Fast, high-resolution in vivo cine magnetic resonance imaging in normal and failing mouse hearts on...

Original Research

Fast, High-Resolution In Vivo Cine MagneticResonance Imaging in Normal and Failing MouseHearts on a Vertical 11.7 T System

Jurgen E. Schneider, PhD,1* Paul J. Cassidy, DPhil,2 Craig Lygate, PhD,1

Damian J. Tyler, PhD,2 Frank Wiesmann, MD,1 Stuart M. Grieve, DPhil,2

Karen Hulbert, BSc,1 Kieran Clarke, PhD,2 and Stefan Neubauer, MD, FRCP1

Purpose: To establish fast, high-resolution in vivo cinemagnetic resonance imaging (cine-MRI) on a vertical 11.7-TMR system and to investigate the stability of normal andfailing mouse hearts in the vertical position.

Materials and Methods: To optimize the method on a high-field system, various MR-related parameters, such as re-laxation times and the need for respiratory gating, werequantitatively investigated. High-resolution cine-MRI wasapplied to normal mice and to a murine heart failure model.Cardiac functional parameters were compared to matchedmice imaged previously on a horizontal MR system.

Results: A T1 of 1.10 � 0.27 seconds and a T2 of 18.5 � 3.9msec were measured for murine myocardial tissue. A quan-titative analysis also proved respiratory gating to be essen-tial for obtaining artifact-free cine images in the verticalposition at this field strength. Cardiac functional parame-ters of mice, obtained within one hour, agreed well withthose from previous studies of mice in the horizontal posi-tion.

Conclusion: This work shows that MR systems with a ver-tical bore design can be used to accurately measure cardiacfunction in both normal and chronically failing mousehearts within one hour. The increased signal-to-noise ratio(SNR) due to the higher field strength could be exploited toobtain higher temporal and spatial resolution compared toprevious studies that were performed on horizontal sys-tems with lower field strengths.

Key Words: mice; MR cine imaging; respiratory gating;relaxation times; cardiac function; heart failureJ. Magn. Reson. Imaging 2003;18:691–701.© 2003 Wiley-Liss, Inc.

GENETICALLY MODIFIED MICE are widely used asmodels for human cardiac disease. Precise, noninvasivecardiac phenotyping of the mouse heart, which weighs�0.1 g, poses a major challenge. High-resolution mag-netic resonance cine imaging (cine-MRI) has been ap-plied successfully to quantify myocardial mass andfunction in normal (1–4) and chronically infarcted mice(5–8). The cine technique also allows the study of de-velopmental changes in cardiac function and massfrom neonatal to adult mice (9). Applications in trans-genic mice have, for example, been shown in a model ofcardiac hypertrophy (10), mice with myocardial overex-pression of tumor necrosis factor-� (11,12), and adultcardiomyocyte-specific vascular endothelial growth fac-tor (VEGF) knockout mice (13).

All previous murine cardiac MR studies have beenperformed on horizontal magnets with field strengths�7 T. However, experimental high-field MR systems arecommonly built with a vertical bore, which is the pre-ferred design for experiments in isolated, perfused or-gans and on aqueous solutions. For in vivo studies onsuch MR systems, animals have to be positioned in anupright position, which may affect cardiovascularphysiology. It is unknown whether left ventricular (LV)volumes and, consequently, cardiac functional param-eters (such as ejection fraction, cardiac output, orstroke volume) are affected by this position. It is wellrecognized that such a study in larger mammals and inhumans would be impractical, as the effect of orthos-tasis would reduce venous return, LV volumes, andcardiac output (e.g., 14). Furthermore, even if normalmice were to maintain stable function in the verticalposition, it is unclear whether this would also hold truefor mouse models of cardiac disease, the most extremeof which is probably the chronically failing heart.

1Department of Cardiovascular Medicine, University of Oxford, Oxford,United Kingdom.2University Laboratory of Physiology, University of Oxford, Oxford,United Kingdom.Contract grant sponsor: British Heart Foundation; Contract grantsponsor: Deutsche Forschungsgemeinschaft; Contract grant number:SFB 355 TPA; Contract grant sponsor: Wellcome Trust; Contract grantsponsor: British Council.*Address reprint requests to: J.E.S., Department of CardiovascularMedicine, University of Oxford, John Radcliffe Hospital, Oxford OX39DU, UK. E-mail: [email protected] June 18, 2003; Accepted July 29, 2003.DOI 10.1002/jmri.10411Published online in Wiley InterScience (www.interscience.wiley.com).

JOURNAL OF MAGNETIC RESONANCE IMAGING 18:691–701 (2003)

© 2003 Wiley-Liss, Inc. 691

Consequently, we implemented high-resolution cine-MRI on an 11.7-T vertical MR system. First, myocardialT1 and T2 relaxation times were measured for contrastoptimization. Next, we demonstrated that in contrast toprevious studies in the horizontal position (e.g., 1,6),respiratory gating is essential in the vertical position forhigh-field systems, thereby enabling virtually artifact-free, high-resolution images to be obtained. To investi-gate whether our method would be generally applicableto mouse models of cardiac disease, we acquired mul-tiframe gradient-echo images with high temporal andspatial resolution throughout the cardiac cycle in nor-mal and chronically failing mouse hearts. Cardiac func-tion was measured and compared with results obtainedusing a horizontal MR system.

MATERIALS AND METHODS

Animal Preparation

Male C57Bl/6J mice (15–35 g) were obtained from acommercial breeder (Harlan, UK) and kept under con-trolled conditions for temperature, humidity, and light,with chow and water available ad libitum. Anesthesiawas induced in an anesthetic chamber using 4% isoflu-rane in 100% oxygen. Animals were then positionedsupine in a purpose-built mouse holder and main-tained at 1.5%–2% isoflurane at 1 liter/minute oxygenflow throughout the MRI experiments. The cradle wasdesigned for positioning mice vertically in the magnetand comprised of an in-built nose cone, lines for thedelivery and scavenging of anesthetic gases, tempera-ture control, and electrocardiogram (ECG) and respira-tory monitoring. Temperature was maintained using ablanket that was heated by air that had flowed through ahome-built heat exchanger placed within the animalholder and above the coil in the magnet. An in-housedeveloped ECG and respiratory gating device was used formonitoring heart and respiration signals, derived fromtwo needles subcutaneously inserted in the front paws.Respiratory signals could also be derived from a looploosely fitted on the chest and abdomen of the animals.Mice were secured within the holder using surgical tape,without compressing their abdomen or chest regions. Allanimals were finally euthanised after the MRI examina-tions, their hearts excised, and the atria and right ventri-cle dissected, blotted, and weighed. Minor differences inLV blotting technique between horizontal and verticalstudies necessitated the application of a correction factorto allow direct comparison. The correction factor was cal-culated as 1.05 � 0.02, measured by experiment in fiveseparate hearts and applied to vertical data.

Infarct Model

Animals were anesthetized with an intraperitoneal in-jection of Hypnorm (total dose � 2.5 �g of fentanylcitrate and 80 �g of fluanisone) and 1.5%–2% isoflu-rane in O2. Mice were intubated and ventilated at 150breaths per minute, stroke volume of 250 �L (Hugo-Sachs MiniVent type 845, Harvard Apparatus, UK). Athoracotomy was performed in the fourth intercostalspace, and a 6/0 Proline suture tied around the leftanterior descending coronary artery a few millimeters

from its origin. Subcutaneous buprenorphine (0.8 mg/kg) was given for pain relief, with subcutaneous salineand post-op warming to aid recovery. Seven mice wereimaged one to two months after infarction.

All investigations conform to Home Office Guidanceon the Operation of the Animals (Scientific Procedures)Act, 1986 (HMSO) and to institutional guidelines.

MRI Setup

Imaging experiments were carried out on an 11.7-T(500-MHz) MR system comprising a vertical magnet(bore size � 123 mm; Magnex Scientific, Oxon, UK), aBruker Avance console (Bruker Medical, Ettlingen, Ger-many), and a shielded gradient system (548 mT/m; risetime � 160 �sec) (Magnex Scientific, Oxon, UK).Quadrature driven birdcage coils with inner diametersof 28 and 40 mm (Rapid Biomedical, Wurzburg, Ger-many) were used to transmit/receive the nuclear mag-netic resonance (NMR) signals.

Relaxation Times Measurement and SequenceOptimization

Five to six short-axis slices (slice thickness � 1 mm,slice gap � 1 mm, field of view (FOV) � 2.56 � 2.56 cm,1282, number of averaged experiments (NAE) � 1) wereacquired. T1 was measured in seven mice by a seg-mented SNAPSHOT-FLASH sequence (TE/TR � 1.5/3.5 msec, � � 8°) (15). Eight phase-encoding (PE) stepsfor one frame were acquired per heart cycle, and thenumber of frames per relaxation curve was maximizedaccording to the respiration rate of the animal. Thus,the inversion times were determined by the heart rate.T2 was measured in six mice by varying the echo timesof a spin echo sequence in eight steps between 3.7 and24 msec. One PE step per heartbeat and the signals offive to six slices were acquired within one respirationcycle. Hence, the repetition time was determined by therespiration rate. The start of both sequences after de-tection of the R-wave was recorded throughout the re-spective experiments to calculate mean inversion times(T1 experiments) and mean repetition times (T2 experi-ments) and to assess stability.

In order to investigate the need for respiration gatingat this field strength, cine imaging was performed in sixmice under various gating and acquisition strategies: 1)ECG-triggered only, without and 2) with averaging(NAE � 2); 3) respiration gated, without and 4) withsteady-state preparation using three heart cycles aftereach respiratory event. The latter two were repeatedwith a segmented PE scheme (16,17) through the res-piration cycle: i.e., 5) without and 6) with steady-statepreparation using three heart cycles after each respira-tion event, respectively.

For all experiments the imaging parameters wereFOV � 25.6 mm2, matrix size � 128 � 128, 1-mm slicethickness, TE/TR � 1.43/4.6 msec, 15° Gaussian ex-citation pulse, NAE � 1 (except case 2). The number offrames per heart cycle varied between 24 and 37 (de-pending on the heart rate), and four or eight PE stepsper respiratory cycle were acquired if respiration gatingwas used (cases 3–6). For quantitative analysis of the

692 Schneider et al.

various strategies, the same experiments were per-formed with the PE gradient turned off.

Cine-MRI

High-resolution cine-MRI was performed in normal andchronically infarcted mice (mean body weight of con-trols � 18.37 � 0.63 g, N � 6; infarcted mice � 30.1 �1.4 g, N � 7) using a 28-mm 1H-imaging probe for thenormal mice and a 40-mm 1H-imaging probe for theinfarcted mice. After scouting for long- and short-axisorientation of the heart, using a segmented cardiac-triggered and respiration-gated FLASH sequence, tun-ing and matching the probe, slice-selective shimmingand flip angle calibration were performed manuallyprior to each experiment. Seven to 10 contiguous slices(slice thickness � 1 mm) were then acquired in short-axis orientation covering the entire heart. The imagingparameters were FOV � 25.6 mm2, matrix size � 256 �256, TE/TR � 1.43/4.6 msec, 15° Gaussian excitationpulse, NAE � 2. The sequence was ECG triggered andrespiratory gated. Between 19 and 28 frames per heartcycle and two to eight PE steps per respiration cyclewere acquired in a segmented fashion (see above andFig. 1), resulting in a total experimental time of approx-imately one hour in the vertical position (including ex-perimental preparation).

Data Analysis

Image reconstruction was performed using purpose-written idl-software (Research Systems International,Crowthorne, Berkshire, UK). For LV volumes and massmeasurements, raw data were isotropically zero-filledby a factor of two and filtered (modified third-orderButterworth filter (18)) prior to Fourier transformation,resulting in an in-plane voxel size of 50 � 50 �m for thevolumetric experiments and 100 � 100 �m in the re-laxation times and contrast experiments. All image datawere exported into TIFF format and loaded into Amira™2.3 (TGS Europe, Merignac Cedex, France). End-dia-stolic and end-systolic frames were selected accordingto maximal and minimal ventricular volume. In both

frames, the epicardial border was outlined first usingthe autotrace function of Amira. The LV cavity was thensegmented by thresholding. The number of voxels ofeach compartment multiplied by the pixel size of0.0025 mm3 yielded the respective volumes. LV masswas obtained by multiplying the volume with the spe-cific gravity of 1.05 g/cm3 (19). Based on end-systolic(ESV) and end-diastolic (EDV) volumes, all parameterscharacterizing cardiac function, such as stroke volume(SV � EDV – ESV), ejection fraction (EF � SV/EDV),and cardiac output (CO � SV � heart rate), were cal-culated.

Endo- and epicardial circumferences were measuredusing purpose-written idl-software to calculate infarctsizes for every slice according to Eq. [1]:

Infarct size

�1

NSLICES �i�1

NSLICES 12 � Iepi

i

Tepii �

Iendoi

Tendoi � � 100% (1)

with TEpi and TEndo being the total endo- and epicardialcircumferences of the left ventricle; and IEpi and IEndo

being the endo- and epicardial length of infarcted tis-sue, respectively.

T1 and T2 maps were computed from relaxation timemeasurements, using a mono-exponential two-param-eter fit (for T2) or three-parameter fit (for T1) in idl. Themean repetition time for T2 experiments and the meaninversion times for T1 experiments, monitored through-out the experiments, were calculated. Points on theinversion curve with corresponding inversion timesshowing a deviation greater than 50 msec, due to irreg-ular respiratory cycle length, were excluded from the fit.

The gating/acquisition strategy was optimized byquantitatively analyzing the cine data sets, acquiredwith the PE gradient turned off. The raw data wereFourier transformed in both dimensions, and the mag-nitude of each frame of the cine-train was summedalong the frequency-encoding direction to give a profilein the PE direction. These profiles were summed over allframes. The ratios of energy of zero frequency/totalimage energy (i.e., sum over the profile) and mean en-ergy of nonperiodic noise/energy of zero frequency werecalculated from the total frequency profile for everyacquisition and gating strategy.

Reproducibility Assessment and StatisticalAnalysis

To assess reproducibility of our method, segmentationof the high-resolution cine data was performed by twoindependent observers (J.E.S. and D.J.T.) as well astwice by one observer (J.E.S.). Autopsy and MRI mea-surements of LV mass were compared using the Stu-dent’s paired t-test. Cardiac function measurements inthe horizontal (1) and in the vertical position (thisstudy) were compared using an unpaired t-test. A valueof P � 0.05 was considered significant. Chronically fail-ing mouse hearts imaged horizontally (5) and verticallywere compared by plotting the ejection fractions vs.infarct sizes and performing a linear regression analy-

Figure 1. Diagram of the acquisition modality applied: thefirst three heart cycles after detection of a respiratory event areused for dummy scanning (DSi), followed by N RR intervalswith varying phase-encoding (PEi) gradients (with N � 2, 4, or8, depending on the ratio of heart rate to respiration rate). ThePE was performed in a segmented fashion. Each PE blockconsisted of 20–30 acquisitions depending on the heart rate.

Mouse Cine-MRI on a Vertical 11.7-T System 693

sis. The 95% confidence interval was calculated forboth slopes according to (20):

m � t0.05,n�2 � sm � M � m � t0.05,n�2 � sm (2)

with m being the estimated slope, sm the standard de-viation of the slope estimate; t0.05,n–2 the Student’s tvalue associated with two-sided 95% confidence, n – 2degrees of freedom, and M the true slope.

RESULTS

Relaxation Time Measurements

Figure 2 shows T1 (Fig. 2a) and T2 (Fig 2b) maps throughtwo different mouse hearts at 11.7 T in a short-axisorientation with the corresponding anatomical images

in the right-hand column (Fig. 2c and d). T1 rangedbetween 300 msec and four seconds and T2 between 3and 200 msec. The mean T1 value was 1.10 � 0.27seconds and the T2 value was 18.5 � 3.9 msec for LVmyocardium, and a T1 of 1.73 � 0.42 seconds and a T2

of 19.8 � 2.1 msec were found for skeletal muscle,respectively (mean � SD). The 10–16 frames acquiredin the inversion recovery experiment covered between1.4 and 2.2 seconds on the relaxation curve. For ani-mals with a regularly beating heart and a constantrespiration rate, the standard deviation of the TI usedfor fitting was typically less than 5 msec. The repetitiontimes in the T2 experiments varied between 2.2 and 3.0seconds in different animals, depending on their respi-ration rate, with a standard deviation of less than 10%for each TR value.

Figure 2. Mid-ventricular T1 map (a) and T2 map (b) in short-axis orientation as measured in two different mice. Mean valuesof T1 � 1.10 � 0.27 seconds and T2 � 18.5 � 3.9 msec were found for LV myocardium, and T1 � 1.73 � 0.42 seconds and T2 �19.8 � 2.1 msec for skeletal muscle. Corresponding anatomical images are shown in (c) and (d).


Acquisition/Gating Strategy

Figure 3 shows the first image of a cine-train, obtainedwithout respiration gating and no averaging (Fig. 3a) oraveraging (Fig. 3b) two experiments. Artifacts in the PE(vertical) direction due to respiratory motion are obvi-ous, resulting in a loss of signal-to-noise and imageresolution. Figure 3c and d shows images with respira-tion gating, acquired using two different PE schemes. Inboth cases, eight PE steps were acquired per respirationcycle. For Figure 3d, three prior heart cycles were usedto reach steady state (dummy scanning) so that in total,11 RR intervals (time between two R-waves) were usedper respiratory cycle. Respiration-gated images weresubstantially improved compared with images acquiredwithout respiratory gating. A residual level of ghostingdue to T1 modulation can be observed in Figure 3c,whereas Figure 3d is completely artifact-free.

The corresponding profiles of the data, acquired withthe PE gradient turned off, are shown in the right col-umn of Figure 3. All graphs were normalized to theiramplitude at zero frequency of the respective strategy.Figure 3e and f show the higher noise levels due torespiratory motion, which can be separated into a pe-riodic part (depending on the ratio of heart rate to res-piration rate) and a nonperiodic part. Quantitativeanalysis of all six hearts yielded a nonperiodic noiselevel of 2.2 � 0.6% of the zero-frequency amplitude forthe ECG-gated images and NAE � 1, and 1.6 � 0.4% forECG gating with NAE � 2, compared to 0.6 � 0.2% withadditional respiratory gating and 0.6 � 0.2% with re-spiratory gating, and a segmented PE scheme withthree heart cycles used for dummy scanning (mean �SD). The periodic peaks in Figure 3g and h were notcaused by respiratory motion, but were the result of T1

modulation of the signal amplitude and depended onthe number of heart cycles used in a respiration period.Using three RR intervals to get into steady state beforedata acquisition reduced this effect (Fig. 3h).

An increased noise level (periodic or nonperiodic) notonly reduced resolution, but also resulted in a loss ofwanted signal energy at zero frequency. This could bequantitatively expressed by the ratio of energy at zerofrequency to total image energy. We found for the vari-ous modalities (mean � SD): 1) ECG only, NAE � 1,24 � 4%; 2) ECG only, NAE � 2, 30 � 4%; 3) respiratorygating, 42 � 8%; and 4) respiratory gating, segmentedPE scheme and dummy scanning, 52 � 8%.

Cardiac Function and Mass in Normal Mice

Based on the above results, respiratory gating with asegmented PE scheme and three heart cycles used fordummy scanning was applied to obtain optimum imagequality.

Volumetric measurements were first validated in aphantom experiment. Short-axis slices (N � 16) wereobtained from a pear-shaped glass phantom, whichwas filled with 254 �L of distilled water and surroundedby a physiological buffer solution containing 2 mM Ga-dodiamide (Omniscan, Amersham, UK) for contrast en-hancement. Image segmentation yielded a volume of250 �L; the deviation from the true volume was there-fore 2%.

Mid-ventricular end-diastolic and end-systolicframes out of cine-trains of 26 or 28 images in short-and long-axis orientation are shown in Figure 4. Thepixel size in both cases is 50 � 50 �m in-plane at a slicethickness of 1 mm. The examples shown, which werecropped for display purposes, reflect routinely obtainedimage quality. No artifacts but excellent contrast be-tween myocardium, blood, and surrounding tissue canbe observed. In particular, a difference in T1 weighting(corresponding to the different T1 times given above)between cardiac and skeletal muscle allowed the sepa-ration of the two muscle types. In the short-axis images(Fig. 4a and b), left and right ventricular walls, as wellas the papillary muscles, can be identified, and theendocardial and epicardial borders are clearly delin-eated. In the long-axis view (Fig. 4c and d), all four heartchambers and the major thoracic vessels are visible. Amean signal-to-noise ration (SNR) of 32 � 3 was mea-sured for blood in the first frame of a mid-ventricularslice through the heart, 12 � 1 for LV muscle, and 7.8 �0.3 for adjacent skeletal muscle, yielding a ratio of 2.8 �0.4 for blood/myocardium and 1.5 � 0.2 for myocardi-um/skeletal muscle (mean � SD).

No statistically significant difference between LVmass determined by autopsy (56 � 4 mg) and MRI(end-diastole: 57 � 4 mg, P � 0.25; end-systole: 59 � 5mg, P � 0.63) was found. Cardiac functional parame-ters and LV mass measurements are summarized inTable 1 (mean � SD). Values that we previously ob-tained on a horizontal system at 7 T in mice that werematched for strain, sex, and body weight (1) are listedfor comparison. The only significant differences werefound for LV mass as measured by autopsy and MRI,and heart rate. As this was seen on autopsy, this dif-ference must have been biological rather than method-ological, and excellent agreement between in vivo andex vivo mass was demonstrated for both horizontal andvertical measurements. The ratio LV mass to bodyweight was (3.2 � 0.2) � 10–3 for the vertical group and(4.1 � 0.5) � 10–3 for the horizontal group (P � 0.002).Quantitative analysis of the images was highly repro-ducible, as illustrated in Table 2, with a low overallintraobserver variability of 3 � 1% and an interobservervariability of 4 � 1% (mean � SD). Similar tools—withineither purpose-written idl-software for analyzing thehorizontal data or the commercially available softwareAmira for analyzing the vertical data—were used inboth studies to measure LV mass and volume, respec-tively. This is also demonstrated by the Bland-Altmanplot for LV mass measurements shown in Figure 5.

Infarct Model

Figure 6a–d shows end-diastolic and end-systolicframes of two chronically infarcted mouse hearts inmid-ventricular short- and long-axis orientations. Thearrows in Figure 6a and b indicate wall thinning in theinfarcted region of the left ventricle. The long-axis viewreveals an anterior wall aneurysm (arrows in Fig. 6cand d). These images show the akinesia of the aneu-rysm with preserved but reduced septal wall thicken-ing.


Figure 3. Left column: End-diastolic frame of a cine-train obtained without respiratory gating, NAE � 1 (a); without respiratorygating, NAE � 2 (b); with conventional respiratory gating, NAE � 1 (c); and with respiratory gating, segmented PE scheme, anddummy scanning after the detection of a respiration event, NAE � 1 (d). Right column: Corresponding rescaled profiles of thesame heart, obtained in experiments without PE and summed over all frames as well as all points acquired in readout direction.The peak at point 63 corresponds to the zero frequency (scaled to 100). All other peaks are mainly caused by motion (e,f) or T1

modulation (g,h).


Quantitative analysis of the cine data (N � 7) demon-strated reduced LV function and dilatation of the leftventricle (Table 3).The mean LV mass, obtained by au-topsy, was 110 � 16 mg; EDV was 147 � 81 �L; andESV was 117 � 85 �L. Accordingly, the mean strokevolume was 31 � 5 �L, and ejection fraction was 28 �16%. Mean heart rate was increased compared to nor-mal mice (504 � 44 bpm), resulting in a mean cardiacoutput of 15 � 2 mL/minute. The mean infarct size asmeasured in the end-diastolic frame of the cine-trainwas 38 � 24%. Data previously obtained in infarctedmice imaged in the horizontal position (5) are shown inTable 3 for comparison. A direct group comparison be-tween studies was not possible due to the large range ofinfarct sizes obtained and the differences in time sinceinfarction (horizontal study: 24 � 12 days; verticalstudy: 46 � 14 days). However, ejection fractions andinfarct sizes were linearly related for both groups (Fig.7: horizontal study: r � 0.95, P � 0.01; vertical study:r � 0.91, P � 0.01), and the obtained slopes were com-

Figure 4. Mid-ventricular end-diastolic (a) and end-systolic (b) frames in the short-axis orientation of a normal mouse heart asused for image segmentation. c,d: End-diastolic and end-systolic frames of a second mouse in the long-axis orientation. Scalebars � 2 mm.

Table 1Cardiac Function and LV Mass of Normal Mice*

Horizontala Verticalb

N 8 6Body weight/(g) 17.8 � 1.0 18.4 � 0.6LV mass (Autopsy)/(mg) 72.8 � 10.0 55.5 � 3.8***LV mass MRI (diastole)/(mg) 76.1 � 6.5 57.1 � 4.2***LV mass MRI (systole)/(mg) 73.1 � 12.6 59.2 � 5.1**Heart rate/(bpm) 366 � 39 429 � 24***EDV/(�l) 45.2 � 9.3 43.0 � 3.9ESV/(�l) 14.6 � 5.5 15.7 � 1.2SV/(�l) 30.5 � 4.6 27.4 � 3.4EF/(%) 68.6 � 6.6 63.5 � 2.9CO/(mL/minute) 11.2 � 2.4 11.4 � 1.2

*Physiological and cardiac functional parameters for normal mousehearts positioned horizontally and vertically.aData taken from Ruff et al (1).bData as measured in this study. **P � 0.05 vs. horizontal; ***P � 0.01.EDV � end-diastolic volume, ESV � end-systolic volume, SV �stroke volume (SV � EDV-ESV), EF � ejection fraction (EF �SV/EDV), CO � cardiac output (CO � SV � heart rate).


parable (mhorizontal � –0.690 � 0.071 vs. mvertical �–0.61 � 0.13). The 95% confidence intervals were(–0.846, –0.534) for the horizontal study and (–0.94,–0.28) for the vertical study.

DISCUSSION

Establishing an MRI protocol for imaging mouse heartsat high field in the vertical position requires optimizingavailable contrast and robustness of the method in or-der to allow the routine application of the method formouse models of cardiac disease. To address this issue,we systematically investigated various method-relatedparameters, such as relaxation times or gating strategy.It was required to verify that cardiac function of healthymice was unaltered by positioning vertically. We hadpreviously demonstrated no significant difference inheart rate, LV end-diastolic pressure, maximal rate ofLV pressure increase, and maximal rate of LV pressuredecrease between horizontal and vertical positions inmice (21). The hemodynamic values were measured in-vasively over one hour by advancing a microtip cathetervia the right carotid artery into the left ventricle. Al-though LV pressure and heart rate showed no signifi-cant difference between vertical and horizontal posi-tions, it was unclear whether LV volumes would beaffected by the upright orientation, and whether thisremained true in the failing heart. Thus, in this studywe investigated normal and failing mouse hearts.

The contrast in bright-blood cine imaging is based onthe inflow effect of blood into the imaging slice, andthus the determination of relaxation times facilitatedmaximizing the contrast between blood and myocar-dium (22). For LV myocardium, we measured a T1 valueof 1.1 � 0.3 seconds and a T2 of 19 � 4 msec. Based onthe assumption that blood and myocardium have thesame relaxation properties, the optimal flip angle wasnumerically estimated to be about �max 25°. This flipangle can be used in experiments designed to investi-gate cardiac function (i.e., LV volumes) only, not car-diac mass, and facilitate manual or automatic imagesegmentation. A lower flip angle is more advantageous

for additionally assessing myocardial mass. We found,by flip angle variation, � of 15° to give optimum contrastsimultaneously between blood/myocardium and skel-etal muscle/myocardium. The contrast between skele-tal muscle and myocardial tissue is based on differentdegrees of T1 weighting, as confirmed by T1 measure-ments (T1

skeletal muscle � 1.7 � 0.4 seconds;T1

myocardium � 1.1 � 0.3 seconds). T2 values were sim-ilar for both tissue types (T2

myocardium � 19 � 4 msec;T2skeletal muscle � 20 � 2 msec). For comparison, Dunnand Zaim-Wadghiri (23) reported a T2 of 28.0 � 0.7msec for skeletal muscle in normal mice at 7 T.

The accuracy of the T1 experiments was essentiallylimited by the number of points on the inversion curve.Choosing TI values beyond the length of the respiratorycycle was found to be impracticable due to the fluctu-ation induced by the varying respiration rate. Not sur-prisingly, the standard deviation of T1

skeletal muscle wasbigger than that of T1

myocardium, as the determinedT1skeletal muscle was in the order of TImax.

Although the standard deviation of each TR wasfound to be less than 10%, a varying respiration ratecould also reduce the accuracy of the T2 experimentsand increase the variability of the measured T2. TR waschosen in the order of approximately 1.5–2 times T1,potentially introducing an additional T1 effect if therespiration rate changed. However, the accuracy for ourexperiments was sufficient, because the contrast wasmainly based on T1.

An optimized sequence design also necessitated theinvestigation of respiratory gating. Previously pub-lished work at lower static magnetic field strength didnot use respiratory gating for cine imaging in mice (1,6).However, it is well known that breathing artifacts arefield dependent (24). Ross et al (6) found at 4.7 T thatrespiratory gating may have enhanced image quality.Thus, this issue needed investigation when implement-ing cardiac imaging in mice on a high-field MR system.In particular, Isoflurane anesthesia causes the animalto breathe impulsively (snatch breathing), which mini-mizes the time within a respiratory cycle used for thebreathing, but increases the motional amplitude. Our

Figure 5. Bland-Altman plot (difference vs. mean) of LV massfrom autopsy and MRI (Œ, diastolic mass; E, systolic mass).The mean of differences was 0.04, and the coefficient of vari-ability (2 SD) was �7.20.

Table 2Inter- and Intra-observer Variability in Measuring Left VentricularVolumes and Masses


Intra-observervariability/

(%)

Inter-observervariability/

(%)

Intra-observervariability/

(%)

Inter-observervariability/

(%)

Diastolic mass 7 � 3 11 � 2 3 � 2 5 � 3Systolic mass 4 � 2 3 � 3 4 � 3 3 � 3EDV 3 � 1 5 � 3 1 � 1 3 � 3ESV 3 � 4 9 � 4 3 � 1 6 � 5SV 6 � 3 2 � 4 3 � 2 4 � 4EF 3 � 2 3 � 2 2 � 1 3 � 2CO 6 � 3 2 � 4 3 � 2 4 � 4Overall 5 � 3 5 � 3 3 � 1 4 � 1

aFor comparison, data from mice positioned horizontally were takenfrom Ruff et al (1).bData as measured in this study.


investigation showed that approximately three RR in-tervals before and after the detection of a respirationevent have to be excluded from data acquisition in orderto obtain virtually artifact-free images.

To investigate this issue quantitatively, several acqui-sition modalities were applied with the PE gradientturned off. In a completely motionless object, the result-ing profile would show the main image energy beingcontained at zero frequency with some energy distrib-uted into random noise (determined by hardware, sam-ple, bandwidth, filter, etc.). Any deviation from the idealcase due to amplitude and/or phase modulationcaused by periodic/nonperiodic motion, sequence tim-ing, etc., could be revealed by a two-dimensional Fou-

rier transformation of the intrinsically one-dimensionaldata. The numbers reported here, determined by quan-titative analysis of these data, clearly emphasized theneed for respiratory gating at high-field MR systems toobtain maximal SNR and image resolution.

The choice of respiratory gating strategy is directlylinked to the tolerable level of residual artifacts. A sim-ple respiration gating (i.e., blanking during respiration)would be the most time-efficient way of acquiring data.However, this would cause a gap in data acquisition of600–900 msec in length (i.e., exclusion of six heart-beats for heart rates ranging between 400 and 600bpm) and result in a loss of steady state. For bright-blood cine imaging, contrast was obtained by saturat-

Figure 6. Mid-ventricular end-diastolic (a) and end-systolic (b) frames in the short-axis orientation of an infarcted mouse heartas used for image segmentation. The arrows indicate wall thinning. c,d: End-diastolic and end-systolic frames of a second mousein long-axis orientation. This view reveals an anterior wall aneurysm as indicated by the arrows. Scale bars � 2 mm.


ing the stationary spins and using the signal of theinflowing spins. A loss of steady state introduces anamplitude modulation and is therefore the source ofanother—motion-unrelated—image artifact, which willdepend on the ratio of acquisition length to respirationcycle length. Maintaining steady state during respira-tion and collecting data between two respiratory eventswould be the most efficient method for the image acqui-sition. Such information was provided in real time byour gating device, but could not be utilized for cineimaging due to timing restrictions in the MR system.Thus, a sequential scheme (i.e., dummy scanning fol-lowed by the data acquisition for every respiratory cy-cle) was implemented.

The best and reproducible image quality was ob-tained by applying an adjustable number of PE stepsper respiration cycle (which had to be a divisor of thetotal number of PE steps of the experiment) and usingthe first three heart cycles after detection of a respira-tory event prior to data acquisition for achieving steadystate. A slightly reduced noise level and more energycentered at zero frequency made the segmented PEscheme more favorable compared to incrementing thePE gradient linearly. According to these findings, alldata were acquired with respiratory gating, a seg-mented PE scheme, and three heart cycles used fordummy scanning.

In fact, spatial and temporal resolution benefitedfrom the increased SNR due to the higher field strength.In our study, the echo time of the sequence was set to1.43 msec as the lipid and water protons have an op-posite phase (25). Flip angle and repetition time werekept constant to provide comparable image contrast indifferent mice and amounted to 15° and 4.6 msec, re-spectively. This was also reflected in the small standarddeviations of the signal ratios of blood/myocardiumand myocardium/skeletal muscle. The number offrames in an RR interval was adapted to the cardiaccycle with a delay of 10–15 msec at the end to allow forsmall variations in the heart rate. No averaging withinthe RR interval was applied. The temporal resolutionwas therefore nearly twice as high as previously re-

ported for the horizontal position (1). The experimentalimage resolution of 100 � 100 �m in-plane also ex-ceeded values reported in previous studies by 15%–50% (e.g., 1,6).

The primary aim of our study was to establish high-resolution, murine cardiac MRI in the vertical positionat 11.7 T, and to investigate whether normal and chron-ically failing mouse hearts were physiologically stablein this position. Both the increased field strength andthe change in position required a modified experimentalsetup as described above. Our work demonstrates thatunder these conditions it is feasible to acquire high-quality images of the beating mouse heart in vivo.

Comparing cardiac function obtained in an uprightposition with our previous measurements performed ona system with a horizontal bore design (1), bodyweight-,sex-, and strain-matched mice were chosen. Table 1showed that functional parameters determined in thevertical position agreed well with values previously ob-tained on a horizontal system (1). Statistically signifi-cant differences were only found for LV mass (as mea-sured by autopsy and MRI), and heart rate. Both groupsshowed good agreement between the different methodsof mass measurements, but differed in absolute valuesfor LV mass. The ratio of body weight to LV mass wasabout one-third lower compared to the horizontalgroup, but in agreement with values reported in theliterature (7,26). The 17% higher heart rate (verticalMRI study vs. horizontal MRI study) was not confirmedby our previous study (21), in which no significant dif-ference in heart rate between upright and horizontalpositions was found. This may therefore be due to phys-iological variation of the mice or most likely due toimproved control of anesthetic levels.

The accuracy of measuring cardiac function wasmainly limited by the resolution in the z-direction (i.e.,slice thickness). Consequently, the resulting partial-volume effect dominated the inter- and intraobservervariability. Therefore, the higher field strength could beused in future experiments to increase resolution in thedirection of the long axis of the heart.

Figure 7. Linear relation between ejection fraction vs. infarctsize as assessed on a horizontal (open symbols, r � 0.95) andon a vertical MR system (filled symbols, r � 0.91). The linesrepresent the respective fit (horizontal data: dashed line, m �–0.69x � 0.61; vertical data: black line, m � –0.61x � 0.51).

Table 3Cardiac Functional Parameters for Failing Mouse Hearts asMeasured Horizontally and Vertically


N 12 7Body weight/(g) 28.9 � 0.9 30.1 � 1.4LV mass (Autopsy)/(mg) 96 � 5 110 � 16Heart rate/(bpm) 444 � 16 504 � 44Infarct size/(%) 47 � 19 38 � 24EDV/(�l) 141 � 11 147 � 81ESV/(�l) 104 � 12 117 � 85SV/(�l) 36.9 � 2.6 30.8 � 5.4EF/(%) 29 � 4 28 � 16CO/(mL/minute) 19.1 � 2.1 15.3 � 1.5

aData taken from Wiesmann et al (5), except infarct sizes, whichwere retrospectively determined for this study.bData as measured in this study.EDV � end-diastolic volume, ESV � end-systolic volume, SV �stroke volume (SV � EDV-ESV), EF � ejection fraction (EF �SV/EDV), CO � cardiac output (CO � SV � heart rate).


After establishing the method in normal mice, it wasapplied to an infarct model as a worst-case scenario ofcompromised cardiac function. This was done to vali-date that cardiac function remained stable during im-aging in the vertical position in severe heart failure.Importantly, stability of failing hearts cannot necessar-ily be assumed even if it is shown for normal mice. Theincreased body weight necessitated the use of a largerradio-frequency (RF) coil for the chronically infarctedanimals. Keeping the spatial resolution and the numberof averages constant, the obtained SNR was decreased(Fig. 6) compared to the study of normal mice (Fig. 4).Although a reduced inflow effect due to the impairedcardiac function additionally diminished the contrast-to-noise ratio, the achieved contrast was still highlysufficient to accurately measure LV volumes. Becausethe slopes of the relationships between LV ejection frac-tion and infarct size were similar for experiments donein vertical and horizontal position, and their confidenceintervals overlapped, our data clearly suggest that thevertical position did not have a major effect on theanalysis of cardiac function in chronically infarctedmouse hearts. Our results, therefore, demonstrate thatnormal and failing mouse hearts can be successfullyimaged using MR systems with a vertical bore.

In conclusion, cardiac imaging was implemented on ahigh-field MR system with high temporal and spatialresolution to examine mice in an upright position. Re-spiratory gating was found to be essential for obtainingartifact-free cine images. Cardiac functional parame-ters of normal and failing mouse hearts agreed well withstudies performed in the horizontal position. Hence, weconclude that MR systems with a vertical bore can gen-erally be used to accurately measure cardiac functionin both normal and failing mouse hearts, within onehour.

ACKNOWLEDGMENTS

We thank Mrs. Yvonne Green for practical assistance inthe laboratory.

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