New MRI, 18F-DOPA and 11C-(+)-alpha-dihydrotetrabenazine templates for Macaca fascicularis neuroimaging: advantages to improve PET quantification.
ABSTRACT Normalization of neuroimaging studies to a stereotaxic space allows the utilization of standard volumes of interest (VOIs) and voxel-based analysis (SPM). Such spatial normalization of PET and MRI studies requires a high quality template image. The aim of this study was to create new MRI and PET templates of (18)F-DOPA and (11)C-(+)-alpha-dihydrotetrabenazine ((11)C-DTBZ) of the Macaca fascicularis brain, an important animal model of Parkinson's disease. MRI template was constructed as a smoothed average of the scans of 15 healthy animals, previously transformed into the space of one representative MRI. In order to create the PET templates, (18)F-DOPA and (11)C-DTBZ PET of the same subjects were acquired in a dedicated small animal PET scanner and transformed to the created MRI template space. To validate these templates for PET quantification, parametric values obtained with a standard VOI-map applied after spatial normalization to each template were statistically compared to results computed using individual VOIs drawn for each animal. The high correlation between both procedures validated the utilization of all the templates, improving the reproducibility of PET analysis. To prove the utility of the templates for voxel-based quantification, dopamine striatal depletion in a representative monkey treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was assessed by SPM analysis of (11)C-DTBZ PET. A symmetric reduction in striatal (11)C-DTBZ uptake was detected in accordance with the induced lesion. In conclusion, templates of M. fascicularis brain have been constructed and validated for reproducible and automated PET quantification. All templates are electronically available via the internet.
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ABSTRACT: Much controversy exists concerning the effect of levodopa on striatal dopaminergic markers in Parkinson´s disease (PD) and its influence on functional neuroimaging. To deal with this issue we studied the impact of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and chronic levodopa administration on striatal (18)F-DOPA uptake (Ki) in an animal model of PD. The levels of several striatal dopaminergic markers and the number of surviving dopaminergic neurons in the substantia nigra (SN) were also assessed. Eleven Macaca fascicularis were included in the study. Eight animals received weekly intravenous injections of MPTP for 7weeks and 3 intact animals served as controls. MPTP-monkeys were divided in two groups. Group I was treated with placebo while Group II received levodopa. Both treatments were maintained for 11months and then followed by a washout period of 6months. (18)F-DOPA positron emission tomography (PET) scans were performed at baseline, after MPTP intoxication, following 11months of treatment, and after a washout period of 1, 3 and 6months. Monkeys were sacrificed 6months after concluding either placebo or levodopa treatment and immediately after the last (18)F-DOPA PET study. Striatal dopamine transporter (DAT), tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) content were assessed. In Group II (18)F-DOPA PET studies performed at 3 and 6months after interrupting levodopa showed a significantly increased Ki in anterior putamen as compared to Group I. Levodopa and placebo treated animals exhibited a similar number of surviving dopaminergic cells in the SN. Striatal DAT content was equally reduced in both groups of animals. Animals in Group I exhibited a significant decrease in TH protein content in all the striatal regions assessed. However, in Group II, TH levels were significantly reduced only in the anterior and posterior putamen. Surprisingly, in the levodopa-treated animals the TH levels in the posterior putamen were significantly lower than in the placebo group. AADC levels in MPTP groups were similar to those of control animals in all striatal areas analyzed. This study shows that chronic levodopa administration to monkeys with partial nigrostriatal degeneration followed by a washout period induces modifications in the functional activity of the dopaminergic nigrostriatal pathway.Neurobiology of Disease 09/2013; · 5.62 Impact Factor
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ABSTRACT: MRI templates and digital atlases are needed for automated and reproducible quantitative analysis of non-human primate PET studies. Segmenting brain images via multiple atlases outperforms single-atlas labelling in humans. We present a set of atlases manually delineated on brain MRI scans of the monkey Macaca fascicularis. We use this multi-atlas dataset to evaluate two automated methods in terms of accuracy, robustness and reliability in segmenting brain structures on MRI and extracting regional PET measures. METHODS: Twelve individual Macaca fascicularis high-resolution 3DT1 MR images were acquired. Four individual atlases were created by manually drawing 42 anatomical structures, including cortical and sub-cortical structures, white matter regions, and ventricles. To create the MRI template, we first chose one MRI to define a reference space, and then performed a two-step iterative procedure: affine registration of individual MRIs to the reference MRI, followed by averaging of the twelve resampled MRIs. Automated segmentation in native space was obtained in two ways: 1) Maximum probability atlases were created by decision fusion of two to four individual atlases in the reference space, and transformation back into the individual native space (MAXPROB)(.) 2) One to four individual atlases were registered directly to the individual native space, and combined by decision fusion (PROPAG). Accuracy was evaluated by computing the Dice similarity index and the volume difference. The robustness and reproducibility of PET regional measurements obtained via automated segmentation was evaluated on four co-registered MRI/PET datasets, which included test-retest data.Results Dice indices were always over 0.7 and reached maximal values of 0.9 for PROPAG with all four individual atlases. There was no significant mean volume bias. The standard deviation of the bias decreased significantly when increasing the number of individual atlases. MAXPROB performed better when increasing the number of atlases used. When all four atlases were used for the MAXPROB creation, the accuracy of morphometric segmentation approached that of the PROPAG method. PET measures extracted either via automatic methods or via the manually defined regions were strongly correlated, with no significant regional differences between methods. Intra-class correlation coefficients for test-retest data were over 0.87. CONCLUSIONS: Compared to single atlas extractions, multi-atlas methods improve the accuracy of region definition. They also perform comparably to manually defined regions for PET quantification. Multiple atlases of Macaca fascicularis brains are now available and allow reproducible and simplified analyses.NeuroImage 03/2013; · 6.25 Impact Factor
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ABSTRACT: Parkinson's disease (PD) is diagnosed when striatal dopamine (DA) loss exceeds a certain threshold and the cardinal motor features become apparent. The presymptomatic compensatory mechanisms underlying the lack of motor manifestations despite progressive striatal depletion are not well understood. Most animal models of PD involve the induction of a severe dopaminergic deficit in an acute manner, which departs from the typical, chronic evolution of PD in humans. We have used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered to monkeys via a slow intoxication protocol to produce a more gradual development of nigral lesion. Twelve control and 38 MPTP-intoxicated monkeys were divided into four groups. The latter included monkeys who were always asymptomatic, monkeys who recovered after showing mild parkinsonian signs, and monkeys with stable, moderate and severe parkinsonism. We found a close correlation between cell loss in the substantia nigra pars compacta (SNc) and striatal dopaminergic depletion and the four motor states. There was an overall negative correlation between the degree of parkinsonism (Kurlan scale) and in vivo PET ((18)F-DOPA K(i) and (11)C-DTBZ binding potential), as well as with TH-immunoreactive cell counts in SNc, striatal dopaminergic markers (TH, DAT and VMAT2) and striatal DA concentration. This intoxication protocol permits to establish a critical threshold of SNc cell loss and dopaminergic innervation distinguishing between the asymptomatic and symptomatic parkinsonian stages. Compensatory changes in nigrostriatal dopaminergic activity occurred in the recovered and parkinsonian monkeys when DA depletion was at least 88% of control, and accordingly may be considered too late to explain compensatory mechanisms in the early asymptomatic period. Our findings suggest the need for further exploration of the role of non-striatal mechanisms in PD prior to the development of motor features.Neurobiology of Disease 06/2012; 48(1):79-91. · 5.62 Impact Factor
New MRI,18F-DOPA and11C-(+)-α-dihydrotetrabenazine templates for Macaca
fascicularis neuroimaging: Advantages to improve PET quantification
M. Collantesa,1, E. Prietob,1, I. Peñuelasa,b,⁎, J. Blesac,d, C. Juric, J.M. Martí-Climentb, G. Quincocesb, J. Arbizub,
M. Riverole, J.L. Zubietaf, M.C. Rodriguez-Orozc,d, M.R. Luquine, J.A. Richterb, J.A. Obesoc,d
aSmall Animal Imaging Research Unit, Center for Applied Medical Research (CIMA) and Clínica Universidad de Navarra, Pamplona, Spain
bDepartment of Nuclear Medicine, Clínica Universidad de Navarra, Pamplona, Spain
cBasal Ganglia and Movement Disorders Group, Neurosciences Division, CIMA, and Department of Neurology and Neurosurgery, Clínica Universidad de Navarra, Pamplona, Spain
dCentro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Pamplona, Spain
eLaboratory of Regenerative Therapy, Center for Applied Medical Research (CIMA) and Clínica Universidad de Navarra, Pamplona, Spain
fDepartment of Radiology, Clínica Universidad de Navarra, Pamplona, Spain
a b s t r a c ta r t i c l ei n f o
Received 11 December 2008
Revised 18 March 2009
Accepted 23 April 2009
Available online 5 May 2009
Normalization of neuroimaging studies to a stereotaxic space allows the utilization of standard volumes of
interest (VOIs) and voxel-based analysis (SPM). Such spatial normalization of PET and MRI studies requires a
high quality template image. The aim of this study was to create new MRI and PET templates of18F-DOPA
model of Parkinson's disease. MRI template was constructed as a smoothed average of the scans of 15
healthy animals, previously transformed into the space of one representative MRI. In order to create the PET
templates,18F-DOPA and11C-DTBZ PET of the same subjects were acquired in a dedicated small animal PET
scanner and transformed to the created MRI template space. To validate these templates for PET
quantification, parametric values obtained with a standard VOI-map applied after spatial normalization to
each template were statistically compared to results computed using individual VOIs drawn for each animal.
The high correlation between both procedures validated the utilization of all the templates, improving the
reproducibility of PET analysis. To prove the utility of the templates for voxel-based quantification, dopamine
striatal depletion in a representative monkey treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) was assessed by SPM analysis of11C-DTBZ PET. A symmetric reduction in striatal11C-DTBZ uptake
was detected in accordance with the induced lesion. In conclusion, templates of M. fascicularis brain have
been constructed and validated for reproducible and automated PET quantification. All templates are
electronically available via the internet.
11C-(+)-α-dihydrotetrabenazine (11C-DTBZ) of the Macaca fascicularis brain, an important animal
© 2009 Elsevier Inc. All rights reserved.
Positron emission tomography (PET) using appropriate radioli-
gands allows imaging of certain components of neurotransmission
such as presynaptic transporters and postsynaptic receptors in living
brains (Ichise et al., 2001). Accurate quantification of the availabilityof
those transporters or receptors can be obtained using complex
mathematical models, which are usually solved with compartmental
approaches. The solution of this mathematical problem can be
undertaken by kinetic, equilibrium or graphical methods such as
Logan (2000), Ichise et al. (2001) or Patlak Plot (Patlak and Blasberg,
1985). Thesemethods initiallyrequire themeasurementof radiotracer
concentration in arterial plasma, an invasive procedure that intro-
duces additional methodological challenges. In practice, assuming the
existence of a reference region without specific uptake of the
radiotracer, measurement of plasma concentration is replaced by the
definition of a volume of interest (VOI) in that particular reference
area and the assessmentof its time-activitycurve (TAC) (Lammertsma
and Hume 1996). Using TAC and dynamic PET study, a parametric
image can be generated which represents a specific biological
parameter for each voxel. This in turn can be measured by drawing
new VOIs in the anatomical areas of interest. Then, in order to obtain
the final parametric value, VOIs have to be defined both in the
reference area and in the areas of interest. The limited spatial
resolution of PET tomographs and the presence of low uptake areas
make it quitedifficult to delimit VOIs accurately fora givenanatomical
brain structure. Consequently, the definition of VOIs is probablyone of
the most critical steps in the quantification procedure and introduces
great intra- and inter-operator variability. This process can be
NeuroImage 47 (2009) 533–539
⁎ Corresponding author. Department of Nuclear Medicine, Clínica Universidad de
Navarra, Avenida de Pío XII, 36, 31008 Pamplona, Spain. Tel.: +34948255400; fax:
E-mail address: email@example.com (I. Peñuelas).
1These authors contributed equally tothis study and should be considered equal first
1053-8119/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ynimg
standardized with the definition of a common stereotaxic coordinate
system, where a template of VOIs can be defined a priori. When
images are spatially normalized and hence forced to match such a
standardized space, those VOIs can be directly applied reducing
observer bias in VOI placement. Moreover, the normalization of
functional images to a common stereotaxic coordinate system allows
to investigate differences across subjects on a voxel basis using
statistical parametric mapping (SPM) (Friston et al., 1995b), a widely
extended method for PET analysis.
In order to perform the spatial normalization for VOI-map or SPM
analysis, a 3D template is required, that is, a smoothed image created
as the average of several subjects to which any other image can be
aligned (Evans et al., 1993). Templates can be created for different
image modalities such as magnetic resonance image (MRI) or PET. It
must be remarked that the choice of the template and the subsequent
normalization strategies may modify the quantification of PET studies
(Gispert et al., 2003). The use of a common stereotaxic coordinate
system to normalize PET studies is widely extended in human PET
analysis (Friston et al.,1995a), but not in the case of primates. In fact,
templates required for spatial normalization are rarely found for these
animals, although MRI templates have already been developed for
some species such as baboons (Black et al., 2001b; Greer et al., 2002)
and macaques (Black et al., 2005; Black et al., 2001a; Marenco et al.,
2004). PET templates are less usual and only a H2
been reported for baboon and Macaca nemestrina (Black et al., 2001a;
Black et al., 2001b). However, PET imaging in primate animal models
is an important and emerging tool to study the pathogenesis and
progression of neurological diseases (Guilarte et al., 2006; Kito et al.,
2001; Strome and Doudet 2007; Venneti et al., 2004) and to test the
efficacy of new radiotracers (Halldin et al., 2003; Halldin et al., 2005;
Schou et al., 2007; Stone-Elander et al., 1997) or therapeutic agents
(Doudet et al., 2004; Melega et al., 2000). These non-human primate
models are especially valuable due to these species' similarities to
Our own research focuses on the cynomolgus monkey (Macaca
fascicularis). This animal is widely used in PET imaging because the
size of the brain is appropriate for dedicated animal PET scanners
(Martí-Climent et al., 2006; Nagai et al., 2007) and the small size of
the animal facilitates its handling and housing. PET images were
performed in these animals for the evaluation of the striatal
dopaminergic system with two different radiotracers: 6-[18F]-fluoro-
L-DOPA (18F-DOPA) and11C-(+)-α-dihydrotetrabenazine (11C-DTBZ).
The aim of this study was to create new MRI template and PET
templates for18F-DOPA and11C-DTBZ of the M. fascicularis brain and
to demonstrate their usefulness to spatially normalize and quantify
PET studies in a fully automated way with a standardized VOI-map,
and to perform voxel-by-voxel statistical analysis.
15O PET template has
Materials and methods
Twenty-four healthy cynomolgus monkeys (M. fascicularis) were
studied (twenty males and four females, 3 to 5 years, weight=
3.6±0.9 kg). Fifteen of those animals (eleven males and four
females, 3 to 5 years, weight=3.4±0.7 kg,) were used to create the
MRI,18F-DOPA and11C-DTBZ templates. PET studies included in the
template generation and18F-DOPA scans of the 9 remaining animals
were used for template evaluation. One additional monkey (male,
5 years, 3.5 kg) treated with the dopaminergic neurotoxin 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied. MPTP
(0.5 mg/kg) was intravenously administered every two weeks to
reach a cumulative dose of 7.20 mg.
On the day of each study, anesthesia was initially induced by
intramuscular injections of ketamine (10 mg/kg) and imidazolam
(1 mg/kg) to allow the animal manage. During the scans anesthesia
was maintained with a mixture of ketamine (5 mg/kg) and
imidazolam (0.5 mg/kg). To block the peripheral decarboxylation of
18F-DOPA, 50 mg of carbidopa was given orally one hour prior to18F-
DOPA PET scans. All procedures were performed according to the
European Council Directive 86/609/EEC as well as in agreement with
the Society for Neuroscience Policy on the Use of Animals in
Neuroscience Research. The experimental design was approved by
the Ethical Committee for Animal Testing of the University of Navarra
MRI image acquisition
Magnetic resonance images were performed in all animals on a
1.5 T Siemens Symphony scanner (Erlangen, Germany). T1 weighted
axial images were acquired using a MPRAGE sequence with the
following acquisition parameters: TE=5.03, TR=2140, flip
angle=15°, slice thickness=1 mm, image matrix=192×192×88
and pixel size=1×1 mm2.
PET acquisition and analysis
Twenty-four healthyanimals underwent18F-DOPA PETstudies and
a subgroup of 15 subjects also had11C-DTBZ PET scans. PET imaging
was performed in a dedicated small animal Philips Mosaic tomograph
(Cleveland, Ohio, USA), with 2 mm resolution, 11.9 cm axial field of
view (FOV) and 12.8 cm transaxial FOV. Under standard anesthesia
animals were placed on the bed in prone position with the head
centered in the FOV. A transmission study prior to the emission scan
was carried out with an external
radiotracer (79±11.9 MBq for11C-DTBZ and 82.7±17.1 MBq for18F-
DOPA) was intravenously injected trough the saphenous vein
simultaneously to the beginning of a list mode study of 40 min for
11C-DTBZ and 100 min for
sinogram of the whole emission study was created and reconstructed
for visual inspection. Dynamic sinograms were also created with 16
frames for11C-DTBZ(7×30″; 4×120″; 5×300″) and23 framesfor18F-
DOPA (10×90″; 9×300″; 4×600″). From these sinograms, dynamic
images were generated containing the information about the
corresponding time intervals. All the images, both summed and
dynamic, were reconstructed in a 128×128 matrix with a
1×1×1 mm3voxel size using the 3D Ramla algorithm (Surti et al.,
2005) with 2 iterations and a relaxation parameter of 0.024. Dead
time, decay, attenuation, random and scattering corrections were
In order to obtainparametric images, PETstudies were analyzed by
suitable tracer kinetic models using PMOD software (PMOD Technol-
ogies Ltd., Adliswil, Switzerland). The computed parameters were the
uptakerate (Ki)for18F-DOPA andthe bindingpotential (BP)of VMAT2
kinetic model requires the PET dynamic data and a TAC obtained by
drawing VOIs over the reference areas. The
calculated using the Patlak graphical analysis, considering the
occipital cortex as the reference area (Whone et al., 2004). The Ichise
Multilinear Reference Tissue Model (Ichise et al., 1996) was used for
11C-DTBZ quantification, using the striatum as transporter-rich region
and occipital cortex as transporter-poor region.
137Cs source (370 MBq). The
18F-DOPA. For each study, a summed
11C-DTBZ. For parametric images generation the
18F-FDOPA Ki was
MRI template construction
Construction of a MRI template has been previously reported
(Black et al., 2001b; Greer et al., 2002) and is described in detail next.
All MRI were skull-stripped using the Brain Surface Extractor (BSE)
within BrainSuite 2 (University of South Carolina, Columbia, South
Carolina, USA) (Shattuck et al., 2001). One of the individual MRI scan
was selected as the representative brain, in terms of the size of the
animal (4.3 kg), the brain size and general shape. This MRI was
M. Collantes et al. / NeuroImage 47 (2009) 533–539
reoriented parallel to the orbitomeatal axis and, in order to remove
unnecessary background data, matrix size was reduced to
88×88×60, maintaining the voxel size (ANALYZE software, Mayo
Clinic, Rochester, Minnesota, USA). Each individual MRI was then
registered to the reference scan using Automated Image Registration
software (AIR 5.0, University of California, Los Angeles, California,
USA) (Woods et al., 1998). For this alignment, the intra-modality
registration tool was used with the standard deviation of ratio
image as cost function and the 3D affine transformation as spatial
model (Greer et al., 2002). The registered images were averaged
voxel-wise and spatially smoothed using a Gaussian filter
(FWHM=2 mm) to get a preliminary template (SPM, Wellcome
Department of Cognitive Neurology, Institute of Neurology, London,
UK). Then each original MRI was automatically normalized to this
preliminary template, to obtain transformed images that were much
more similar to each other. These ones were again averaged and
smoothed in order to achieve a higher quality template. To obtain
the final template this process was repeated one more time (third
iteration) without visually appreciable changes.
18F-DOPA and11C-DTBZ template construction
For each PET study, a summed PET image across frames was
generated and registered with the corresponding MRI (with skull and
scalp) using an automated algorithm based on mutual information
(PMOD fusion tool).
Normalization of each individual PET image to the standard
stereotaxic space was performed, calculating the geometric transfor-
mation for MRI normalization and applying that transformation over
the registered PET. Normalized PET images were averaged and
smoothed (FWHM=2 mm) to obtain the
the11C-DTBZ template (SPM2) in the common stereotaxic space.
18F-DOPA template and
To create a standard VOI-map, anatomical structures were
identified over the reference MRI with the help of a M. fascicularis
brain atlas (Martinet al.,2000). VOIs werehand drawnin thestriatum
(VOI size 400 mm3) and occipital lobe (VOI size 310 mm3) on axial
MRI slices based on anatomical borders (striatum) or position
(occipital region). These VOIs were drawn by common consent
between two skilled operators.
Templates were evaluated by performing a comparison analysis of
regional values of BP (n=15) and Ki (n=24) measured with11C-
performed using the manual method used regularly in our center and
the automated methods using different templates for spatial normal-
ization. The different quantification methods are described in detail
18F-DOPA PET respectively. This PET quantification was
1. Manual method without spatial normalization: Summed PET
study was registered automatically to its original MRI (PMOD
fusion tool) and the transformation was applied over the
dynamic study. Individual VOIs were drawn manually for each
subject over its PET summed image, with the anatomical
reference of the MRI.
2. Automated method with anatomical normalization to the MRI
template: Summed PET study was registered automatically to
its MRI (before skull-stripping) using PMOD fusion tool and the
transformation parameters were applied over the dynamic
study. Individual PET study was spatially normalized using the
skull-stripped MRI for the estimation of the geometric
Fig.1. Representative axial, sagittal and coronal slices of MRI template (A),18F-DOPA PET template (B) and11C-DTBZ PET template (C) of the Macaca fascicularis brain obtained in the
common stereotaxic coordinate space. A volume of interest, drawn as an iso-contour over MRI, has been applied over three images to show the correspondence between the three
M. Collantes et al. / NeuroImage 47 (2009) 533–539
transformation (SPM2). The normalized VOI-map previously
defined in the standard stereotaxic coordinate system was
directly applied, with slight repositioning in some cases.
3. Automated method with functional normalization to the PET
template: Spatial normalization of each PET image to the PET
template was performed in two steps: an initial rigid alignment
to the PET template (PMOD fusion tool) and the SPM2 non-rigid
normalization. The normalized VOI-map was directly applied,
and in only a few cases VOIs were slightly repositioned over the
In all methods, parametric images were generated (PMOD) using
the suitable reference regions and the biological parameter was
calculated in the striatal regions. As a final value, the data of both
striata were averaged. Therefore, for each PET study three different
parametric values were obtained and statistically compared (SPSS Inc,
Chicago, USA). First, parametric values obtained foreach method were
checked for normality using the Kolmogorov–Smirnov test. Further,
statistical differences between methods were evaluated by means of
the paired Student's t-test in the case of normally distributed
variables, and the Wilcoxon rank test for paired samples with no
normal distribution. A p-value less than 0.05 was considered to
indicate statistically significant differences between samples. Correla-
tion between samples was also assessed by the Pearson's correlation
Lastly, a SPM analysis was performed using a11C-DTBZ PET image
of a monkey with severe dopaminergic depletion after MPTP systemic
treatment. A two-sample t-test was performed in order to assess
regional differences between this animal and the11C-DTBZ control
group used for the creation of the templates. For this purpose, the
parametric PET image of MPTP-treated monkey was normalized using
both MRI and11C-DTBZ templates. All PET images were smoothed
using a 6 mm Gaussian kernel.
Using the methodology described above, we created a high
resolution MRI template of the M. fascicularis brain (Fig. 1). The MRI
template obtained allows spatial normalization to a common space of
individual MRI studies, obtaining an excellent spatial fit between
images with automatic procedures. PET templates of summed images
werealso constructedin the samestereotaxic spacewithtwodifferent
radiotracers (11C-DTBZ and18F-DOPA). Representative sections of the
templates are shown in Fig.1. The11C-DTBZ and18F-FDOPA templates
were excellent in quality based on visual inspection. Although18F-
FDOPA template presents some extracerebral uptake, this pattern is
representative of all the summed PET images that were averaged.
These templates are also available on the internet and can be
Template evaluation: normalization
All PET studies were spatially normalized by two different
automated methods: anatomical normalization using the MRI
template after PET to MRI registration, and functional normalization
using the corresponding PET template. Most normalization operations
worked correctly in one iteration, showing good spatial alignment to
the common stereotaxic space. Only in 2 out of 39 images was the
normalization carried out twice because the first alignment was
considered inaccurate. As an example, MRI and summed18F-DOPA
spatially normalized to the MRI template are shown in Fig. 2. The
MRI template is also presented in order to demonstrate the correct
spatial alignment between them. The VOI-map for striatum and
occipital cortex, pre-defined over the standard coordinates system,
was directly applied over the MRI or PET images of this particular
monkey. A precise matching between the VOI-map and the
anatomical structures of interest can be noticed. A similar, accurate
normalization was also carried out using PET templates (data not
shown). In some cases, using both modality templates, minor VOIs
repositioning was needed.
11C-DTBZ PET images acquired for the same monkey and
Template evaluation: comparison of quantification values
PETstudies permitted calculation of the Ki for18F-DOPA and BP for
11C-DTBZ in the striatum for each subject. The three different
Fig. 2. MRI template image showing the standardized VOI-map on the striatum and
occipital regions (A). The same VOIs were applied over MRI (B) and summed18F-DOPA
(C) and11C-DTBZ PET (D) images from the same monkey spatially normalized to the
MRI template. For graphical purposes, skull from18F-DOPA image was removed in this
figure, using the brain mask obtained from the registered MRI of the animal.
Results (mean±SD) of PET quantification using the three methodologies.
Manual MRI templatePET template
18F-DOPA Ki n=24
11C-DTBZ BP n=15
M. Collantes et al. / NeuroImage 47 (2009) 533–539
quantification approaches described previously were used for each
PET study. Mean and standard deviation of data obtained for each
procedure are shown in Table 1.
Data obtained using the three different methods (manual, MRI
template and PETtemplate)wereanalyzed statistically toexaminethe
correspondence between them. The Ki values for18F-DOPA obtained
with the manual method were not normally distributed (pb0.05 in
the Kolmogorov–Smirnov test), whereas for the other two methodol-
ogies data followed a normal distribution. For18F-DOPA, significant
differences were found using the Wilcoxon non-parametric test in
comparisons between the automated and the manual methods
(pb0.05), while there were not statistical differences between MRI
and PET templates methods. Data obtained with different methods
were highlycorrelated (Fig. 3), with a Pearson's coefficient higher that
0.87. On the other hand, the BP values obtained for11C-DTBZ with any
of the considered methods were normally distributed across subjects,
allowing the use of a parametric t-test. Comparisons yielded non-
significant differences between all methods. The correlation between
data was also evaluated, showing a very high Pearson's coefficient,
greater than 0.95 (Fig. 3).
As an illustration of statistical voxel-based analysis, a SPM
comparison of the MPTP-treated monkey vs. the control group was
performed using the MRI template or11C-DTBZ template for normal-
ization (Fig. 4). A symmetric reduction in
11C-DTBZ uptake in the
Fig. 3. Correlation analysis of Ki and BP values for18F-DOPA and11C-DTBZ studies comparing the three different methods: manual vs. automated using MRI template, manual vs.
automated using PET template and automated method using MRI template or PET template. r: Pearson's correlation coefficient.
Fig. 4. Effect of MPTP-induced dopaminergic depletion. Statistical parametric maps showing significant reduction of11C-DTBZ uptake in the striatum rendered over the normalized
MRI template. Results of the SPM analysis were obtained using MRI template (A) or11C-DTBZ template (B) for images normalization.
M. Collantes et al. / NeuroImage 47 (2009) 533–539
striatum was detected in both analyses, but the significant cluster
observed using the11C-DTBZ template had a greater extension. The
11C-DTBZ uptake in the MPTP PET study was also assessed using VOI-
map analysis, showing a clear dopaminergic depletion (BP=0.28).
The SPM results were thus in agreement with the VOIs analysis.
The objective of this study was to create M. fascicularis brain
templates in order to quantify PET studies in an automated and
reproducible manner. Particularly, to study the dopaminergic system
we utilize two PET radiotracers:18F-DOPA and11C-DTBZ.18F-DOPA
estimates the synthesis and storage capacity of dopamine and is
classically used in clinical studies of Parkinson's disease (Morrish et
al., 1996; Volkow et al., 1996).11C-DTBZ is a marker for monoamine
transporter VMAT2, located in the presynaptic vesicles of the
dopaminergic neurons (Koeppe et al.,1996) and is also used to assess
presynaptic dopaminergic terminals (Collantes et al., 2008; Kumar et
al., 2003; Lee et al., 2000). Both radiotracers are very useful tools for
evaluating the striatal dopamine deficit caused by MPTP in monkeys,
which is one of the best animal models of Parkinson's disease
MRI templates for other primate species such as baboons and M.
nemestrina have been previously reported and they are digitally
available (Black et al., 2001a; Black et al., 2001b; Greer et al., 2002).
Black developed a MRI template for the M. fascicularis (Black et al.,
2005) but with some limitations. Firstly, the template has to be
created from a representative group of the population under study
and in Black's abstract the general characteristics of the animals are
not well described. Furthermore, a MRI template without extracer-
ebral structures should be used for an accurate registration of
subsequent MRI. Black's template was created as average of T1-
weighted MRI including skull and scalp and a mask was later offered
in order to extract non-brain structures from the template. This mask
was drawn manually over the template, resulting in a loose mask
which does not eliminate completely the undesired anatomical
structures. In our template, the extraction was performed automati-
cally for each scan using BrainSuite 2 software, which provides a mask
that closely fits the brain surface. Consequently, non-brain structures
are completely removed in the final MRI template.
Regarding the procedure of creation of templates we have used
several programs widely extended and already validated. For inter-
subject registration without template we have used AIR, which has
been proven to be even better than SPM for this application (Zhilkin
and Alexander 2004). For intra-modality registration, we have used
the normalized mutual information method of PMOD, which is
already experimentally validated (Studholme et al., 1999). For non-
linear spatial normalization to the template SPM was used because it
is clearly the standard software to this aim (Friston et al., 1995a).
Although normalization based on MRI templates is better than for
any other template due to their high quality anatomical information
and high spatial resolution, the main limitation of this kind of
template is that its utilization requires the acquisition of a MRI for
each animal and the MRI-PET registration. The creation of PET
templates for specific radiotracers allows the normalization of
individual PET images directly to the PET template, avoiding the
MRI-PET registration. However, we were unable to find any published
PET template for M. fascicularis. This fact prompted us to develop our
own PET templates for the two radiotracers that are being used most
frequently in our animal studies:18F-DOPA and11C-DTBZ. Moreover,
images used for the creation of these PET templates wereobtained in a
dedicated small animal PET scanner with a 2 mm resolution, resulting
in high quality templates.
Our different templates allow spatial normalization by automatic
procedures of all PET studies to a common space, obtaining a very
good spatial fit between images (Fig.1). Once PET images are spatially
normalized, we are able to use a standard VOI-map (Fig. 2) permitting
totally automated quantification of PET studies and reducing the
processing time. This standardization reduces intra- and inter-
operator variability derived from the uncertainty in drawing VOIs
manually, not only because the position can be slightly different but
also because VOIs may have different shape and size. Although we are
working with healthy animals, it must be noted that the application of
the pre-defined VOI-map would be especially useful in injured
animals, due to the fact that regions of interest have a deficit in the
radiotracer uptake and the definition of VOIs is more uncertain.
Results obtained comparing manual and automated quantification
were strongly correlated. Although we are unable to check which
method yields more real quantitative values, we have validated the
utilization of any of the methodologies on an equal basis in healthy
animals. Moreover,11C-DTBZ analysis exhibited no statistical differ-
ences between methods, demonstrating the equivalence between
them. The statistical differences between methods observed for18F-
DOPA might be explained by the possible inaccurate registration of
these PET images with different extrastriatal uptake. As previously
noted,the complete inhibitionof the peripheral aromatic L-amino acid
decarboxylase enzyme with carbidopa is not attainable in primates
because of their high levels of this enzyme (Chan et al., 1995).
Another important advantage of the normalization is that the
definition of a standard coordinate system gives us the chance to
statistically compare images on a voxel basis (Fig. 4). As an example,
we have compared the dopaminergic activity in an MPTP-treated
monkey with the control group using SPM. As expected, a decrease
was detected in the striatum using both anatomical and functional
normalization with MRI and11C-DTBZ templates respectively. How-
ever, using MRI template, the decreased uptake areas were less
extended and more adjusted to the anatomical areas of interest. The
highest spatial specificity of SPM analysis using anatomical normal-
ization was already recognized (Gispert et al., 2003) and can be
justified by the fact that MRI spatial normalization is more accurate
than that performed using functional PET images, given the better
anatomical information and higher spatial resolution of MRI. In any
case, quantification of
using the VOI-map analysis also revealed a clear reduction in
dopaminergic activity in the same area, supporting the SPM results.
The main limitation of our study is that the three templates and
consequently all normalized images are not aligned to any atlas such
as that published by Martin et al. (2000). In fact, images are
oriented parallel to the orbitomeatal axis, for analogy with the usual
human orientation. The development of a digital atlas is therefore a
convenient next step.
In summary, templates of M. fascicularis brain have been
constructed for MRI and PET and have been used to spatially
normalize18F-DOPA and11C-DTBZ PET studies. This spatial normal-
ization allows the utilization of an automated and reproducible
procedure for PET quantification using a pre-defined VOI-map, and
has been validated by comparison with manual methods used
previously. Furthermore, the utilization of spatial normalization offers
the chance to conduct image analysis further by means of SPM. These
templates are available for the scientific community on the website of
11C-DTBZ uptake in MPTP-treated monkeys
This work was partially supported by the Plan Nacional de
Investigación (SAF2005-08416), Ministerio de Investigación y Ciencia
and by the agreement UTE-CIMA of the University of Navarra. CJ is
supported by the Programme ALBAN, the European Union Programme
of High Level Scholarships for Latin America, scholarship No.
M. Collantes et al. / NeuroImage 47 (2009) 533–539
We thank the excellent work of the Cyclotron Unit staff for
radiotracer production and Margarita Ecay, Izaskun Bilbao and Elena
Iglesias for the acquisition of the PET studies. Monkeys were housed
and cared at the CIFA Primate Unit.
Black, K.J., Koller, J.M., Perlmutter, J.S., 2005. Template Images for Neuroimaging in
Macaca fascicularis. Program No. 454.18. 2005 Abstract Viewer and Itinerary
Planner. Society for Neuroscience, Washington DC.
Black, K.J., Koller, J.M., Snyder, A.Z., Perlmutter, J.S., 2001a. Template images for
nonhuman primate neuroimaging: 2. Macaque. Neuroimage 14 (3), 744–748.
Black, K.J., Snyder, A.Z., Koller, J.M., Gado, M.H., Perlmutter, J.S., 2001b. Template
images for nonhuman primate neuroimaging: 1. Baboon. Neuroimage 14 (3),
Chan, G.L., Doudet, D.J., Dobko, T., Hewitt, K.A., Schofield, P., Pate, B.D., Ruth, T.J., 1995.
Routes of administration and effect of carbidopa pretreatment on 6-[18F]fluoro-L-
dopa/PET scans in non-human primates. Life Sci. 56 (21), 1759–1766.
Collantes, M., Penuelas, I., Alvarez-Erviti, L., Blesa, J., Marti-Climent, J.M., Quincoces, G.,
Delgado, M., Ecay, M., Martinez, A., Arbizu, J., et al., 2008. Use of 11C-(+)-alpha-
dihydrotetrabenazine for the assessment of dopaminergic innervation in animal
models of Parkinson's disease. Rev.Esp.Med.Nucl. 27 (2), 103–111.
Doudet, D.J., Cornfeldt, M.L., Honey, C.R., Schweikert, A.W., Allen, R.C., 2004. PET
imaging of implanted human retinal pigment epithelial cells in the MPTP-induced
primate model of Parkinson's disease. Exp.Neurol. 189 (2), 361–368.
Emborg, M.E., 2007. Nonhuman primate models of Parkinson's disease. ILAR J. 48 (4),
Evans, A., Collins, D., Mills, S., Brown, E., Kelly, R., Peters, T., 1993. 3D statistical
neuroanatomical models from 305 MRI volumes. Nuclear Science Symposium and
Medical Imaging Conference, IEEE Conference Record 1813–1817.
Friston, K.J., Ashburner, J., Frith, C.D., Poline, J.B., Heather, J.D., Frackowiak, R.S.J., 1995a.
Spatial registration and normalization of images. Hum. Brain Mapp. 2, 165–189.
Friston, K.J., Holmes, A., Worsley, K., Poline, J., Frith, C., Frackowiak, R.S.J., 1995b.
Statistical parametric maps in functional imaging: a general linear approach. Hum.
Brain Mapp. 2 (4), 189–210.
Gispert, J.D., Pascau, J., Reig, S., Martinez-Lazaro, R., Molina, V., Garcia-Barreno, P., Desco,
M., 2003. Influence of the normalization template on the outcome of statistical
parametric mapping of PET scans. Neuroimage 19 (3), 601–612.
Greer, P.J., Villemagne, V.L., Ruszkiewicz, J., Graves, A.K., Meltzer, C.C., Mathis, C.A., Price,
J.C., 2002. MR atlas of the baboon brain for functional neuroimaging. Brain Res. Bull.
58 (4), 429–438.
Guilarte, T.R., Chen, M.K., McGlothan, J.L., Verina, T., Wong, D.F., Zhou, Y., Alexander, M.,
Rohde, C.A., Syversen, T., Decamp, E., et al., 2006. Nigrostriatal dopamine system
dysfunction and subtle motor deficits in manganese-exposed non-humanprimates.
Exp. Neurol. 202 (2), 381–390.
Halldin, C., Lundberg, J., Sovago, J., Gulyas, B., Guilloteau, D., Vercouillie, J., Emond, P.,
Chalon, S., Tarkiainen, J., Hiltunen, J., et al., 2005. (11)C]MADAM, a new serotonin
transporter radioligand characterized in the monkey brain by PET. Synapse 58 (3),
Halldin, C., Erixon-Lindroth, N., Pauli, S., Chou, Y.H., Okubo, Y., Karlsson, P., Lundkvist, C.,
Olsson, H., Guilloteau, D., Emond, P., et al., 2003. (11)C]PE2I: a highly selective
radioligand for PET examination of the dopamine transporter in monkey and
human brain. Eur. J. Nucl. Med. Mol. Imaging 30 (9), 1220–1230.
Ichise, M., Ballinger, J.R., Golan, H., Vines, D., Luong, A., Tsai, S., Kung, H.F., 1996.
Noninvasive quantification of dopamine D2 receptors with iodine-123-IBF. SPECT
Ichise, M., Meyer, J.H., Yonekura, Y., 2001. An introduction to PET and SPECT
neuroreceptor quantification models. J. Nucl. Med. 42 (5), 755–763.
Kito, G., Nishimura, A., Susumu, T., Nagata, R., Kuge, Y., Yokota, C., Minematsu, K., 2001.
Experimental thromboembolic stroke in cynomolgus monkey. J. Neurosci. Methods
105 (1), 45–53.
Koeppe, R.A., Frey, K.A., Vander Borght, T.M., Karlamangla, A., Jewett, D.M., Lee, L.C.,
Kilbourn, M.R., Kuhl, D.E., 1996. Kinetic evaluation of [11C]dihydrotetrabenazine by
dynamic PET: measurement of vesicular monoamine transporter. J. Cereb. Blood
Flow Metab. 16 (6), 1288–1299.
Kumar, A., Mann, S., Sossi, V., Ruth, T.J., Stoessl, A.J., Schulzer, M., Lee, C.S., 2003. [11C]
DTBZ-PET correlates of levodopa responses in asymmetric Parkinson's disease.
Brain 126, 2648–2655.
Lammertsma, A.A., Hume, S.P.,1996. Simplified reference tissue model for PET receptor
studies. Neuroimage 4, 153–158.
Lee, C.S., Samii, A., Sossi, V., Ruth, T.J., Schulzer, M., Holden, J.E., Wudel, J., Pal, P.K., de la
Fuente-Fernandez, R., Calne, D.B., et al., 2000. In vivo positron emission
tomographic evidence for compensatory changes in presynaptic dopaminergic
nerve terminals in Parkinson's disease. Ann. Neurol. 47 (4), 493–503.
Logan, J., 2000. Graphical analysis of PET data applied to reversible and irreversible
tracers. Nucl. Med. Biol. 27 (7), 661–670.
Marenco, S., Carson, R.E., Berman, K.F., Herscovitch, P., Weinberger, D.R., 2004. Nicotine-
induced dopamine release in primates measured with [11C]raclopride PET.
Neuropsychopharmacology 29 (2), 259–268.
Martí-Climent, J.M., Collantes, M., Peñuelas, I., Álvarez-Erviti, L., Blesa, F.J., Ecay, M.,
Arbizu, J., Obeso, J.A., Richter, J.A., 2006.
fascicularis monkeys: methodological development for a mosaic microPET scanner.
Eur. J. Nucl. Med. Mol. Imaging 33 (Supplement 2), S118.
Martin, R.F., Bowden, D.M., Wu, J., Dubach, M.F., Robertson, J.E., 2000. Primate Brain
Maps: Structure of the Macaque Brain. Elsevier Science, Amsterdam.
Melega, W.P., Lacan, G., Desalles, A.A., Phelps, M.E., 2000. Long-term methampheta-
mine-induced decreases of [(11)C]WIN 35,428 binding in striatum are reduced by
GDNF: PET studies in the vervet monkey. Synapse 35 (4), 243–249.
Morrish, P.K., Sawle, G.V., Brooks, D.J., 1996. An [18F]DOPA-PET and clinical study of the
rate of progression in Parkinson's disease. Brain 119, 585–591.
Nagai, Y., Obayashi, S., Ando, K., Inaji, M., Maeda, J., Okauchi, T., Ito, H., Suhara, T., 2007.
Progressive changes of pre- and post-synaptic dopaminergic biomarkers in
conscious MPTP-treated cynomolgus monkeys measured by positron emission
tomography. Synapse 61 (10), 809–819.
Patlak, C.S., Blasberg, R.G., 1985. Graphical evaluation of blood-to-brain transfer
constants from multiple-time uptake data. Generalizations. J. Cereb. Blood Flow
Metab. 5 (4), 584–590.
Schou, M., Pike, V.W., Sovago, J., Gulyas, B., Gallagher, P.T., Dobson, D.R., Walter, M.W.,
Rudyk, H., Farde, L., Halldin, C., 2007. Synthesis of11C-labelled (R)-OHDMI and
CFMME and their evaluation as candidate radioligands for imaging central
norepinephrine transporters with PET. Bioorg. Med. Chem. 15 (2), 616–625.
Shattuck, D.W., Sandor-Leahy, S.R., Schaper, K.A., Rottenberg, D.A., Leahy, R.M., 2001.
Magnetic resonance image tissue classification using a partial volume model.
Neuroimage 13 (5), 856–876.
Stone-Elander, S., Thorell, J.O., Eriksson, L., Fredholm, B.B., Ingvar, M., 1997. In vivo
biodistribution of [N-11C-methyl]KF 17837 using 3-D-PET: evaluation as a ligand for
the study of adenosine A2A receptors. Nucl. Med. Biol. 24 (2), 187–191.
Strome, E.M., Doudet, D.J., 2007. Animal models of neurodegenerative disease: insights
from in vivo imaging studies. Mol. Imaging Biol. 9 (4), 186–195.
Studholme, C., Hill, D., Hawkes, D., 1999. An overlap invariant entropy measure of 3D
medical image alignment. Pattern Recognit. 32 (1), 71–86.
Surti, S., Karp, J.S., Perkins, A.E., Cardi, C.A., Daube-Witherspoon, M.E., Kuhn, A.,
Muehllehner, G., 2005. Imaging performance of A-PET: a small animal PET camera.
IEEE Trans. Med. Imaging 24 (7), 844–852.
Venneti, S., Lopresti, B.J., Wang, G., Bissel, S.J., Mathis, C.A., Meltzer, C.C., Boada, F.,
Capuano, S., Kress, G.J., Davis, D.K., et al., 2004. PET imaging of brain macrophages
using the peripheral benzodiazepine receptor in a macaque model of neuroAIDS.
J. Clin. Invest. 113 (7), 981–989.
Volkow, N.D., Fowler, J.S., Gatley, S.J., Logan, J., Wang, G.J., Ding, Y.S., Dewey, S., 1996. PET
Whone, A.L., Bailey, D.L., Remy, P., Pavese, N., Brooks, D.J., 2004. A technique for
standardized central analysis of 6-(18)F-fluoro-L-DOPA PET data from a multi-
center study. J. Nucl. Med. 45 (7), 1135–1145.
Woods, R.P., Grafton, S.T., Watson, J.D., Sicotte, N.L., Mazziotta, J.C., 1998. Automated
image registration: II. Intersubject validation of linear and nonlinear models.
J. Comput. Assist. Tomogr. 22 (1), 153–165.
Zhilkin, P., Alexander, M.E., 2004. Affine registration: a comparison of several programs.
Magn. Reson. Imaging 22 (1), 55–66.
18FDOPA striatal uptake in Macaca
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