Dual Contrast Molecular Imaging Allows Noninvasive Characterization of Myocardial Ischemia/Reperfusion Injury After Coronary Vessel Occlusion in Mice by mri running title
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- In vitro static and flow chamber adhesion assay
- Gadolinium application
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Materials & Methods Contrast agents Platelet-specific contrast agent (LIBS-MPIO) The monoclonal “anti-LIBS”-antibody binds to ligand-induced binding sites (LIBS) on the GPIIb/IIIa-receptor only in its active conformation, and demonstrates strong binding to ADP- activated platelets in the presence of fibrinogen 19 . Cloning, generation and production of the “anti- LIBS” single-chain antibody has previously been described in detail 20 . To obtain a non-functional antibody for control purposes, exchange of the arginine in the RXD motif of the heavy chain CDR3 region of a single-chain antibody was performed. Generation and purification of the antibody was performed as described elsewhere 15, 19, 21 . For construction of the contrast agent, schemia/reperfusion injury as well as the clinical relevance of the respective the e r
rape p p u ut ut ic ic ic r r r ec ec ec ep ep to nhibition was confirmed in dual contrast MRI and histology in P2Y 12 knockout mice undergoing e e
mp mpor
or or ar ar y y y LA LA LA D D li li li g ga gation. Ma Ma Mate te teri ri ri al al s s s & & & Me Me eth h hod od d s s Contrast age ge ge nt nt nt s s by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from DOI: 10.1161/CIRCULATIONAHA.113.008157 5 cobalt-functionalised auto-fluorescent MPIOs with a diameter of 1 m were conjugated to the histidine-tag of the anti-LIBS/control single-chain antibody as described in the protocol of the manufacturer (Dynal Biotech, Oslo, Norway) and previously published studies 22-24 . Throughout the manuscript, MPIOs conjugated to the anti-LIBS-antibody will be referred to as “LIBS-MPIO”, MPIOs conjugated to control antibody are named “control-MPIO”. Injection of the LIBS-MPIO and control-MPIO contrast agent (each with 4x10 8 particles in 50μl saline) was via an 80 cm long tube and the tail vein catheter with the animal positioned in the MR scanner. Flushing the tube with 100μl saline assured full injection. In vitro static and flow chamber adhesion assay Static assays were performed using small Petri dishes coated with washed platelets. Platelets were activated with 20μM ADP and incubated with MPIOs. In vitro flow chamber adhesion assays were performed using collagen coated glass capillaries 25 . Microthrombi were formed by perfusion of whole blood into the capillary using a syringe pump (PhD 2000, Harvard Apparatus, USA). MPIOs were perfused through the capillary for 5 min. Both experiments were observed with IX81 Olympus microscope (Olympus, Tokyo, Japan), and Cell^P 1692 (ANALYsis Image Processing) software, using DIC with a 20x objective. Binding was quantified using Image Pro Plus software. Gadolinium application Multihance™ (gadoben acid dimeglumin, Bracco Suisse) was used as paramagnetic Gadolinium based contrast agent for the late enhancement examination. A concentration of 0.4ml per kg body weight was diluted in 50μl of saline, and was administered via the tail vein catheter as described above.
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d wi wi ith th I I I X X8 81 Olympus micr cr r os os os co co o pe pe pe ( ( ( Ol O O ym ym m pu pu pu s, s, , T T T ok ok k yo yo o , , Ja Ja Ja pa pa pa n) n) n) , , an an an d d d Ce C C ll ll l ^P ^P P 1 1 1 69 69 9 2 2 2 (A (A A NA NA NA LY LY LY si si si s s Im Im Im ag ag ag e e e Pr P P ocessing ) by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from DOI: 10.1161/CIRCULATIONAHA.113.008157 6 receptor-deficient (P2Y 12 -/-
) mice were kindly provided by J.-M. Boeynaems and B. Robaye (IRIBHM and Erasme Hospital, Université Libre de Bruxelles, Bruxelles, Belgium) 26, 27 . P2Y
12 -/-
mice were also on a C57BL/6N background and all mice were housed in the local animal facility prior to the experiments. All experiments were conducted strictly according to the German animal protection law and in accordance with good animal practice as defined by FELASA (www.felasa.eu) and the national animal welfare body GV-SOLAS (www. gv-solas.de). The examinations undertaken in this study were approved by the Federal Authorities in Freiburg/IRB through the animal experiment permission 35/9185.81/G-09/47.
Mice were anesthetized with Ketamine (125 mg/kg, Pfizer Pharmacia GmbH, Berlin, Germany), Xylazine (6 mg/kg, Bayer Vital GmbH, Leverkusen, Germany) and 2% isoflurane (Abbott, Wiesbaden, Germany), and maintained at 37°C. After oro-tracheal intubation, mice were ventilated with a maximal end-inspiratory pressure of 10 cm H 2 O, at a respiratory rate of 110/min, and an I/E ratio of 1/1.5 with a small animal respirator (TSE Systems, Bad Homburg, Germany). Left lateral thoracotomy in the third intercostal space was performed after right lateral positioning of the animal. The medial LAD was identified after pericardiotomy and ligated with an 8-0 prolene suture. Complete ligation was confirmed by paling of the anterior wall of the left ventricle and ST-elevation in 3-lead electrocardiography, and removed after 50 minutes to allow reperfusion. Pneumothorax was evacuated, and chest and skin were closed with a 6-0 prolene suture. Analgesia was provided with buprenorphine (0.1mg/kg s.c.) and mice were henceforth kept in individual clean cages. Throughout the procedure, oxygen saturation, respiratory rate, and heart rate were continually monitored with a MouseOX system (Starr Life Sciences, Oakmont, PA, USA) and maintained within the normal ranges. Mice were anesthetized with Ketamine (125 mg/kg, Pfizer Pharmacia GmbH, Be Be e rl rlin
in n , Ge Ge Ge rm rm rm an an a y) y),
Xylazine (6 mg/kg, Bayer Vital GmbH, Leverkusen, Germany) and 2% isoflurane (Abbott, Wi Wi es es es ba ba bade de de n, n, n, G
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Germany). Le Le e ft ft ft l l at at at er er er al al l t t t ho ho ra ra ra co co c to to o my my my i i n th th th e e e th th th ir ir ir d d d in in n te te t rc rc r os s s ta ta ta l l l sp sp sp ac ac ac e e e wa wa wa s s pe pe pe rf rf rf or or or me me e d d d af af af te te te r r r right latera a a by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from DOI: 10.1161/CIRCULATIONAHA.113.008157 7
All magnetic resonance experiments were performed on a dedicated small animal MRI system (BioSpec70/20, Bruker Germany), run with AVANCE III electronics, Paravision 5.1 software, and employing a two channel cryogenically cooled mouse head surface coil. The animals were placed head first in supine position onto the cryo-coil animal bed supported by a cotton pad underneath the spine, in order to assure the mouse chest to fully fill the sensitive coil volume. For animal monitoring and sequence triggering, neonatal ECG electrodes were attached to the left front and the right hind paw of the mice. In addition, a breathing sensor pad was placed beneath the animal and animal temperature was maintained by warm-water supported heating of the animal cradle. During MRI, anesthesia was maintained via slowly introducing isoflurane up to a maximum of 2 Vol % in oxygen, stabilizing the animals at a breathing rate of approximately 70 breaths per minute. The MRI protocols consisted of three parts. First, a retrospectively gated multi slice Intragate-Flash pilot scan was performed in order to verify the animal position and to place the reference slice for transmitter gain adjustments of the cryo-coil. Planning of the final cardiac pseudo short axis slice position was performed using an ECG and respiration triggered standard FLASH sequence (TE/TR: 2.8/35 ms) acquiring three parallel slices. On an axial image, three sagittal slices through the left ventricle were tilted towards the coronary plane until parallel to the septum. Perpendicular on these images, three further slices were planned parallel to the imaginary line connecting the mid of the basis and the apex of the left ventricle. Finally, on these pseudo four chamber view images, the three left ventricular pseudo short axis slices were planned. For these, a respiration- and ECG-triggered FLASH sequence tailored to gain T 2 * and
placed beneath the animal and animal temperature was maintained by warm-wat at e e er s s s up up p po po po rt rt rt ed ed ed
heating of the animal cradle. During MRI, anesthesia was maintained via slowly introducing s s of of f lu lu lura ra ra ne ne e u u u p p to a a a m m maximum of 2 Vol % in oxyge ge gen,
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DOI: 10.1161/CIRCULATIONAHA.113.008157 8 T 1 contrast while maintaining sufficient signal to noise was employed. In order to obtain images from the late diastolic phase, the trigger delay was chosen deducting TR and an additional 20ms from the ECG gating derived period time. Blood signal attenuation was achieved through a 7mm saturation slice placed parallel to the imaging slices across the atrium. Images were acquired once pre contrast agent application, and minimum 5 scans lasting ~50 minutes after injection of LIBS-MPIO/control-MPIO, in order to monitor platelet invasion. Subsequently, injection of MultiHance™ and further 4-5 scans followed for monitoring late enhancement through gadolinium uptake. With four averages the sequence parameters included a TE/TR of 2.8ms/35ms, a flip angle of 50°, a bandwidth of 81.5kHz, a slice thickness of 0.6mm, a Field of View of 25x25mm, and a matrix size of 256x256 resulting in a resolution of 100μm in plane. To achieve the best signal to noise ratio, we refrained from obtaining cine-like movies with the necessity of repeated excitation pulses, but focused on the recording of late diastolic single frames.
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