it will be of interest to explore if such enhancement patterns might be supportive for our vague notion that A549 grafts might display a better vascular response, while only MC-38 show oxygen-induced T1 shortening as observed in normoxia.
Our preliminary experiment shows that different tumor grafts planted on the CAM can be distinguished non-destructively in ovo using MRI. We show that a functional gas challenge is feasible through the CAM, and affects MRI signals associated with vascular reactivity and oxygenation status of the graft. The CAM assay may thus help qualifying such MRI markers to discern distinct vascular functional and oxygenation phenotypes.
 Zuo et al. (2014) NMR Biomed 28: 440-447
 Baudelet et al. (2006) NMR Biomed 19: 69-76
 O’Connor et al. (2015) Cancer Res 76(4) : 787-95
Representative MRI images of MC-38 colon carcinoma cell graft in ovo.
A. Tumor cells planted into the middle of a supportive plastic ring on top of the CAM, shown 7 days after grafting. B. Experimental setup with gas challenge delivery tube and surface coil placed over the egg shell window. C. Graft outlined on T1w and T2w anatomical reference images and on quantitative color-coded T1 and T2* maps obtained while the graft was exposed to medical air and carbogen, respectively.
Fig. 2: Quantitative analyses of changes in T1 and T2* upon carbogen gas challenge
in A549 lung adenocarcinoma cell grafts and in MC-38 colon carcinoma cell grafts. While no significant response was observed in neither, qT1 and qT2* in both graft types, in A549 lung adenocarcinoma cell grafts T2* values almost doubled upon carbogen exposure (p < 0.06, Wilcoxon test), while T1 displayed a decreasing trend in MC-38 colon carcinoma cell grafts (p < 0.11, Wilcoxon test). No consistent trend was observed in T1 for A549 lung adenocarcinoma grafts and in T2* in MC-38 colon carcinoma grafts.
Keywords: Chorioallantoic membrane (CAM), magnetic resonance imaging (MRI), MC-38 colon carcinoma cells graft, A549 lung adeno-carcinoma cell graft, oxygenation-sensitive MRI
An Extracorporeal Circulation Mouse Model for Simultaneous Measurements of DCE-MRI Arterial Input Functions and Radiotracer Blood Concentrations (#321)
Philipp Backhaus1, 2, 3, Florian Büther1, 2, Lydia Wachsmuth3, Lynn Frohwein1, 2, Klaus Schäfers1, 2, Sven Hermann1, 2, Michael Schäfers1, 2, Cornelius Faber3
1 University Hospital Münster, Department of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany
2 University of Münster, European Institute for Molecular Imaging - EIMI, Münster, North Rhine-Westphalia, Germany
3 University of Münster, Translational Research Imaging Center - TRIC, Münster, North Rhine-Westphalia, Germany
DCE-MRI can provide quantitative estimates of blood-brain barrier integrity. Dynamic PET measurements allow to quantify molecular processes. The calculation is based on contrast agent (CA) / radiotracer dynamics in tissue and in blood (AIF). Measurement of the AIF however is challenging in both small animal MRI and PET. Only few examples of direct AIF-measurements of MR CA in mice are published in the literature1-4, each featuring significant limitations.
Intracranial tumor-bearing mice received an extracorporeal shunt from the femoral artery to the tail vein. MRI scanning was performed using a 9.4 T MRI (Bruker BioSpec) and a cryo-cooled surface coil. The extracorporeal line featured two reservoirs which resided in the MRI field of view. A MRI-compatible measuring chamber for a β-Microprobe (biospace lab) was included in the circulation. Dynamic MRI scanning of the head was performed for 15 minutes using a 3D FLASH with a spatial resolution of 0.175 x 0.175 x 1 mm and a temporal resolution of 4.015 s. A 100 µl solution containing 10-20 MBq F-18-PSMA-1007 and CA (Gadovist, 35 mM) was injected intravenously at 1 ml/min. Dispersion correction for MRI CA was performed based on the recorded dispersion effect at the two interspaced reservoirs.
The CA AIFs of nine recorded mice show little noise and typical AIF curve shapes after dispersion correction (Figure 2, A & C). Eight of nine mice show a close range of peak concentrations (0.55 – 0.85 µmol/ml) and shunt flow velocities (34-58 µl/min). β-emitting radioactive tracer AIFs can be simultaneously recorded using a MR-compatible β-Microprobe (Figure 2 A) and mice were transferred into the PET-scanner immediately after DCE-MRI (Figure 2 B). Significant inverse correlation between AIF maxima and the delays between the CA-influx into the two reservoirs was observed (r = -0.84). The time constant τ for monoexponential deconvolution was significantly positively correlated with the delay (r = 0.98). The results of mass spectrometry (MS) validation show a systematic and consistent underestimation of the image-derived concentrations (MS-quantification / MR-quantfication: 1.57-1.8).
We present a novel approach for DCE-measurements of the AIF in mice with conceivable potential compared to so far published methods. Moreover, we present the first dual recordings of AIFs of a MR CA and a PET tracer in mice. This supports evaluation approaches to deduce the CA/PET tracer AIF from one another. Further, it might provide the basis for simultaneous and integrated modeling of PET tracer and CA kinetics in mice,
1.Loveless ME, Halliday J, Liess C, et al. A quantitative comparison of the influence of individual versus population-derived vascular input functions on dynamic contrast enhanced-MRI in small animals. Magn Reson Med. 2012;67:226–236.
2. Moroz J, Wong CL, Yung AC, et al. Rapid measurement of arterial input function in mouse tail from projection phases. Magn Reson Med. 2014;71:238–245.
3. Kim J-H, Im GH, Yang J, et al. Quantitative dynamic contrast-enhanced MRI for mouse models using automatic detection of the arterial input function. NMR Biomed. 2012;25:674–684.
4. Barnes SL, Whisenant JG, Loveless ME, et al. Practical Dynamic Contrast Enhanced MRI in Small Animal Models of Cancer: Data Acquisition, Data Analysis, and Interpretation. Pharmaceutics. 2012;4:442–478.
Extracorporeal shunt imaged in DCE-MRI
A) The extracorporeal volume between artery, 1st reservoir (S1), 2nd reservoir (S2) and β-microprobe are of equal volume. A tube (“Fix”) filled with defined concentration of CA (1 µmol/ml) and F-18 is centrally above the head. DCE images (same animal as in Figure 2 A & B) before Gd-injection (B) at 40 s p.i. (C, S1 filled with contrast) and 101 s p.i. (D, S1 and S2 filled). (E) Corresponding T2wi.
Parallel dynamics of CA and radiotracer
A) Dynamic cGd and CF-18-PSMA-1007 curves after simultaneous CA and tracer injection at 60 s. The green curve shows the deconvolved S1 curve with τ derived from the estimated convolution between S1 and S2. (B) Subsequent PET-image 23-40 minutes after injection fused to T2wi. (C) Dynamic cGd blood curves of another animal with plotted results of mass spectrometry validation measurements.
Keywords: DCE-MRI, PET/MRI, Arterial Input Function
Assessment of tumor hypoxia with Blood (BOLD) and tissue oxygenation level-dependent (TOLD) MRI measurements as prognostic biomarkers for breast cancer aggressiveness (#256)
Viktoria Ehret1, Joachim Friske1, Lubos Budinsky1, Katja Pinker-Domenig1, Vanessa Fröhlich1, Daniela Laimer-Gruber1, Thomas Helbich1
1 Medical University Vienna, Department of Biomedical Imaging and Image-guided Therapy, Division of Gender and Molecular Imaging, Preclinical Imaging Laboratory, Vienna, Wien, Austria
In tumors, hypoxia reflects an imbalance of oxygen delivery and consumption  and is therefore a prognostic biomarker of aggressiveness, local recurrence and metastasis [2‐4]. To quantify the oxygenation level within a tumor, two different techniques namely blood- (BOLD) [5‐6] and tissue oxygenation level‐dependent (TOLD)  can be evaluated combining different intrinsic MRI contrast mechanisms (T1, T2*). Our main goal is to assess both techniques to characterize the aggressiveness of different breast cancer models (MCF-7, SKBR-3, MDA-MB-231).
Human breast cancer cells of three different levels of aggressiveness were injected into the mammary gland of female nude mice. MRI measurements were performed using a 9.4T magnet system combined with a 1H volume coil (Bruker) when breast tumours reached a diameter of at least 10mm. The MR protocol includes a T1‐RARE sequence (TOLD) and T2* MGE/T2* EPI sequences (BOLD). A baseline measurement was acquired while the animal breathed air. The oxygenation challenge was performed in two different ways: A stepwise increase of the oxygen level (50%, 80%, 100%) and a direct increase to 100%. T1 and T2* maps were calculated using the ISA Tool fit routine. Data analysis of the calculated maps was performed on a voxel-by-voxel basis using Matlab. Imaging findings were compared with HIF-1alpha.
First results and a comparison of the two different oxygen challenges and the different aggressive breast cancer models will be presented. TOLD and BOLD provide different temporal and spatial information on breast cancer oxygenation. Typical results of TOLD/BOLD measurements for breast cancer of high aggressiveness are shown in figure 2, giving evidence of elevated hypoxia levels. For BOLD, the signal does not change heavily after the 3rd timepoint of the oxygenation challenge therefore we may shorten the measurement protocol accordingly. A stepwise oxygenation or starting the measurement after oxygen saturation could lead to different information. To make the technique even more efficient, T2* EPI might also be a potential sequence for fast BOLD measurements. Even if the scan protocol can still be optimized, the first measurements already provide impetus to use non-invasive BOLD/TOLD MRI for monitoring breast cancer oxygenation and drawing conclusions regarding aggressiveness.
Results give evidence of elevated hypoxia levels in breast cancer of high aggressiveness. First measurements provide further impetus to use non-invasive BOLD/TOLD MRI for monitoring tumor oxygenation and drawing conclusions regarding aggressiveness.
 O´Connor, J. P. B., et al. Int. J. Radiation Oncology Biol. Phys., 2009, 75(4):1209‐1215
 Christen, T., et al. AJNR Am J Neuroradiol., 2013, 34:1113‐1123
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 Zhao, D., et al., Magnetic Resonance in Medicine, 2009, 62:357‐364
 Baudelet, C., et al., Magnetic Resonance Imaging, 2004, 22:905‐912
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This work is supported by the EU project Hallmarks of Cancer (667211‐HYPMED H2020‐PHC) and the Siemens project CA-ID C00220910 and covered by the ethics (66.009/0284‐WF/V/3b/2017). In part supported by a research grant from Bruker.
Figure 1: Scan protocol of the BOLD/TOLD MRI
∆SI(%) for the TOLD measurement after oxygen challenge (left) and of the BOLD measurement (right) for five different time points during tumour oxygenation (B-F). Difference between the single time points is approximately 10 min. Tumour position in the BOLD images is shown in (A). The increases in ∆SI during oxygenation challenge are clearly visible.
Keywords: MRI, BOLD, TOLD, breast cancer
Severity assessment – Influence of repeated MRI measurements on animal welfare (#170)
Jasmin Baier1, Anne Rix1, Milita Darguzyte1, Maike Baues1, Jan-Niklas May1, Diana Möckel1, Rupert Palme3, René H. Tolba2, Fabian Kiessling1
1 RWTH Aachen University, Institute for Experimental Molecular Imaging, Aachen, North Rhine-Westphalia, Germany
2 RWTH Aachen University, Institute for Laboratory Animal Science & Experimental Surgery and Central Laboratory for Laboratory Animal Science, Aachen, North Rhine-Westphalia, Germany
3 University of Veterinary Medicine Vienna, Department of Biomedical Sciences, Wien, Wien, Austria
Non-invasive imaging is often used to observe physiological processes in small animals and most imaging devices are considered to be safe nowadays. However, the handling1 of animals, necessary anesthesia or the imaging procedures themselves can induce stress and therefore have an effect on animal welfare. Magnetic resonance imaging (MRI) measurements in particular can influence the behavior2 of animals due to the magnetic field. In the following study we investigate the influence of repeated MRI measurements on the welfare and different physiological parameters in mice.
For this purpose healthy, female BALB/C mice were exposed to 1T or 7T MRI field strength three times per week over a four weeks period. Imaging was performed under isoflurane anesthesia for 30 minutes using an established MRI protocol which is often applied in cancer research. A behavioral Rotarod test as well as blood pressure measurements were performed twice per week. Stress hormone levels in the feces and blood, the food intake and the nest-building behavior were examined. In addition, the general condition of the animals was evaluated daily using a score sheet. At the end of the experiment organs like brain, liver, spleen, lung, intestines, heart and kidneys were removed for histological examinations.
The investigations have shown that even after repeated MRI measurements there was no change in the behavior of the animals compared to different control groups (no imaging and no anesthesia or only anesthesia). Neither the Rotarod tests nor the blood pressure measurements showed any difference between the groups. Also, no differences were seen in the nest-building behavior, the food intake or the organ weights of the investigated groups. The evaluation of the stress hormone level (corticosterone) in feces and blood showed no increase in the animals exposed to MRI compared to the control groups. Based on the score sheets, the burden of the animals measured with MRI is not significantly different from the burden of the control animals.
The results show that the burden of the animals during MRI imaging can be considered as mild. In conclusion, we evaluate single as well as repeated MRI measurements under isoflurane anesthesia as a safe tool for the non-invasive evaluation of physiological processes in laboratory mice.
1 Balcombe J P, Barnard N D, Sandusky C. Laboratory Routines Cause Animal Stress. Contemporary Topics. 2004, 43 (6): 42 - 51
2 Houpt T A, Pittman D W, Barranco J M, Brooks E H, Smith J C. Behavioral Effects of High-Strength Static Magnetic Fields on Rats. The Journal of Neuroscience. 2003, 23 (4): 1498 - 1505
This research was funded by the Deutsche Forschungsgesellschaft (DFG) within the framework of the research group FOR2591.
Influence of repeated MRI measurements on behavior and stress levels in mice
A control group, an anesthesia group (Isoflurane for 30 min), and two MRI groups (1T or 7T under Isoflurane anesthesia for 30 min) were examined.
a. Changes in Rotarod performance after MRI measurements compared to the baseline
b. Mean blood pressure after MRT measurements
c. Stress hormone (corticosterone) levels measured from feces throughout the experiment
Scores throughout the experiment after repeated 7T MRI measurements
Mice were scored daily. Parameters such as the appearance of the fur and eyes as well as the weight of the animals, the heart rate and the behavior were assessed. If an animal was evaluated with a total score of 1 - 9, it was exposed to a mild level of stress.
Keywords: animal handling standardisation, stress level assessment, behavior evaluation, imaging influence
Strategies to avoid isoflurane chemical shift artefacts in high sensitivity in vivo19F MRI (#258)
Alexander H. Staal1, Andor Veltien2, N. Koen van Riessen1, Arend Heerschap2, Mangala Srinivas1
1 Radboud University Medical Centre, Tumor Immunology Lab, Nijmegen, Netherlands
2 Radboud University Medical Centre, Department of Radiology, Nijmegen, Netherlands
19F MRI is an increasingly popular imaging technique exploiting the benefits of background free imaging with the stable 19F isotope in imaging agents mainly consisting of inert perfluorocarbons. Isoflurane (ISO) is the anaesthetic of choice for preclinical imaging studies, however, as this compound contains 19F, it can be a complicating factor, and may result in chemical shift artefacts (CSA). Currently, ISO artefacts are avoided by short imaging times, injection anaesthesia or chemical shirt imaging. Here, we show three distinct, easy to implement, strategies for avoiding ISO artefacts.
A phantom consisting of 50% PFCE and 50% ISO was imaged as a proof of concept. For in vivo imaging, C57Bl/6 mice were injected with 20mg PFCE containing nanoparticles dissolved in 400µl 0,9% NaCl and imaged 2 days after injection. MRI was performed on an 11.7T BioSpec (Bruker, Ettlingen, Germany). 19F pulse-acquire with bandwidth=30ppm and points=8000. A RARE sequence was used with 4 different parameter sets. 1) standard: TE=15.2ms, TR=5000ms 2) out of plane shift with an extremely small bandwidth 3) suppression pulse 4) 3D selective excitation. In vitro imaging took <20 sec for all scans, in vivo imaging time was 12:48min. For in vivo1H reference scans a respiratory gated FLASH was used.
In vitro NMR spectra of the phantom show a sharp ISO and PFCE peak (Fig 1a), in vivoNMR spectra show multiple broad ISO peaks and a single sharp PFCE peak(Fig 1b). In vitro 19F MRI shows the CSA when using a standard sequence(Fig 1c), that can be shifted out of plane by using a very narrow acquisition bandwidth (Fig 2d). Figure 2e shows the two resonance frequencies of ISO and PFCE a fair distance apart, either signal can be supressed using a frequency selective 90°pulse before the excitation pulse. In vivo19F MRI with the standard sequence shows the isoflurane problem; ISO CSA creates ghosts that confound image interpretation and quantification (Fig 2a). Shifting these signals out of plane is feasible in vivo,but results in aggravated susceptibility artefacts(Fig 2b). Using our ISO suppression pulse, results in effective in vivosuppression of ISO signals(Fig 2c). Selective 3D slab excitation with a narrow excitation bandwidth results in artefact free images(Fig 2d).
Preclinical 19F MRI scan times are limed by isoflurane chemical shift artefacts, complicating imaging analysis and quantification. Here we show three strategies to avoid these chemical shift artefacts while maintaining a high signal to noise ratio. Having 3 different approaches allows for flexibility in sequence design.
1. M. Srinivas, A. Heerschap, E.T. Ahrens, C.G. Figdor, I.J.M. de Vries. 19F MRI for quantitative in vivo cell tracking. Trends in Biotechnol 2010; 28(7): 363-70.
2. S.M. Fox, J.M. Gaudet and P.J. Foster, Fluorine-19 Mri Contrast Agents for Cell Tracking and Lung Imaging’, Magnetic Resonance Insights. 2015; 8(1).
3. E. Swider, A. H. J. Staal, N. K. van Riessen, et al. Design of triphasic poly(lactic-co-glycolic acid) nanoparticles containing a perfluorocarbon phase for biomedical applications. RSC Adv. 2018; 8: 6460-6470
19F-spectra, (a) in vitro (b) in vivo. c-e In vitro ISO avoidance strategies, (c) standard imaging, (d) shift ISO out of plane (e) frequency specific suppression pulse. Note: in all images only one phantom is present
Figure 2: in vivo imaging
in vivo imaging 1H reference image in grey scale 19F image in red hot. (a) standard imaging with ISO ghosts overlapping the liver (b) ISO artefact is shifted out of plane, clear 19F susceptibility artefacts of the reference and liver projecting outside the animal, (c) ISO suppression pulse and (d) selective excitation 3D-RARE both resulting in an absence of ISO artefacts.
Keywords: 19F MRI, isoflurane, in vivo, chemical shift artifacts
In vivo Fast Field Cycling Relaxometry reports on the extra- and intracellular localization of iron oxide particles in tumour mice models. (#62)
Maria Rosaria Ruggiero1, Simona Baroni1, Valeria Bitonto1, Smeralda Rapisarda1, Silvio Aime1, Simonetta Geninatti Crich1
1 Università degli studi di Torino, torino, Italy
The relationship“immune system/tumour”is considered an important hallmark of cancer1. Tumour associated macrophages (TAMs) adopt an anti-inflammatory phenotype and secrete factors to promote angiogenesis and tumor invasion.The use of Ultra Small Iron Oxides nanoparticles (USPIO) has been already proposed to the TAM detection generating contrast in T2-weighted images indipendently of extra and intracellular localization of the NPs. While, T1 at different fields appear dependent on localization, especially at low field, of the NPs allowing an unambiguous TAM quantification.
In vitro studies have been carried out on a murine monocyte-derived macrophage cell line (J774) to evaluate the relaxivity changes due to the intracellular localization of ferumoxytol, clinical negative contrast agent. T1 were acquired on a FFC relaxometer able to switch over a large range of field stranghts (0.01-20MHz). In order to host a mouse, the commercially available relaxometer (Stelar, Mede, Italy) has been modified with the implementation of a 40 mm 0.5T Field Cycling magnet and a dedicated 11mm solenoid detection coil placed around the anatomical region of interest2. The tumour xenografts were prepared by injecting three tumour cell lines (B16 melanoma, 4T1 and 168FARN breast carcinoma) in the hindlimb muscle.
The relaxivity peak at ca. 8-10 MHz observed in water on ferumoxytol is shifted to lower magnetic field strengths (at 0.5-1 MHz) when the NPs were entrapped in macrophages (Fig.1). For in vivo model, the selected types of tumours (168FARN, 4T1 and B16) are characterized by different amount of necrotic zones and macrophages infiltrating the tumor stroma. Ferumoxytol was injected at a dose of 0.5 mmol/kg of Fe. The profile obtained 3h and 24h after the injection were significantly different. (Fig.2) The profile observed at 24h displays a bell-shaped profile with a maximum around 0.4-0.5 MHz similar to one found for ferumoxytol labelled macrophages. This finding clearly indicated the intracellular localization of ferumoxytol as confirmed by histological analysis by the Pearls assay.
The measured T1 at different field immediately reports on the intra- or extra-cellular localization of the investigated contrast agent. This information could be open new horizons for cell tracking applications. Despite the herein used prototype FFC-NMR, FFC has recently been applied to MRI, largely thanks to the work of the Lurie group at Aberdeen University where two prototype human whole-body sized FFC-MRI scanners have been built3.
 Morita Y, Zhang R, Leslie M, Adhikari S, Hasan N, Chervoneva I, Rui H, Tanaka T. Oncol Lett. 2017;14:2111-2118.
 Ruggiero MR, Baroni S, Pezzana S, Ferrante G, Geninatti Crich S, Aime S. Angew Chem Int Ed Engl. 2018, 57:7468-7472.
 Pine KJ, Davies GR, Lurie DJ. Magn Reson Med., 2010, 63, 1698-702.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 668119
NMRD profiles of J774 cells incubated for 24 h with different ferumoxytol concentrations or with ferumoxytol added to the external buffer. The indicated concentrations refer to the [Fe] in the measured cell pellets
A) NMRD profiles of B16 tumour bearing mouse leg 3 and 24h after (POST) the i.v. injection of ferumoxytol subtracted by the corresponding PRE profile (acquired before ferumoxytol injection). B) Different initial slopes of the NMRD profiles
Keywords: Iron oxide NPs, labelled macrophage, FFC-NMR
Hyperpolarisation of clinical agents using SABRE and SABRE-relay (#16)
Ben Tickner1, Alexandra Olaru1, Peter Rayner1, Soumya Roy1, Wissam Iali1, Aneurin Kennerley1, Simon Duckett1
1 Center for Hyperpolarisation in Magnetic Resonance, Department of Chemistry, University of York, York, United Kingdom
Magnetic Resonance (MR) techniques are insensitive as signal intensities are derived from small population differences across nuclear spin energy levels. Hyperpolarisation techniques can be used to increase the number of spins that contribute to the MR signal. Here we use Signal Amplification By Reversible Exchange (SABRE) to make low concentration biomolecules visible to MR. This technique takes para-hydrogen (p-H2), a spin isomer of hydrogen, and catalytically transfers its latent hyperpolarisation into a target substrate using an iridium catalyst.
SABRE traditionally necessitates substrate hyperpolarization through coordination to an iridium catalyst, limiting substrate scope to N-heterocycles. A recent variation termed SABRE-Relay allows molecules to become hyperpolarised by exchange of hyperpolarised nuclei. Amines can act as hyperpolarization carriers by coordinating to the SABRE catalyst and relaying polarisation onto other functional groups including alcohols. This rapidly expands the substrate scope of SABRE by hyperpolarising molecules without the need for direct catalyst interaction. Here, we create active SABRE catalysts of the form [Ir(H)2(NHC)(Amine)3] to hyperpolarise molecules via SABRE-relay or metal complexes of the form [Ir(H)2(NHC)(α-carboxyimine)(amine)] which can be hyperpolarised by SABRE.
We use SABRE and SABRE-relay to produce 1H and 13C signals enhanced by 4 orders of magnitude for a range of different molecules including alcohols and pyruvate. The determining factor in achieving high alcohol enhancements is the level of carrier NH polarisation. The alcohol and carrier amounts should be equal for optimal alcohol enhancement; the best carrier NH3 has been identified. Such hyperpolarised molecules including pyruvate have been imaged in vitro. We can also create iridium α-carboxyimine complexes from the in situ reaction between pyruvate and amines. These complexes undergo rapid p-H2 exchange and isotopic labelling techniques were used to achieve 13C signal enhancements of 750-fold and a long-lived 13C2 state can be created in the first instance, the lifetimes of which approach 18 s. Such states may prove highly beneficial in the future when looking for intermediates in chemical reactions or as flow probes.
We present the hyperpolarisation of a wide range of molecules including organics (alcohols), biomolecules (pyruvate and ethyl lactate) and transition metal complexes (iridium α-carboxyimines). We show that SABRE can be used as a route to create hyperpolarised molecules in a fast, cheap, and refreshable manner. Current research is directed at improving biocompatibility and we expect SABRE will provide unique opportunities for molecular imaging.
- R. W. Adams, J. A. Aguilar, K. D. Atkinson, M. J. Cowley, P. I. Elliott, S. B. Duckett, G. G. Green, I. G. Khazal, J. López-Serrano and D. C. Williamson, Science, 2009, 323, 1708-171
- W. Iali, P. J. Rayner and S. B. Duckett, Sci. Adv., 2018, 4, eaao6250.
- B. J. Tickner, W. Iali, S. S. Roy, A. C. Whitwood and S. B. Duckett, ChemPhysChem, 2018.
The Wellcome Trust (092506 and 098335), MRC (R16249), and the ESPRC IAA G00251 (B.J.T. studentship) are thanked for supporting this work.
A substrate can become hyperpolarised by SABRE when it and para-hydrogen are in reversible exchange with an iridium catalyst. In a second step, polarisation can be relayed onto other molecules by exchange of nuclei
Keywords: SABRE, Hyperpolarisation, Para-hydrogen, Pyruvate
A spin echo pulse sequence with unpaired adiabatic refocusing pulse and a 3D cone readout for hyperpolarized 13C imaging (#181)
Vencel Somai1, Alan Wright1, Kevin M. Brindle1, 2
1 University of Cambridge, Cancer Research UK, Cambridge Institute, Cambridge, United Kingdom
2 University of Cambridge, Department of Biochemistry, Cambridge, United Kingdom
Dynamic nuclear spin polarization has enabled dynamic measurements of tissue metabolism in vivo using 13C magnetic resonance spectroscopic imaging. However, this is demanding in terms of imaging speed due to the short lifetime of the hyperpolarization, hence the shortest possible sequence with the highest possible SNR is desirable for dynamic measurements. We have developed a single spin-echo sequence, which improves on that described in , with a shorter readout, better potential SNR efficiency and an isotropic PSF that does not degrade the nominal resolution.
The sequence uses a spatial spectral pulse set to the resonant frequency of the individual metabolites and a HS adiabatic refocusing pulse without slice selection. The gradient readout uses a 3D cone trajectory , which is built from 13 cones. Each of the cones is fully refocused with time optimal rewinders and the 7th cone is refocused at the time of the spin-echo. The total readout time is 18.576ms. The nominal isotropic resolution and FOV are 2 mm and 3.2 cm respectively. The total pulse sequence length is 43.73 ms. PSF of the sequence was simulated according to .
The pulse sequence was tested on two cylindrical phantoms filled and half-filled with thermally polarized 5M [1-13C]lactate and 3M [1-13C]acetate respectively, placed in the magnet isocentre and parallel with the z-axis.
The immunity of the proposed sequence to hardware imperfections was tested by measuring the resulting k-space, as described in . Despite the slew-rate limited regime and high gradient amplitudes, no measurable delay was observable and the amplitude of the waveforms matched the design values within the detection noise. Simulations showed that the PSF of the sampling trajectory is isotropic and maintains a resolution of <4 mm in the x, y and z directions. The simulation results were validated with measurements on two phantoms containing 13C-labelled lactate and acetate using a 7T scanner (Agilent, Palo Alto, CA), with a 42 mm diameter bird‐cage volume coil for 1H transmission and reception, and a similar volume coil for 13C transmission and a 20 mm diameter surface coil for 13C detection (Rapid Biomedical, Rimpar, Germany). The sequence was designed with a maximum gradient strength of 0.4T/m and slew rate of 2000T/m/s.
The proposed sequence gave better resolution, potentially higher SNR efficiency and a shorter readout and TR when compared with the sequence described in . The main advantage of the sequence is its isotropic sampling PSF.
- Wang J, Hesketh RL, Wright AJ, Brindle KM. Hyperpolarized 13C spectroscopic imaging using single‐shot 3D sequences with unpaired adiabatic refocusing pulses. NMR in Biomedicine. 2018;31:e4004. https://doi.org/10.1002/nbm.4004
- Gurney, P. T., Hargreaves, B. A. and Nishimura, D. G. (2006), Design and analysis of a practical 3D cones trajectory. Magn. Reson. Med., 55: 575-582. doi:10.1002/mrm.20796
- Wang, J. , Wright, A. J., Hu, D. , Hesketh, R. and Brindle, K. M. (2017), Single shot three‐dimensional pulse sequence for hyperpolarized 13C MRI. Magn. Reson. Med., 77: 740-752. doi:10.1002/mrm.26168
- Takahashi, A. and Peters, T. (1995), Compensation of multi‐dimensional selective excitation pulses using measured k‐space trajectories. Magn. Reson. Med., 34: 446-456. doi:10.1002/mrm.1910340323
The work was supported by a Cancer Research UK Programme grant (17242), by the CRUK-EPSRC Imaging Centre in Cambridge and Manchester (16465) and by the CRUK Cambridge Centre.
The pulse sequence uses a fly-back SpSp excitation (10.056ms), HS adiabatic refocusing pulse (10ms) and a 3D cones k-space trajectory. The excitation pulse has a bandwidth at half maximum of 350 Hz and a period of excitation bands of 1645 Hz. To reduce signal loss due to the quadratic phase imparted by the adiabatic pulse, the encoding gradients are turned off on all three axes during refocusing.
Central slice of the images of the acetate (without 1H decoupling) and lactate phantoms. Due to 1H coupling and NOE the quartet spectrum of the acetate leads to inherently smaller signal compared to the singlet lactate. The reference 1H images were acquired using a 2D FSE sequence with the same isotropic 3.2 cm FOV, 2 mm slice thickness and 128x128 in-plane resolution. The echo train had 8 echoes.
Keywords: hyperpolarizaton, 13C, 3D single shot, spin-echo, 3D cones
Detection of T cell activation through metabolic changes using hyperpolarized 13C magnetic resonance (#268)
Emine Can1, Mor Mishkovsky1, Hikari Yoshihara1, Ulf Petrausch4, Marie-Agnès Doucey5, Arnaud Comment3, 2
1 EPFL, Lausanne, Switzerland
2 University of Cambridge, Cambridge, United Kingdom
3 GE Healthcare, Chalfont St Giles, United Kingdom
4 OnkoZentrum, Zurich, Switzerland
5 UNIL, Lausanne, Switzerland
Upon activation, T cells rapidly increase their requirements in metabolic substrates to sustain their proliferation and function. While memory and naïve T cells mostly rely on the β-oxidation of fatty acids to produce their ATP, it has been shown that the higher need in energy and biosynthetic material required by activated T cells leads to an upregulation of the catabolism of glucose1. This has been observed through both an increase in glucose transporters as well as an upregulation in enzymes involved in glycolysis2. We aimed at detecting this metabolic shift by hyperpolarized 13C MRS.
CD4+T cells were isolated from healthy human peripheral mononuclear blood cells by immune magnetic selection. T cells were activated through a 5-day incubation in anti-TCR/CD3 (10µg/ml) coated tissue culture plates containing soluble anti-CD28 (1µg/ml) and 100U/ml of IL-2. At the end of the stimulation, ~5·106 T cells were transferred into a 5-mm NMR tube containing 30mM unlabeled lactate. Another 5-mm tube was prepared as control with non-activated T cells. The two tubes were placed in a dual 13C NMR probe and loaded in a 9.4T/31cm horizontal bore magnet at 37°C. Hyperpolarized [1-13C]pyruvate solution (300µl per tube) prepared in a 7T polarizer was injected in parallel using a flow splitter, and interleaved 13C MRS measurements were acquired on both samples (4s Trep, 5o flip angle).
In both non-activated and activated T cells, the formation of [1-13C]lactate was detected following the injection of hyperpolarized [1-13C]pyruvate. The lactate-to-pyruvate signal ratio recorded in activated T cells was about 3 times larger than in non-activated cells. This can be explained by the upregulation of glycolysis triggered by the activation, which leads to an increase in 13C labelling of the lactate pool through LDH activity. Although the observed lactate signal is the sum of both intra- and extracellular lactate since the line width was too large to differentiate the two pools3, most of the detected signal likely originated from extracellular lactate. It is in agreement with recent experiments showing that extracellular lactate increases upon T cell activation4. Despite the fairly high signal-to-noise ratio, no bicarbonate signal could be observed, which shows that the flux through PDH was low and that most of the injected pyruvate was not used to feed the TCA cycle.
We demonstrated that T cell activation can be detected by hyperpolarized 13C MRS. This method can complement the standard ECAR measurements (Seahorse) and could be used to analyze the behavior of T cells in presence of antigens. Since it is well known that cancer cells will also compete for pyruvate5, it is not the ideal substrate to detect T cell activation in the tumor microenvironment and alternative hyperpolarized 13C tracers shall be tested.
- Pearce E.L., Poffenberger M.C., Chang C.H., Jones R.G. (2013). Fueling immunity: insights into metabolism and lymphocyte function. Science 342: 1242454.
- Ghesquiere B., Wong B.W., Kuchnio A., Carmeliet P. (2014). Metabolism of stromal and immune cells in health and disease. Nature 511: 167-76.
- Breukels V. et al. (2015). Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with 13C hyperpolarised NMR. NMR Biomed. 28: 1040–1048.
- Grist J.T. et al. (2018). Extracellular lactate : a novel measure of T cell proliferation. J. Immun. 200:1220-1226.
- Comment A. and Merritt M.E. (2014). Hyperpolarized Magnetic Resonance as a Sensitive Detector of Metabolic Function. Biochem. 53: 7333-7357.
This work is part of a project that has received funding from the European Union’s Horizon 2020 European Research Council (ERC Consolidator Grant) under grant agreement no. 682574 (ASSIMILES).
Keywords: NMR, mri, hyperpolarization, immunology, metabolism
Active targeting of epicardium-derived progenitor cells (EPDC) by EPDC-specific peptides (#432)
Tamara Straub1, Julia Nave1, Pascal Bouvain1, Julia Kistner1, Zaoping Ding1, Aseel Marzoq1, Stefanie Stepanow2, Siva S. K. Dasa3, Brent A. French4, Julia Hesse1, Karl Köhrer2, Ulrich Flögel1, Jürgen Schrader1, Sebastian Temme1
1 Heinrich Heine University, Department of Molecular Cardiology, Düsseldorf, North Rhine-Westphalia, Germany
2 Heinrich Heine University, Genomics & Transcriptomics Laboratory, Düsseldorf, North Rhine-Westphalia, Germany
3 University of Virginia, Cardiovascular Research Center, Charlottesville, Virginia, United States of America
4 University of Virginia, Department of Biomedical Engineering, Charlottesville, Virginia, United States of America
Epicardium-derived progenitor cells (EPDC) play an important role during heart development . After myocardial infarction (MI), EPDC are reactivated, proliferate and migrate into the damaged tissue where they can differentiate into fibroblasts, endothelial cells and possibly also to cardiomyocytes1. How EPDC precisely contribute to the healing process after MI is still unclear. To further study the role of EPDC after MI, the aim of this study was to identify EPDC-specific peptides to enable the in vivo visualization by 1H/19F magnetic resonance imaging (MRI).
About 1010 clones of a Ph7 phage-library (linear 7mer peptides) were incubated for 1.5 h at 37 °C on cultured rat EPDC, isolated 5 d after MI. Cells were washed, bound phages were eluted and subsequently incubated with rat whole blood to eliminate clones that bind to blood cells. Phage titer was determined by qPCR. Identification of individual peptide sequences was performed with deep sequencing (Illumina Platform) and subsequent bioinformatics analysis (PHASTPep). Peptides were commercially manufactured and equipped with a carboxyfluorescein (flow cytometry) and a cystein for coupling to maleimide-perfluorocarbons (PFC). MR-experiments were performed at a 9.4 T Bruker AVANCEIII Wide Bore NMR spectrometer. Data were acquired using a 25 mm birdcage resonator tuneable to 1H and 19F.
Sequential panning of the phage library on EPDC and blood followed by deep-sequencing and bioinformatics analysis (Fig.1) revealed a total of 78,300 ± 31,900 different peptide-insertion sequences. Five peptides (EP1-EP5) showed an increased abundance of more than 70 in all samples and flow cytometry revealed that EP1-EP5 strongly bind to EPDC (MFI range: 16,000–51,000; Fig.2A) but not to blood immune cells (MFI: 970±610; Fig.2A). EP5 displayed the best binding affinity followed by EP2, EP4, EP1 and EP3. EP-peptides also showed strong binding to non-cultured EPDC freshly isolated from infarct hearts. Coupling EP5 to maleimid-containing PFC nanoemulsions (EP5-PFCs), incubation with EPDC and subsequent FACS analysis revealed strong targeting of EP5-PFC to EPDC in comparison to control-PFCs without peptide. Furthermore, 1H/19F MRI showed a more than two-fold higher 19F-signal of EPDC treated with EP5-PFCs compared to controls (14.1±2.8 vs 6.8±2.9; Fig.2B).
In the present study, we identified linear peptides which specifically bind to rat EPDC but not to circulating immune cells. Coupling of these peptides to PFC nanoemulsions enabled the targeting of EP-PFCs to EPDC and the visualization by 19F MRI in vitro. Therefore, this approach holds the potential to specifically track EPDC also in vivo by 1H/19F MRI to further unravel their role in the healing phase after MI.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ST 1209/1-1, FL 303/6-1 and the Sonderforschungsbereich SFB 1116.
1. Smart N, Riley PR. The epicardium as a candidate for heart regeneration. Future Cardiology. 2012;8(1):53–69.
Fig. 1: Schematic structure of the phage-display with rat EPDC.
First, a Ph7 phage-library was incubated on rat EPDC. The cells were washed, bound phages were eluted and incubated on rat whole-blood. The blood was subsequently centrifuged to split plasma and serum. Plasma phages were subjected to a PCR amplification. The PCR products were used for deep-sequencing and subsequent bioinformatics revealed differend peptide sequences.
Fig. 2: Binding affinity of EPDC-peptides to rat EPDC
A) The peptides bind strongly to EPDC but not to immune cells. The MFI for blood immune cells is around 970±610. EP5 has the best binding affinity to EPDC with a MFI of 59246, whereas EP3 shows the lowest binding with a MFI of 16742. n = 6
B) 1H/19F MRI showed a more than two-fold higher 19F-signal of EPDCs treated with EP5-PFCs compared to controls (14.1±2.8 vs 6.8±2.9). n = 2
Keywords: myocardial infarction, magnetic resonance imaging, 19F MRI, Epicardium-derived progenitor cells, phage-display
Examination of photo-CIDNP-based 19F MR hyperpolarization in dependence of the temperature (#490)
Frederike Euchner1, Rainer Ringleb1, Christian Bruns1, Joachim Bargon2, Johannes Bernarding1, Markus Plaumann1
1 Otto-von-Guericke University Magdeburg, Institute for Biometrics and Medical Informatics, Magdeburg, Saxony-Anhalt, Germany
2 University of Bonn, Institute for Physical and Theoretical Chemistry, Bonn, North Rhine-Westphalia, Germany
MR signal enhancements can be achieved by using the hyperpolarization technique photo-Chemical Induced Dynamic Nuclear Polarization (photo-CIDNP).[1,2] Previous studies have shown that 19F MR hyperpolarization in aqueous solutions is a challenge.[3,4] Metabolism examination of fluorinated drugs as well as their intermolecular interactions are of high interest. To date, only photo-chemically induced dynamic nuclear polarization (photo-CIDNP) allows the 19F hyperpolarization in pure water.[4,5] Here, a LED-based set-up[6,7] was used and we investigate the effect of a temperature change.
The investigated sample contained 3-fluoro-DL-tyrosine (2mM) and riboflavin 5’-monophosphate sodium salt hydrate (0.21mM) dissolved in physiologic salt solution. NMR-spectroscopic measurements were performed in a 5mm NMR tube on a 7T MR system (Bruker WB-300 Ultrashield). In all examinations an optical fiber is connected to a Cree XP E high power LED (455nm) and was centrally positioned in the solution. Irradiation times between 0s and 15s were chosen and a 90° pulse was used for the detection of 19F (P1=32.5µs, PL1=17W) NMR spectra. For measuring the temperature effect on the photo-CIDNP, the temperature unit of the NMR spectrometer was used and spectra were acquired at defined temperature in the range of 300-318K. Temperatures up to 330K were set for chemical shift change determination.
Photo-CIDNP enables the hyperpolarization of fluorine nuclei in aqueous solution (Figure 1). The 19F MR spectra show that with increasing temperature the signal enhancement decreases slightly. Nevertheless, the increase at 318 K is still sufficient to allow use in the MRI. In addition, the measurable change in chemical shift of the 19F signal to lower fields is of high importance. It provides a very accurate determination of the temperature within an MRI system (within high magnetic fields) using the graphical plot and the determined trend line (Figure 2).
While e.g. the hyperpolarization method such as PHIP does not enable a 19F hyperpolarization in pure water, the hyperpolarization technique photo-CIDNP allows a repetitive hyperpolarization without adding new substrates. In comparison to the laser-based CIDNP (standard method), the LED set-up allows irradiation without warming the sample.
The photo-CIDNP measurements show significant enhancements of the 19F NMR signal of 3-fluoro-DL-tyrosine in a biocompatible system using flavins as photosensitizers. Here, the 19F MR hyperpolarization occurs directly at 7 T using a low-cost and easy-to-handle LED-based set-up. At higher temperatures, both the MR signal and the signal gain decrease slightly. However, the detected enhanced signal can still be used for imaging.
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 U. Flögel, E. Ahrens, 1. Ed. , Fluorine Magnetic Resonance Imaging, Pan Stanford Publishing, Singapore, 2017.
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG BE 1824/12-1).
Figure 1: 19F Hyperpolarization
The 19F NMR spectra of hyperpolarized 3-fluoro-DL-tyrosine dissolved in aqueous solution. The signal enhancement is dependent from the irradiation time.
Figure 2: Plot of the chemical shift vs. temperature
The plot shows the dependence of the chemical shift of the 19F signal (3-fluoro-D,L-tyrosine) when changing the temperature.
Keywords: hyperpolarization, 19F, photo-CIDNP, 3-fluoro-DL-tyrosine, riboflavin