First-pass perfusion CMR two days after infarction predicts severity of functional impairment six weeks later in the rat heart.

Abstract

In humans, dynamic contrast CMR of the first pass of a bolus infusion of Gadolinium-based contrast agent has become a standard technique to identify under-perfused regions of the heart and can accurately demonstrate the severity of myocardial infarction. Despite the clinical importance of this method, it has rarely been applied in small animal models of cardiac disease. In order to identify perfusion delays in the infarcted rat heart, here we present a method in which a T1 weighted MR image has been acquired during each cardiac cycle.
In isolated perfused rat hearts, contrast agent infusion gave uniform signal enhancement throughout the myocardium. Occlusion of the left anterior descending coronary artery significantly reduced the rate of signal enhancement in anterior regions of the heart, demonstrating that the first-pass method was sensitive to perfusion deficits. In vivo measurements of myocardial morphology, function, perfusion and viability were made at 2 and 8 days after infarction. Morphology and function were further assessed using cine-MRI at 42 days. The perfusion delay was larger in rat hearts that went on to develop greater functional impairment, demonstrating that first-pass CMR can be used as an early indicator of infarct severity. First-pass CMR at 2 and 8 days following infarction better predicted outcome than cardiac ejection fraction, end diastolic volume or end systolic volume.
First-pass CMR provides a predictive measure of the severity of myocardial impairment caused by infarction in a rodent model of heart failure.

Full text

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RESEARCHOpen Access
First-pass perfusion CMR two days after infarction
predicts severity of functional impairment six
weeks later in the rat heart
Daniel J Stuckey1,2*, Carolyn A Carr1, Stephanie J Meader1, Damian J Tyler1, Mark A Cole1and Kieran Clarke1
Abstract
Background: In humans, dynamic contrast CMR of the first pass of a bolus infusion of Gadolinium-based contrast
agent has become a standard technique to identify under-perfused regions of the heart and can accurately
demonstrate the severity of myocardial infarction. Despite the clinical importance of this method, it has rarely been
applied in small animal models of cardiac disease. In order to identify perfusion delays in the infarcted rat heart,
here we present a method in which a T1weighted MR image has been acquired during each cardiac cycle.
Methods and results: In isolated perfused rat hearts, contrast agent infusion gave uniform signal enhancement
throughout the myocardium. Occlusion of the left anterior descending coronary artery significantly reduced the
rate of signal enhancement in anterior regions of the heart, demonstrating that the first-pass method was sensitive
to perfusion deficits. In vivo measurements of myocardial morphology, function, perfusion and viability were made
at 2 and 8 days after infarction. Morphology and function were further assessed using cine-MRI at 42 days. The
perfusion delay was larger in rat hearts that went on to develop greater functional impairment, demonstrating that
first-pass CMR can be used as an early indicator of infarct severity. First-pass CMR at 2 and 8 days following
infarction better predicted outcome than cardiac ejection fraction, end diastolic volume or end systolic volume.
Conclusion: First-pass CMR provides a predictive measure of the severity of myocardial impairment caused by
infarction in a rodent model of heart failure.
Background
Non-invasive, in vivo measurements of cardiac morphol-
ogy, viability and function have revolutionised our
understanding of cardiac disease [1-3]. In combination,
echocardiography, angiography, PET, SPECT, CT and
CMR can be used to measure wall and valve motion,
cavity volumes, aortic and coronary blood flows, tissue
perfusion and cardiac metabolism [1-5]. CMR offers
excellent soft tissue contrast, coupled with high tem-
poral and spatial resolution, allowing accurate measure-
ment of cavity volumes, wall thickness and ejection
fraction. Further, infarct size and transmurality, mea-
sured using CMR shortly after systemic infusion of the
contrast agent gadolinium-diethylenetriaminepentaace-
tate (DTPA), strongly correlates with progression to
heart failure and patient mortality [2,5-8]. MR imaging
of the first-pass of a bolus infusion of Gd-DTPA has
become a standard clinical method for identifying
under-perfused regions of the human heart and accu-
rately predicts the severity of myocardial ischemia
[9-14].
Scaling down imaging techniques to study the ever
increasing number of small animal disease models has
been a formidable challenge [15]. The rat model of myo-
cardial infarction, induced by occlusion of the left ante-
rior descending coronary artery, is widely used and has
provided data essential for the development of surgical
and pharmacological therapy [16]. However, the left
ventricular mass of the rat heart (~500 mg) is approxi-
mately three hundred times smaller than the human left
ventricle (~150 g), and contracts 5 to 10 times faster,
making it necessary to use high field strengths, smaller
voxel sizes and rapid gating strategies.
* Correspondence: daniel.stuckey1@imperial.ac.uk
1Department of Physiology, Anatomy and Genetics, University of Oxford,
Parks Road, Oxford, UK
Full list of author information is available at the end of the article
Stuckey et al. Journal of Cardiovascular Magnetic Resonance 2011, 13:38
http://www.jcmr-online.com/content/13/1/38
© 2011 Stuckey et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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Small animal cine-MRI has identified increased left
ventricular mass, end diastolic and end systolic volumes
in the infarcted heart during progressive cardiac remo-
deling [17]. Pharmocologiical and biological therapies
have been extensively tested in this model system
[16,18]. However, owing to the variability of infarct size
induced by experimental myocardial infarction, it is use-
ful to have a baseline measure of infarcted heart func-
tion prior to administration of the therapeutic agent.
Acute changes to left ventricular mass and volume, typi-
cally made using echocardiography, are not good predic-
tors of functional outcome, making these frequently
used experimental methods for assessing acute infarct
severity inaccurate. A more robust early measure of dis-
ease severity may be obtained from late gadolinium
enhancement (LGE) CMR [19] or perfusion imaging.
LGE-CMR identifies tissue damage due to increased
contrast agent retention within infarcted areas of caused
by cell membrane rupture and increased interstitial
space [3,5]. These processes occur within hours of
infarction, so offer good early predictors of infarct sever-
ity. An even earlier indicator of the extent of functional
impairment may be offered by perfusion imaging
because it is the reduced perfusion that ultimately
results in cell necrosis, membrane rupture, reduced con-
tractility, remodeling and heart failure [10]. Myocardial
perfusion has been imaged using arterial spin labeling
and T1mapping [20-23], but imaging the first-pass of a
Gd-bolus infusion has rarely been used to study small
animal models of myocardial infarction [24,25], probably
owing to the need for ultra-fast and high-resolution
imaging [20].
Here, we have developed a method to identify perfu-
sion deficits in infarcted rat hearts during bolus infusion
by acquiring a T1weighted MR image every cardiac
cycle, both in the isolated perfused heart and in vivo.
Perfusion delay and myocardial viability, morphology
and function were measured in vivo at 2 and 8 days
after infarction to compare with cardiac function mea-
sured in the same animals at 42 days.
Methods
Experimental design
For ex vivo MRI studies of isolated, perfused hearts,
control female Wistar rat hearts (n = 12) were isolated,
perfused and imaged as described below. Hearts were
then removed from the magnet, the coronary artery
occluded, the heart repositioned and re-imaged (n = 8).
For in vivo MRI studies, baseline cine-MRI was per-
formed prior to surgery. Female Wistar rats (n = 34)
underwent myocardial infarction followed by in vivo
first-pass imaging, cine- and LGE-CMR at 2 and 8 days.
Problems with i.v. infusions, owing to unsuccessful tail
vein cannulation, movement of cannulae during
positioning of rats within the magnet and blood clots or
air bubbles in the drug line, meant that first-pass ima-
ging could not be performed on all 34 rats at each time
point (2 days, n = 21; 8 days, n = 27; both 2 and 8 days,
n = 14). After 9 days, 7 of the rats that were imaged at
2 and 8 days were sacrificed; hearts were isolated, per-
fused and imaged ex vivo. The remaining 27 rats
(14 and 20 of which had had first-pass imaging at 2 and
8 days respectively) underwent cine-MRI at 42 days.
The University of Oxford Animal Ethics Review Com-
mittees and the Home Office (London, UK) approved all
procedures.
Ex vivo MRI of isolated, perfused hearts
Hearts were excised from control (n = 12) or infarcted
(n = 7) rats, arrested in ice-cold Krebs-Henseleit buffer
and attached to a cannula by the ascending aorta.
Hearts were perfused at 85 mmHg with filtered Krebs-
Henseleit buffer containing 11 mM glucose and 1.8 mM
pyruvate. Buffer was maintained at 37°C and pH 7.4,
aerated with 95% O2/5% CO2and not re-circulated, to
avoid contrast agent accumulation. A balloon connected
to a pressure transducer was placed in the left ventricu-
lar (LV) cavity, inflated to an end diastolic pressure of
4 mmHg and used to measure cardiac function. The
heart was placed in a 20 mm NMR tube and lowered
into the centre of a vertical-bore, 11.7 T MR magnet
(Magnex Scientific, Oxon, United Kingdom) with a
20 mm1H birdcage coil (Rapid Biomedical, Würzburg,
Germany) and a Bruker console (Bruker Medical, Ettlin-
gen, Germany) running Paravision 2.1.1. Perfusion was
then switched to a constant flow of 15-20 ml/min, based
on each heart’s coronary flow at 85 mmHg.
First-pass perfusion imaging was performed to cover
the entire LV with repeated acquisitions using a T1
weighted fast gradient echo sequence that acquired one
image every 128 ms (TE/TR, 0.8/2 ms; 60° pulse; field of
view 20 × 20 mm; slice thickness 1 mm; matrix size 64 ×
64, zero filled to 256 × 256). It was determined that rapid
image acquisition was preferential to greater spatial reso-
lution as this allowed more images to be acquired during
the first pass of contrast agent, gave greater signal to
noise and reduced motion artifacts. A bolus of 25 μl of
0.5 mmol/ml Gd-DTPA-BMA (Gadodiamine, Omniscan)
was rapidly infused after approximately 10 of the 128
images were acquired. First-pass imaging was repeated
during multiple bolus infusions to cover the entire LV.
Control hearts were then removed from the magnet, the
LAD was permanently occluded using a 5-0 silk suture
(successful in 8 hearts) and first-pass imaging was
repeated during multiple bolus infusions to cover the
entire LV. Saturated gradient echo images were also
acquired to determine whether LGE could be detected in
the infarcted, perfused heart.
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Coronary occlusion model of myocardial infarction
Rats (n = 34) were anesthetized with 2.5% isoflurane in
O2, intubated, and a thoracotomy was performed. The
pericardium was removed and the heart was infarcted
by ligation of the left anterior descending coronary
artery (LAD), approximately 2 mm from the origin,
using a 5-0 proline suture [26].
In vivo CMR
Rats were anesthetized with 2.5% isoflurane in O2and
positioned supine in a purpose-built cradle. ECG elec-
trodes were inserted into the forepaws, a respiration
loop was taped across the chest and heart and respira-
tion signals were monitored using a custom-built phy-
siological motion gating device [27]. The cradle was
lowered into a vertical-bore, 11.7 T MR system with a
52-mm birdcage coil (Rapid Biomedical, Würzburg, Ger-
many) and a Bruker console running Paravision 2.1.1.
First-pass CMR was performed on a single short axis
slice, which encompassed the papillary muscles, using a
fast gradient echo sequence that acquired one image per
heart beat (TE/TR, 0.8/2 ms; ~60° pulse; field of view 40 ×
40 mm; slice thickness 1.5 mm; matrix size 64 × 64, zero
filled to 256 × 256). The flip angle was adjusted to null sig-
nal in the myocardium before bolus infusion. A bolus of
0.5 mg/kg Gd in 200-270 μl saline was infused via the tail
vein in 2 seconds during image acquisition. This volume,
derived from ex vivo data, was ten times greater than that
used in ex vivo experiments as in vivo cardiac output (75-
100 ml/min) was 5 times greater than ex vivo perfusion
rate (15 - 20 ml/min) and infusion time was 2 seconds
in vivo and less than 1 second ex vivo
Cardiac cine-MRI was performed 10 to 25 min after Gd
infusion as described [28]. A stack of contiguous 1.5 mm
thick true short-axis ECG and respiration-gated cine-
FLASH images (TE/TR 1.43/4.6 ms; 25° pulse; field of
view 51.2 × 51.2 mm; matrix size 256 × 256; voxel size
200 × 200 × 1500 μm; 25 to 35 frames per cardiac cycle
(mean heart rate 360 ± 20 bpm), were acquired to cover
the entire left ventricle. Long-axis two-chamber and
four-chamber images were also acquired. The relatively
high flip angle (25°) of the cine-MRI acquisition allowed
LGE-CMR to be assessed from the same stack of cine
images, similar to the method recently described and
validated by Protti et al [29]. The entire in vivo imaging
protocol was performed in approximately 60 minutes.
MRI data analysis
Image analysis was performed by an operator blinded to
the surgical procedure performed on the rats using Ima-
geJ (NIH Image, Bethesda, MD). Left ventricular
volumes and ejection fractions were calculated from the
stack of cine images as described [18]. To determine the
area of akinetic scar tissue, the average endocardial and
epicardial length of akinetic tissue in cine images were
measured in each slice, summed across all slices and
multiplied by slice thickness (1.5 mm) to give the abso-
lute scar size as described by Nahrendorf et al [17] Per-
fusion was assessed by analyzing the change in signal
intensity during Gd infusion in regions of interest
(ROIs) selected in the infarcted anterior myocardium,
adjacent to the infarct in the peri-infarcted lateral region
and in the remote tissue of the inferior wall of the left
ventricle. The relative rate of perfusion delay was mea-
sured as the time from inflow of contrast agent to the
LV cavity, to 90% of the maximum signal intensity in
the ROI. From ex vivo perfusion measurements, it was
possible to measure the rate of contrast inflow and
efflux from the gradient of the signal intensity/time
curve. The area of reduced perfusion on the stack of ex
vivo images was identified as myocardium with signal
intensity greater than two standard deviations below
that of remote myocardium.
In vivo LGE-MR images were thresholded to two stan-
dard deviations above the mean signal intensity from
remote tissue. The volume and perimeter (endocardial
length of the enhanced region) of the hyperenhanced
region was traced manually and expressed as a percen-
tage of total LV volume or perimeter.
Data and statistical analysis
Results are shown as means ± standard errors. Differ-
ences were considered significant at P < 0.05 using
unpaired Student t-tests or ANOVAs for repeated mea-
sures. Dependence between two measures was assessed
using Pearson’s Correlations.
Results
First-pass cardiac perfusion imaging in isolated, perfused
rat hearts
Repeated imaging of bolus injections of Gd were per-
formed to determine the contrast agent infusion volume
and duration that resulted in maximal contrast within
perfused control rat hearts. At a buffer flow rate of
15 ml/min, a rapid (approximately 1 second) injection
of 25 μl of 0.5 mmol/ml Gd, directly proximal to the
aorta, led to uniform enhancement throughout the myo-
cardium (Figure 1a, b), with time to 90% maximum
enhancement similar for inferior, lateral and anterior
regions. The rapid clearance of Gd from the tissue
allowed bolus injections to be repeated every 90 sec-
onds. Larger bolus infusions (> 50 μl) resulted in non
uniform signal enhancement, and smaller infusion
volumes (10 μl) did not enhance at all (Figure 1b). It
was also possible to measure the gradient of the signal
intensity/time curves at the time of Gd inflow. Again,
the rate of signal intensity increase was similar for infer-
ior, lateral and anterior regions (Figure 1d). The rate of
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clearance of the Gd bolus from the myocardium was
calculated as the gradient of the signal intensity/time
curve from 10 to 15 seconds after bolus infusion. No
differences in Gd efflux between regions were observed
(Figure 1d).
Perfused hearts were then removed from the magnet
and the LAD occluded (successful occlusion in 8 of 12
hearts). Areas of low perfusion were identified below the
occluded LAD as non-enhanced regions on MR images
acquired during the Gd bolus infusion (Figure 1a). Perfu-
sion delay (PD), calculated as the delay between time to
90% enhancement in an ROI compared with the remote
inferior wall (Figure 1c), was significantly longer in the
infarcted anterior wall (3.47 ± 0.65 s, p < 0.001) and the
peri-infarcted lateral wall (0.81 ± 0.49 s, p < 0.05). The
gradient of signal intensity increase was significantly
lower in the infarcted (0.40 ± 0.02 a.u./s, p < 0.05) and
peri infarcted (3.00 ± 0.20 a.u./s, p < 0.05) regions com-
pared with remote regions (4.65 ± 0.30 a.u./s, Figure 1d).
The rate of Gd clearance was also significantly lower in
anterior myocardial regions affected by LAD occlusion
(infarct, -0.28 ± 0.04, peri-infarct -1.00 ± 0.20, remote
-1.40 ± 0.68 a.u./s, Figure 1d). Saturated gradient echo
images, acquired up to 50 min after coronary occlusion
found no evidence of LGE in the perfused infarcted
hearts.
In vivo first-pass perfusion imaging of infarcted rat hearts
Cardiac morphology and function
Cine-MRI measurements of cardiac morphology and
function were made serially on rats prior to and at 2, 8
and 42 days after infarction (Table 1). Left ventricular
mass, end diastolic volume (EDV), end systolic volume
(ESV) and body weight were significantly increased at 8
and 42 days after infarction. Ejection fraction (EF) was
reduced by 2 days, whereas stroke volume and cardiac
output were depressed at 2 days, but returned to normal
levels by 8 days. The percentage of akinetic myocardium
and heart rates did not change from 2 to 42 days.
First-pass cardiac perfusion imaging 2 and 8 days post
infarction
Infusion of Gd initially reduced signal in the LV cavity
owing to T1 and T2 shortening of blood from the high
concentration of contrast agent (Figure 2a). This made
it impossible to measure an arterial input function.
However, signal in the myocardium became enhanced
shortly after cavity hypoenhancement, as contrast agent
perfused through the coronary circulation. Slight cyclical
elevations in myocardial signal intensity occurred due to
respiratory motion. Even though the image acquisition
time continued through much of the cardiac cycle, car-
diac motion did not prevent measurement of contrast
inflow. Imaging of first-pass perfusion in rat hearts
10 ?l Gd
0
5
10
15
05 101520
Inferior
(remote)
Lateral
(peri)
Anterior
(occluded)
time (s)
signal intensity (a.u.)
90% enhancement
25 ?l Gd
50 ?l Gd
efflux
influx
??
?
?
?
?
?
?
??????????????
control occluded
Change in SI / sec
control
occluded
sec
b
0
1.53.04.5 6.07.5 9.010.512 13.51516.5
*
*
??????????????
a
c
d
inf
ant
sep
lat
Figure 1 First-pass imaging of the isolated, perfused rat heart. (a) 12 of the 128 images acquired prior to, during and after bolus Gd
infusion from control and ex vivo occluded perfused hearts: (b) Images showing effect of different Gd bolus volumes; (c) Signal intensity/time
curves from inferior (inf), lateral (lat) and anterior (ant) regions of a heart in which the coronary artery was occluded on the rig. Arrow indicates
time of bolus infusion: (d) Graph of rates of change in signal intensity in different regions of control (n = 12) and ex vivo occluded (n = 8)
hearts. Positive values calculated from contrast influx, negative values calculated from contrast efflux. *p < 0.05 compared with lateral remote
tissue.
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identified areas of perfusion deficit in the region affected
by the LAD coronary artery occlusion (Figure 2a, b).
The average perfusion delay in infarct and peri-infarct
compared with remote tissue was 4.0 ± 0.7 and 1.7 ±
0.4 s at 2 days post infarction and 3.3 ± 0.5 and 0.5 ±
0.3 s at 8 days post infarction.
Late Gadolinium enhancement imaging 2 and 8 days post
infarction
LGE-CMR was performed 10-25 min after first-pass
imaging. Hyperenhancement was present in 18 ± 2% of
the LV volume and 19 ± 2% of LV endocardial peri-
meter at 2 days post infarction, and 11 ± 1% of the LV
volume and 16 ± 2% of LV endocardial perimeter at 8
days post infarction (Figure 2c).
For the sub-group of infarcted rats (n = 14) in which
tail vein canulation was successful at both time points,
first-pass CMR and LGE-CMR were performed at both
2 and 8 days post infarction. Perfusion delay (measured
using first-pass CMR) and LGE-CMR endocardial scar
perimeter were unchanged from 2 to 8 days (Table 2).
Table 1 In vivo measurements of rat heart morphology and function before and at 2, 8 and 42 days after infarction (n = 27)
Pre2 days8 days42 days
586 ± 16*#
428 ± 24*#+
185 ± 19*#+
243 ± 11#
58 ± 2*#+
28 ± 6*#+
11 ± 2*
346 ± 26
80 ± 3#
245 ± 19
Left ventricular mass (mg)
End diastolic volume (μl)
End systolic volume (μl)
Stroke volume (μl)
Ejection fraction (%)
Akinetic myocardium (mm2)
Akinetic myocardium (%)
Heart rate (bpm)
Cardiac output (ml/min)
Weight (g)
492 ± 35
265 ± 12
55 ± 8
210 ± 9
80 ± 2
-
-
404 ± 11
85 ± 5
234 ± 11
543 ± 18*
282 ± 11
108 ± 5*
174 ± 8*
62 ± 1*
16 ± 4*
9 ± 2*
361 ± 9
61 ± 3*
214 ± 3
571 ± 15*
395 ± 15*#
154 ± 13*#
241 ± 7#
62 ± 2*
23 ± 5*#
10 ± 2*
344 ± 27
83 ± 3#
230 ± 5
* p < 0.05 compared with pre-infarction.#p < 0.05 compared with 2 days.+p < 0.05 compared with 8 days,
control
infarct
sec
b
0
1.53.04.56.0 7.59.010.512 13.51516.5
0
5
10
15
05 101520
time (s)
signal intensity (a.u.)
infarctremote peri
cine LGE-CMR first-pass ex vivo
9d
Infarct 8d Infarct 2d sham 2d
90% enhancement
a
c
inf
ant
sep
lat
Figure 2 In vivo first-pass imaging. (a) 12 of the 128 images acquired prior to, during and after bolus Gd infusion from control and in vivo
infarcted rat hearts: (b) signal intensity/time curves from infarcted, peri-infarcted and remote regions of an infarcted heart. Arrow indicates time
of bolus infusion: (c) in vivo cine-CMR, LGE-CMR and first-pass images from a sham operated rat and an infarcted rat at 2 and 8 days post
surgery. First-pass images from the same isolated, perfused hearts are presented on the far right.
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However, there was a significant reduction in LGE-CMR
scar size (measured as % LV volume) between 2
and 8 days (19 ± 3 and 10 ± 2% respectively, p < 0.01;
Figure 3 and Table 2) and in absolute scar size (100 ±
18 μl at 2 days vs. 56 ± 11 μl at 8 days, p < 0.01).
Correlations between MRI measurements at 2 and 8 days
and ejection fractions and end systolic volumes at 42 days
Perfusion delays measured at 2 days post infarction cor-
related with left ventricular end systolic volumes (R =
0.84, P < 0.0001) and ejection fractions (R = 0.85, P <
0.001) measured at 42 days (Figure 4), suggesting that
rats with acute perfusion deficit went on to develop
greater chronic cardiac impairment. As anticipated,
infarct size measured using LGE-CMR at 2 days post
infarction correlated with left ventricular end systolic
volumes (R = 0.67, P < 0.01) and ejection fractions (R =
0.74, P < 0.01) measured at 42 days (Figure 4). However,
correlations between infarct size and EF or ESV were
lower than those found using first-pass CMR. At 2 days,
perfusion delay gave a better prediction of outcome
than cine MRI measurements of morphology and func-
tion, with no correlation between 2 day end systolic
volumes and ejection fractions and the end systolic
volumes and ejection fractions measured at 42 days.
Perfusion delay measured at 8 days post infarction also
correlated with ESV (R = 0.76, P < 0.05) and EF (R =
0.63, P < 0.05) at 42 days. Again, these correlations were
stronger than those between LGE-CMR, ESV and EF at
8 days and the resulting ejection fractions and end sys-
tolic volumes measured at 42 days (data not shown).
First-pass cardiac perfusion imaging in isolated, perfused
infarcted rat hearts
Nine days after infarction, 7 rat hearts were excised,
perfused in Langendorff mode and imaged. Infusion of
25 μl Gd into the perfusate proximal to the aorta gave
uniform signal enhancement throughout the remote
myocardium, whereas infarcted regions showed only
limited enhancement (Figure 2c). Signal intensity/time
curves plotted from images acquired at the same loca-
tion in vivo and ex vivo showed similar characteristics
and resulted in similar measurements of perfusion delay
(in vivo = 2.9 ± 2.1s, ex vivo = 2.8 ± 0.8s, correlation;
R2= 0.7. p < 0.05). The gradient of signal intensity
increase was significantly lower in the infarcted and peri
infarcted regions compared with remote tissue (infarct,
0.38 ± 0.03, peri 2.36 ± 0.42, remote 4.31 ± 0.91, p <
0.01), while the rate of Gd clearance was significantly
lower in infarcted regions compared with remote
(infarct, 0.05 ± 0.04, peri 0.25 ± 0.08, remote 0.44 ±
0.05). These experiments also indicated that the volume
of reduced perfusion measured ex vivo matched the
volume of LGE measured in vivo (Additional file 1).
Interestingly, saturated gradient echo images performed
10 to 50 minutes after Gd infusion found no evidence
of LGE in the perfused infarcted hearts. This probably
occurred because Gd was infused as a single 25 μl bolus
and heart perfusate was not circulated, preventing accu-
mulation of Gd in the infarcted tissue
Discussion
Clinical studies have demonstrated that the extent of
myocardial perfusion deficit after infarction is a strong
predictor of functional outcome, with patients who pre-
sent with large microvascular obstructions (MVO) hav-
ing poor prognosis [10]. MR imaging of the first-pass of
a bolus infusion of Gd is now a well established techni-
que for accurate identification and localisation of MVOs
[5,11-13,30]. Despite its importance, first-pass MR ima-
ging has only very recently been used in small animal
models of myocardial infarction [24,25]. Here we devel-
oped a CMR method which acquired one image every
heart cycle and was sensitive to the inflow of Gd into
the myocardium, thus providing a rapid technique for
assessing tissue perfusion.
Several methods have been used to assess myocardial
perfusion in small animal models. Arterial spin labelling
and T1mapping can provide quantitative measurement
Table 2 In vivo measurements of cardiac perfusion and
viability in infarcted rat hearts (n = 14) at 2 and 8 days
after infarction
2 days 8 days
Perfusion delay (s)
LGE-CMR % perimeter
LGE-CMR % volume
LGE-CMR volume (μl)
3.57 ± 0.71
18 ± 3
19 ± 3
100 ± 18
3.51 ± 0.58
15 ± 3
10 ± 2#
56 ± 11#
#p < 0.01 compared with 2 days.
Figure 3 Repeated measures of LGE-CMR volume made in the
same 14 infarcted rat hearts that had successful tail vein
cannulation at 2 and 8 days after infarction. * p < 0.05
compared with 2 day data.
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of blood flow post infarction in rats and mice [20-23].
However, the accuracy of these techniques is decreased
by rapid and variable heart rates, artefacts produced by
respiratory motion during acquisition and a single slice
acquisition takes tens of minutes. PET imaging of radio-
tracers can be used to measure myocardial perfusion,
but low spatial resolution, attenuation artefacts and the
need for a local cyclotron to produce tracers, such as
82Rb or H215O, limit the application of this technology,
especially when applied to small animals [5,11-13,30,31].
We initially developed and optimised the first-pass
method using isolated perfused rat hearts. This prepara-
tion permitted repeated contrast infusions in the same
heart so that Gd bolus volume, concentration and infu-
sion time could be optimised in a controlled environ-
ment. Repeated infusions indicated good reproducibility
and uniform enhancement in anterior, lateral and infer-
ior myocardial regions. To determine the sensitivity of
the first-pass technique to regional perfusion deficits the
LAD coronary artery was occluded and first-pass ima-
ging was repeated. Signal intensity was measured in
regions of interest placed in infarcted, peri-infarcted and
remote tissue of all 128 images acquired during bolus
infusion. Two methods were used to quantify regional
perfusion changes. Firstly, perfusion delay was calculated
as the time delay, in seconds, between 90% max
enhancement in infarcted regions compared with remote
regions. Secondly, in ex vivo perfused hearts it was
possible to assess contrast influx rate from the linear
portion of the positive gradient of the signal intensity/
time curve and contrast efflux rate from the signal
intensity/time curve 10-15 seconds after infusion. As
both of these analysis methods relate changes in
infarcted regions with those of remote regions of the
same infusion any effects of slight differences in bolus
transit time and volume can be reduced, providing more
reproducible data.
The first-pass imaging method had sufficient T1
weighting and temporal and spatial resolution to permit
in vivo measurement of contrast inflow into the myocar-
dium. The requirement for a rapid imaging sequence that
could acquire an image every heart beat favoured a fast
gradient echo acquisition rather than the more tradi-
tional inversion recovery sequence. The fast gradient
echo acquisition resulted in signal saturation within the
myocardium prior to contrast arrival, but did not null
signal in the blood pool due to the rapid influx. Contrast
transit through the ventricular cavities reduced signal
intensity owing to the susceptibility effects of high Gd
concentration at 11.7 T. Importantly the Gd bolus
resulted in signal enhancement within the perfused myo-
cardial tissue. As with the isolated perfused heart experi-
ments, signal intensity was measured in regions of
interest placed in infarcted, peri-infarcted and remote tis-
sue of all 128 images acquired during bolus infusion and
perfusion delay was calculated as the time delay in
Figure 4 Correlations between 2 day measurements of perfusion delay, LGE-CMR, ESV or EF, and resulting ESV or EF measured in the
same animals at 42 days post infarction (n - 14 animals that had successful tail vein cannulation at 2 days and were imaged at
42 days).
Stuckey et al. Journal of Cardiovascular Magnetic Resonance 2011, 13:38
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seconds between 90% max enhancement in infarcted
regions compared with remote regions. Perfusion delay
in the infarcted regions was significantly longer than in
remote tissue. Owing to the need for acquisition of high
temporally resolved images, the in vivo spatial resolution
of the first-pass technique is limited. Although regions of
interest could be selected within the infarct territory, it
was not possible to accurately assess the area of the per-
fusion deficit. This is augmented in the thinned scar tis-
sue of the chronically infarcted heart and can make
delineation of the endocardial border problematic. A
further limitation of the method is that it cannot be per-
formed at multiple slice positions during a single Gd
bolus, while repeated bolus imaging is complicated by
LGE in infarcted regions.
The severity of perfusion delay and extent of delayed
enhancement measured at 2 and 8 days strongly corre-
lated with cardiac function measured in the same rats
42 days after infarction. These correlations were stronger
than those between ejection fraction and end systolic
volume measured at 2 or 8 days and ejection fraction or
end systolic volume at 42 days. Acutely after infarction,
myocardial stunning, edema, and elevated catecholamine
levels can strongly influence EF and ESV, masking the
extent of systolic dysfunction. A combination of these
factors, coupled with the variability of infarct size typi-
cally induced by experimental myocardial infarction,
makes acute measurement of EF and ESV a poor early
predictor of outcome and suggests that the common
practice of dividing infarcted animals into experimental
groups based on EF measured made principle by echo-
cardiography in the first week post infarction can lead to
wide variability in extent of functional impairment
between groups. The first-pass and LGE-CMR techni-
ques presented here gave better predictors of outcome
cine-MRI, provided a more accurate assessment of myo-
cardial viability acutely after infarction and may offer a
useful baseline measure of infarct severity prior to
administration of the therapeutic agent.
Ex vivo first-pass imaging was performed on isolated,
perfused infarcted rat hearts that had undergone in vivo
imaging at 2 and 8 days post infarction. In vivo and ex vivo
measurements of perfusion delay correlated, as did
in vivo LGE-CMR volume and ex vivo low perfusion
volume and Gd clearance time. This supports the hypoth-
esis that LGE-CMR is due to reduced washout and hence
greater retention of Gd in infarcted regions [32].
The volume of LGE significantly decreased from 2 days
to 8 days post infarction, and was unquantifiable at 42
days. Similar results were reported in canine models [33]
and in two recent clinical studies which found a decrease
in LGE from 12 to 8% of the left ventricle between 1 and
8 days after infarction [34] and from 27 to 22% between
24 hours to 6 months [35]. This decrease is probably due
to hyperenhancement of oedematous, inflamed, viable
tissue within the infarct border zone at 2 days post
infarction, and thinning of the scar, infarct resorption
and hypertrophy of remote tissue at 8 days [33,34]. How-
ever, it draws attention to possible limitations of using
LGE-CMR in small animal models of chronic infarction
where transmurality and tissue remodelling can make
infarct assessment difficult. Few studies have used LGE-
CMR chronically after infarction, and those that have did
not study animals with large transmural infarcts [36],
The first-pass CMR measurements of perfusion delays
measured in this study did not change from 2 to 8 days
post infarction, but we predict that the spatial resolution
of the technique may be inadequate for measuring perfu-
sion in established transmural scar tissue.
The resolution of the first-pass method may be
improved through parallel imaging using multi channel
phase array coils and accelerated image acquisitions.
Schneider et al recently demonstrated the feasibility of
using parallel imaging to achieve threefold acceleration of
cine image acquisition in mice [37]. By using similar stra-
tegies, it may be possible to perform multi slice first-pass
imaging with a spatial resolution that permits accurate
delineation of underperfused areas of myocardium, or to
assess changes in perfusion in peri-infarct areas in
response to dobutamine stress or after experimental ther-
apy with angiogenic cytokines or stem cells. A novel ther-
apeutic strategy for replacing myocardium lost during
infarction is grafting of engineered heart tissue [38,39]. It
is currently difficult to assess in vivo whether the graft is
incorporated into the host’s vasculature. First-pass CMR
may be able to detect contrast agent inflow into the engi-
neered tissue and determine successful integration.
Conclusions
In summary, we have presented a novel, simple and use-
ful MR method for identification of under-perfused tis-
sue in the infarcted rat heart and demonstrate the high
prognostic value of the technique compared with other
non-invasive measures of cardiac function and viability.
This method is a useful tool for assessing the severity of
myocardial ischemia acutely after infarction and could
provide important data on treatment strategies designed
to revascularise and regenerate the infarcted heart.
Additional material
Additional file 1: (a) Plot showing similarity in percent volume of
LGE-MRI in slices from base to apex measured in vivo 8 days post
infarction, and percent volume of myocardium with low perfusion
measured from base to apex measured using first-pass MRI of
isolated, perfused rat hearts 9 days post infarction: (b) Correlation
between LGE-MRI volumes measured in vivo and low perfusion
volumes measured using first-pass MRI in the same slice in
isolated, perfused hearts (R = 0.80. P < 0.01).
Stuckey et al. Journal of Cardiovascular Magnetic Resonance 2011, 13:38
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Abbreviations
LGE-CMR: late gadolinium enhancement cardiac magnetic resonance; EF:
ejection fraction; EDV: end diastolic volume; ESV: end systolic volume; FLASH:
fast low angle shot; Gd-DTPA: gadolinium diethylenetriaminepentaacetate;
LAD: left anterior descending coronary artery; LV: left ventricle; MVO:
microvascular obstruction; PD: pefusion delay.
Acknowledgements
This work was supported by The British Heart Foundation (grant number
PG/07/059/23259).
Author details
1Department of Physiology, Anatomy and Genetics, University of Oxford,
Parks Road, Oxford, UK.2Biological Imaging Centre, Imperial College,
Hammersmith Hospital, Du Cane Road, London W12 0NN, UK.
Authors’ contributions
DJS conceived and designed the study, performed all MRI and analysis, and
drafted the manuscript; CAC performed all surgical procedures and assisted
with MRI; SJM and MAC assisted with perfused heart experiments, DJT
assisted with MRI; KC assisted with study design, manuscript preparation and
funding. All authors read and approved the final manuscript
Competing interests
The authors declare that they have no competing interests.
Received: 21 February 2011 Accepted: 3 August 2011
Published: 3 August 2011
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doi:10.1186/1532-429X-13-38
Cite this article as: Stuckey et al.: First-pass perfusion CMR two days
after infarction predicts severity of functional impairment six weeks
later in the rat heart. Journal of Cardiovascular Magnetic Resonance 2011
13:38.
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