Assessment of left ventricular perfusion by intraperitoneal administration of MiBI-Tc using
high-resolution single photon detector
Abstract – The possible use of stem cell therapy
for coronary heart disease has generated a lot of
interest in the research community and in general
public. Clinical trials have been performed but
the results are contradictory. More basic studies
with small animals have to be performed.
Radionuclides imaging techniques have a special
role for their metabolic and functional capability
and for their high sensitivity. Nevertheless
studying cardiovascular diseases by means of
animal models is very challenging. In fact mice
have to be used for the availability of models and
this is very complicate both from animal handling
and for the characteristics required for the
detectors. Submillimeter spatial resolution, good
energy resolution and high sensitivity are
required. Moreover, using mice is particularly
challenging in situations that require injection of
radiotracers many times, possibly for week or
even months. Alternative routes of delivering the
radiotracer in tail vein have to be used. We
demonstrated that it is possible to inject the
radiotracer (MIBI-Tc) in the peritoneum with
reasonable uptake in the myocardium. This makes
possible to start studying the possible effects of
stem cell and monitoring the diffusion and the fate
of them.
1. Introduction
A novel treatment strategy, the delivery of foetal
or neonatal cardiomyocites, skeletal myoblasts, or
embryonic or bone marrow derived stem cells has
been used recently for research with animals and
even for clinical trials [1]. This strategy aims at
enhancing cardiac function by repopulating the
infarcted region with viable cardiomyocytes and,
therefore, bears great promise for the cure of this
disease. Initial nonrandomized trials showed slight
improvement in the left ventricular ejection
fraction. Later randomized, controlled trials,
however, suggested a less significant effect [2].
Open question remains. Further basic researches
with animals are needed; specific imaging
techniques that allow for in vivo visualization
of the therapeutic cells are mandatory.
Molecular imaging is the technique of choice.
Among different techniques the ones using
radio nuclides are very powerful for the high
sensitivity that allows seeing biological
processes in vivo at pico molar level.
Nevertheless, the technique is challenging
because of the concurrent requirements in
terms of submm spatial resolution and high
sensitivity. SPECT techniques have limited
sensitivity due to the collimation but the PET
has intrinsic limitations in terms of spatial
resolution [4]. SPECT detectors have a special
role in small animal imaging research. A large
spectrum of SPECT radiotracers is accessible,
and, provided the detector has good energy
resolution, multi-labelling allows studying
different phenomena simultaneously. For
example, dual tracer small animal SPECT would
allow simultaneous imaging of 99mTc-labeled
stem cells to assess myocardial perfusion and
of 111In labelled stem cells to delineate stem
cells engraftment. One of the processes to be
studied is the reduction of perfusion (and the
relative decrease in LV ejection fraction)
consequent to the infarction. This can be detected
with SPECT [5-7]. Multilabeling would be very
useful [3,5].
It has been shown [14] that after careful
calibration, using standard nuclear medicine
software, perfusion ECG gated SPECT in mice
permits quantification of LV volumes and motion.
This would allow evaluating the effects of therapy
in the limit of the sensitivity attained by the
system. This is very important because of the
smallness of the effects shown in the human trial
studies. Monitoring trafficking and homing of
stem cells after injection will be another task in
this challenging study.
The paper describes the research studies of our
collaboration for optimizing an imaging system
for cardiovascular diseases studies in mice.
Nevertheless in order to study the perfusion
decrease and possible recovery after stem cell
therapy repetitive injections (for weeks or months)
of the radiotracer are needed. This would be
impossible for mice modelled with infarction. A
different route of delivery of the radiotracer has to
be found. We decided to inject the tracer trough
the peritoneum and verify if the uptake by
myocardium was big enough to allow imaging the
perfusion. We have demonstrated that this is
possible, with some reduction in the uptake by the
myocardium. This makes possible to start
studying the effects of stem cell therapy and
monitoring the diffusion and the fate of them.
2. Material and Methods
The ideal system for these cardiovascular studies
is an “open” detector, to be integrated in the
future, with significant modifications (see later) in
a multimodality system with optical and MRI
detector [3]. To design such a system and the
animal handling issues we decided to use a single
head high-resolution detector prototype.
2.1 Detector layout
High spatial resolution and sensitivity as well as
good energy resolution are needed. The need for
trade off spatial resolution vs. sensitivity suggests
that an optimized SPECT detector system for
cardiovascular studies in mice should be made of
8 detectors (10 x 10 cm2) around the animal
allowing, with a magnification M=3, a FOV of 3.3
cm2, imaging the part of the mouse body relevant
for studying the stem cell trafficking and the
perfusion.
The calculations of Fig.1 were performed with ri
(intrinsic spatial resolution) =1 mm.
A resolution of ~ 0.5 mm is needed for the
research planned. It has been shown [13] that in
order to obtain this in the SPECT with
multipinholes, with 3D reconstruction, a sufficient
number of “resolution elements” has to be used.
This translates in the need of 120 pixels in 100
mm, that means an intrinsic spatial resolution of
ri=0.8 mm. Scintillator arrays of very small pixels
have to be used and identifying so small pixels is
challenging. It will require to fully exploit the
characteristics of the electronics we have designed
and built, capable of reading out up to ~ 4096
channels individually at 20 KHz [9, 16].
2.2 Micro SPECT System prototype.
Our prototype high resolution Single Photon
Emission Computed Tomography (SPECT)
system showed to be able to obtain good images
of perfusion by injecting MIBI-Tc on tail vein of
mice [7-12]. For this reason we have started our
research with a 100 x 100 mm2 NaI (Tl)
scintillator array (6700 pixels of 1.5 mm pitch, the
smallest to our best knowledge, used in this kind
of detectors). This allows obtaining a spatial
resolution largely sufficient for our scope also
because the readout electronics doesn’t allow, at
the moment, to perform gated SPECT.
The prototype SPECT system is a detector
equipped with a 2.5-cm-diameter acrylic
cylindrical bed-holder (3 mm thick) that keeps the
mouse horizontal (see Fig. 2).
Fig. 1 (a) Spatial resolution vs. efficiency for
different FOV and hole diameter.
Fig.1 shows the performances that can be obtained
with 1 detector 100 x 100 mm2, with intrinsic
spatial resolution of 1 mm. Fig 2 shows what can
be gained in spatial resolution by improving the
intrinsic spatial resolution (hole = 0.5 mm).
Fig. 3 The Spect system prototype
Fig. 2 Spatial resolution vs. intrinsic spatial
resolution.
Two detectors are mounted on a motorized gantry
that can rotate (only one detector was used for the
measurement described here). The bed holder stay
fixed. The system could be manually adjusted to
optimize the distance between the pinhole and the
axis of rotation, giving the possibility to resize the
camera parameters depending on measurements
requirements.
The detector characteristic and performance
parameters are listed in Tab1.
Tab.1
Pinhole Diameter (mm)
NaI (Tl) Scintillator:
- pitch (mm)
- Thickness (mm)
0.5
1.5
6
- Dimension (mm)
100 × 100
Photomultiplier Array
(2 × 2) H8500
Resolution (mm)
Efficiency (cps/MBq)
Magnification Factor
Field of View (mm)
sec/projection. The same procedure was used for the
second mouse but was injected with 6.7 mCi of 99mTcMIBI intraperitoneally. To assure high-resolution and
artefacts free SPECT image reconstruction, mechanical
calibration of detector was needed. For this reason,
tomographic acquisition of a set of 2-point sources (~ 1
mm in size) positioned as far as possible both along the
axis of rotation and away of it, was also performed.
< 0.8
35
3
33
2.3 PHANTOM STUDIES
To test the tomographic spatial resolution
capabilities of our detector, a miniature acrylic
resolution phantom was manufactured, as shown
in Fig. 2. It consists of 6 sectors, each containing
equally sized sets of capillaries (0.8, 0.9, 1.0, 1.1,
1.2, 1.3 mm). The overall phantom diameter was
2.5 mm. The total activity in all filled capillary
was ~ 4.5 mCi of 99mTc. The single pinhole
projection data were acquired in 60 angular
intervals over 360 degrees at 2 min/projection.
2.4 Animal procedures, Anaesthesia, and
Tracer administration
Two adult mice three-month-old VFB/N male
mice, weighted 30 g, were intraperitoneally
anesthetized. For one of the mice, the radiotracer
(MIBI-99mTc) was injected into the tail vein. Care
was taken to minimize, as much as possible, the
volume of injected tracers around 0.02-0.05 ml to
avoid significant changes in the whole blood
volume of the mice. The single pinhole projection
data were acquired in 60 angular intervals over
360 degrees.
Thoracic bone scan was performed to evaluate system
image quality (mouse was injected with 2 mCi of
99m
Tc-MDP).
Tomographic acquisitions started 2 hour after tracer
administration. Projection data were acquired at 2
min/projection. Myocardial perfusion scan was
performed. Live mouse was injected with 6.7 mCi of
99m
Tc-MIBI; acquisitions started 1 hour after tracer
administration to ensure a better contrast of heart to
soft tissues. Projection data were acquired at 60
2.5 Image reconstruction technique
Upper head projection data were reconstructed
using a 3D pinhole OS-EM algorithmError!
Reference source not found. Which takes into
account
system
geometric
misalignment
parameters, including the centre-of-rotation error,
the tilt angles between the axis-of-rotation and the
detector plane in the 3D space. Reconstruction
matrix size was 90°×°90°×°90 with a voxel size of
0.25 mm. A 3D Butterworth filter was used for
the post-reconstruction
2.6 Myocardial Perfusion Analysis
There is no true standard for quantification of
SPECT [15]. We used the Standardized uptake
value, SUV, also referred to as the dose uptake
ratio, DUR, calculated as a ratio of tissue
radioactivity concentration (in units kBq/ml) at
time T, CPET(T) and injected dose (in units MBq)
at the time of injection divided by body weight (in
units kg).
SUV=CPET(T)/(Injected dose / Patient's weight).
If the is radiotracer uniformly distributed, taking
into account the delay time, we calculated it for
the region of interest (heart) (Regional Uptake
value (RUV) RUV=[(counts)eλt)inROI/(Volume
ROI/CPET).
3. RESULTS
3.1 Phantom
Sensitivity
studies,
Spatial
Resolution,
For the resolution phantom as well as for
myocardial perfusion we used a FOV of 33
mm. The spatial resolution of the system is
~0.8 mm. The intrinsic spatial resolution of
the detector is 1.5 mm.
Tab.2
1.3 mm
Peritoneum
Tail vein
Dose (MBq)
170
130
Weight (g)
37
37
Age (week)
12
12
Duration/view
(min)
1
1
Transverse
0.62
1.09
Coronal
0.51
1.22
Sagittal
0.53
1.31
0.8 mm
1.2 mm
0.9 mm
1.1 mm
1.0 mm
Fig. 3 Miniature acrylic resolution phantom (left),
and reconstructed image (right), sum of 21 transaxial slices. 0.8 mm capillaries are clearly
separated on image
The sensitivity of the system was ~ 35 cps/MBq.
It was measured by using a source 370 kBq of Co57 in the centre of the FOV at a distance of 10
mm. The energy resolution was 14%.
3.2 Perfusion images
In Fig. 4 we show images of the mouse-injected
trough the tail vein; transverse, sagittal and
coronal views are shown.
Fig.4 Transverse, sagittal and coronal heart views. Tail
vein injection
The other mouse had the radiotracer injected
peritoneally. The procedures were the same. Fig 5
shows the perfusion images.
Fig. 4. The same as Fig. 3 for the mouse injected
peritoneal.
3.3 Uptake
Tab.2 shows the results for the two delivery
modalities.
A significant reduction of uptake occurred.
Ventricular cavities are identified in both cases.
4. SUMMARY CONCLUSIONS AND OUTLOOK
A single head high-resolution detector prototype
has been built for studying a SPECT system for
studying the diffusion of stem cells, their fate and
the effect of the therapy. The scope was to
determine the detector characteristics and the
study animal handling issues.
The spatial resolution of the prototype showed to
be largely sufficient for perfusion studies. The
energy resolution is sufficient for studies with
dual tracers techniques.
The sensitivity should have been significantly
higher using a larger dimension of the hole, but, in
any case, due to the difficulties of injecting many
times for weeks or months infarcted mice an
appropriate route of delivery of radiotracers has to
be found.
We demonstrated that injecting the radiotracer
through the peritoneum instead of the tail vein
allows obtaining an image of perfusion.
The price to be paled is a significant reduction of
the uptake by the heart.
So the sensitivity of the system has to be
increased to compensate the reduction of uptake
due to the intraperitoneal injection first.
Moreover to detect even small effects of the
therapy and to monitor the trafficking homing and
fate of stem cell injected great increase of the
sensitivity will be needed. This can be obtained by
fine-tuning the parameters (the hole dimension
and the magnification of the pinhole) of the
pinhole first, and then using the multipinhole
technique and finally adding as much as possible
detectors to the system.
It has to be underscored that in order to fully
accomplish the objectives of the study will
probably require the integration of other
modalities, essentially optical and MRI with
significant modifications of the layout, and of the
materials and components starting with
substitution
of
PSPMT’s
with
Silicon
Photomultipliers (SiPm) insensitive to the
magnetic fields.
Research in this sense is ongoing in the
collaboration.
References
[1] Orlic, D., et al., Bone marrow cells regenerate
infarcted myocardium. Nature, 2001. 410(6829):
p. 701-5.
Zhang, S., et al., Purified human bone marrow
multipotent mesenchymal stem cells regenerate
infarcite myocardium in experimental rats. Cell
Transplant, 2005. 14(10): p. 787-98.
Assmus, B., et al., Transcoronary transplantation
of progenitor cells after myocardial infarction.
N.Engl J Med, 2006. 355(12): p. 1222-32.
Wollert, K.C., et al., Intracoronary autologous
bone-marrow cell transfer after myocardia
infarction: the BOOST randomised controlled
Segers Vincent F.M. & Richard T. Lee, Stem cell
therapy for cardiac diseases, Nature, Vol. 451, 21
February 2008, doi:10.1038/nature06800
[2] reference to be provided by G. Marano
[3] Bengel, F.M., V. Schachinger, and S.
Dimmeler, Cell-based therapies and imaging in
cardiology.
Eur J Nucl Med Mol Imaging, 2005. 32 Suppl 2:
p. S404-16.
[4] T.Lewellen, Phys.Med.Biol.53(2008)R287–
R317
[5] Acton, P.D. and R. Zhou, Imaging reporter
genes for cell tracking with PET and SPECT. Q J
Nucl Med Mol Imaging, 2005. 49(4): p. 349-60.
[6] R. Meikle et al. Small animal SPECT and its
place in the matrix of molecular Imging
Technologies, Phys. Med. Biol. 50 (2005) R45R61)
[7] F. Garibaldi et al. Small animal imaging by
single photon emission using pinhole and coded
apertures collimation, IEEE TNS 52(3)2005,. 573579
[8] F. Garibaldi et al. NIM A 569, 2006, 286 – 290
[9] E. Cisbani et al. NIM A 571, 2007, 169-172
[10] F. Cusanno et al. NIM A 569, 2006, 193-196, and
references quoted therein
[11] F. Garibaldi et al. Nucl. Instr. Meth A 471, 2001,
222-228
[12] M.L. Magliozzi et al. Proceedings of 9th ICATPP
Conference, Como 2005)
[13]Y Wang and BMW Tsui, IEEE Trans
Med Imag clinical trial. Lancet, 2004. 364(9429):
p. 141-86(3), 298 (2007).
[14] A. Costantinesco et al. Assessment of
ventricular Perfusion, Volumes and Motion in
Mice Using Pinhole Gated SPECT, JNM
2005; 46:1005-1011
[15] Seo et al. JNM, Vol. 45, N. 9, September
2004
[16] A.G. Argentieri et al., "A Novel Modular
and Flexible Readou Electronics for Photon
imaging Applications", in proceedings of the
IEEE NSS-MIC08 conference, 2008