Evaluation of the novel TSPO radiotracer [18F] VUIIS1008 in a preclinical model of cerebral ischemia in rats
Pulagam et al. EJNMMI Research
Evaluation of the novel TSPO radiotracer 18 [ F] VUIIS1008 in a preclinical model of cerebral ischemia in rats
Krishna R. Pulagam 2
Lorena Colás 0
Daniel Padro 1
Sandra Plaza-García 1
Vanessa Gómez-Vallejo 2
Makoto Higuchi 3
Jordi Llop 2
Abraham Martín 0
0 Experimental Molecular Imaging, Molecular Imaging Unit, CIC biomaGUNE , P° Miramon 182, San Sebastian , Spain
1 Magnetic Resonance Imaging, Molecular Imaging Unit, CIC biomaGUNE , P° Miramon 182, San Sebastian , Spain
2 Radiochemistry and Nuclear Imaging, Molecular Imaging Unit, CIC biomaGUNE , P° Miramon 182, San Sebastian , Spain
3 National Institutes for Quantum and Radiological Science and Technology, National Institute of Radiological Sciences , Chiba , Japan
Background: In vivo positron-emission tomography (PET) imaging of transporter protein (TSPO) expression is an attractive and indispensable tool for the diagnosis and therapy evaluation of neuroinflammation after cerebral ischemia. Despite several radiotracers have shown an excellent capacity to image neuroinflammation, novel radiotracers such as [18F] VUIIS1008 have shown promising properties to visualize and quantify the in vivo expression of TSPO. Methods: Longitudinal in vivo magnetic resonance (MRI) and PET imaging studies with the novel TSPO radiotracer 2-(5,7-diethyl-2-(4-(2-[18F] fluoroethoxy) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl)-N, N-diethylacetamide ([18F] VUIIS1008), and (N, N-diethyl-2-(2-[4-(2-fluoroethoxy)-phenyl]-5,7-dimethyl-pyrazolo [1,5-a] yrimidin-3-yl)-acetamide ([18F] DPA-714) were carried out before and at days 1, 3, 7, 14, 21, and 28 following the transient middle cerebral artery occlusion (MCAO) in rats. Results: MRI images showed the extension and evolution of the brain infarction after ischemic stroke in rats. PET imaging with [18F] VUIIS1008 and [18F] DPA714 showed a progressive increase in the ischemic brain hemisphere during the first week, peaking at day 7 and followed by a decline from days 14 to 28 after cerebral ischemia. [18F] DPA714 uptake showed a mild uptake increase compared to [18F] VUIIS1008 in TSPO-rich ischemic brain regions. In vivo [18F] VUIIS1008 binding displacement with VUIIS1008 was more efficient than DPA714. Finally, immunohistochemistry confirmed a high expression of TSPO in microglial cells at day 7 after the MCAO in rats. Conclusions: Altogether, these results suggest that [18F] VUIIS1008 could become a valuable tool for the diagnosis and treatment evaluation of neuroinflammation following ischemic stroke.
T2W-MRI; [18F] VUIIS1008; [18F] DPA-714; PET; Cerebral ischemia
Cerebral ischemia induces the death of ischemic neurons
and especially the release of necrotic debris that triggers
the inflammatory reaction resulting in a strong activation of
the resident glial cells and leucocyte infiltration [
]. As the
damage progresses, microglia undergo progressive changes,
including altered expression of cell surface markers and
inflammation-related genes, process retraction and the
acquisition of an ameboid morphology, cell body migration,
and increasing phagocytic ability [
]. The inflammatory
reaction promotes a dramatic increase of a mitochondrial
transmembrane protein, the translocator protein (TSPO),
considered as the hallmark of neuroinflammation in brain
]. Despite this, TSPO overexpression mainly
reflects microglia, infiltrated macrophages, and astrocytes
activation and proliferation rather than neuroinflammation
in its broader sense [
]. Nevertheless, over the last decade,
TSPO has become an attractive target for
positronemission tomography (PET) imaging of cerebral
inflammation due to its high expression after pathologic situations
despite the low expression showed in the healthy cerebral
]. TSPO expression in a wide diversity of
neurologic and neurodegenerative diseases has been
monitored by PET with a large variety of first generation
] or second and third generation
of radioligands such as [11C] SSR180575 [
], [11C] DAA1106 [
], [11C] CLINME
, [18F] FEAC [
], [18F] FEDAC [
], [18F] FEPPA
], [18F] DPA-714 [
3, 17, 18
], [18F] PBR111 ,
[18F] PBR06 [
], and [18F] GE-180 [
others, evidencing the interest aroused by TSPO imaging
from the bench to the bedside. In fact, the second and third
generations of TSPO radioligands have been synthesized to
improve the limitations presented by [11C] PK11195,
including the high level of non-specific binding and the
poor signal-to-noise ratio . In particular, [18F] DPA-714
and [18F] GE-180 have been directly compared with [11C]
PK11195 after cerebral ischemia in rats confirming the
better signal-to-noise ratio in the ischemic lesion and the
low level of non-specific binding in the contralateral brain
3, 22, 24
]. In this sense, a novel TSPO
radioligand, 2-(5,7-diethyl-2-(4-(2-[18F] fluoroethoxy) phenyl)
pyrazolo [1,5-a] pyrimidin-3-yl)-N, N-diethylacetamide
([18F] VUIIS1008), has shown a 36-fold enhancement in
binding activity (Ki = 0.3 nM) compared to its parent
compound, DPA-714 (Ki = 10.9 nM) [
]. The purpose
of the present study was to investigate the usefulness of the
novel radiotracer [18F] VUIIS1008 to monitor TSPO
expression following a preclinical model of cerebral ischemia.
For this reason, ischemic rats were subjected to PET
studies with [18F] VUIIS1008 and [18F] DPA-714 from 1 to
28 days after the experimental middle cerebral artery
(MCA) occlusion in rats. PET was conducted in parallel
with immunohistochemistry of TSPO. The results reported
here are particularly relevant for providing novel
information on the suitability of [18F] VUIIS1008 for TSPO
imaging after neurologic and neurodegenerative diseases.
Adult male Sprague-Dawley rats (296+/− 9, 11 g body
weight; Janvier, France) (n = 28) were used. Animal studies
were approved by the animal ethics committee of CIC
biomaGUNE and local authorities and were conducted in
accordance with the Directives of the European Union on
animal ethics and welfare. Transient focal ischemia was
produced by a 90-min intraluminal occlusion of the
middle cerebral artery (MCA) followed by reperfusion
as described elsewhere [
]. Briefly, rats were anesthetized
with 4% isoflurane in 100% O2, and a 2.6-cm length of 4-0
monofilament nylon suture was introduced into the right
external carotid artery up to the level where the MCA
branches out. Animals were then sutured and placed in
their cages with free access to water and food. After
90 min, the animals were re-anesthetized, and the filament
was removed to allow reperfusion. Sixteen rats were
repeatedly examined with PET before (day 0) and at 1, 3,
7, 14, 21, and 28 days after ischemia to evaluate TSPO
binding. Eleven animals were subjected to displacement
PET studies at day 7 after the MCA occlusion. The
animals studied at day 0 have been considered as the baseline
control group. One rat from the displacement study was
used to perform ex vivo immunohistochemistry for TSPO
receptor expression at day 7 after cerebral ischemia.
Magnetic resonance imaging
T2-weighted (T2W) MRI scans were performed before
(day 0) to measure the size of the infarction (n = 16) and
at 1, 3, 7, 14, 21, and 28 days after the middle cerebral
artery occlusion (MCAO) to co-register PET signal data
(n = 2, one for radiotracer). Before the scans, anesthesia
was induced with 4% isoflurane and maintained by 2–2.5%
of isoflurane in 100% O2 during the scan. Animals were
placed into a rat holder compatible with the MRI
acquisition systems and maintained normothermia using a
water-based heating blanket at 37 °C. MRI experiments
were performed on a 7 Tesla Bruker Biospec 70/30 MRI
system (Bruker Biospin GmbH, Ettlingen, Germany) and
interfaced to an AVANCE III console. The BGA12-S
imaging gradient (maximum gradient strength 400 mT/m
switchable within 80 μs), an 82-mm-inner-diameter
quadrature volume resonator for transmission and surface rat
brain coil for the reception were used. T2W images were
acquired with a rapid acquisition with relaxation
enhancement (RARE) sequence with the following parameters:
RARE factor 2, TR/TE = 4400/40 ms, FOV = 25 × 25 mm,
ACQ Matrix =256 × 256, slice thickness = 1 mm, 2
averages and 24 contiguous slices. Contiguous slices covering
all the infarcted volume were acquired and fat suppression
Magnetic resonance imaging image analysis
MRI (T2W) images at 1 day after ischemia were used to
calculate the lesion volume. Regions of interest (ROIs) were
manually defined using the Open Source software 3D Slicer
image analysis software (Version 3.6.3 www.slicer.org) for
each rat on the region of increased signal in the ipsilateral
hemisphere. The total lesion volume was calculated by
summing the area of the infarcted regions of all slices
affected by the lesion.
The synthesis of radiotracers N,
N-diethyl-2-(2-(4(2-fluoroethoxy) phenyl)-5,7-dimethylpyrazolo [1,5-a]
pyrimidin-3-yl) acetamide ([18F] DPA-714) and
2(5,7-diethyl-2-(4-(2-fluoroethoxy) phenyl) pyrazolo [1,5-a]
pyrimidin-3-yl)-N, N-diethylacetamide ([18F] VUIIS1008)
were prepared from their corresponding tosylate
precursors according to previously reported procedures using a
TRACERlab FXFN synthesis module (GE Healthcare) [
Briefly, once transferred into a dedicated (ventilated and
lead-shielded) hot cell, [18F] fluoride was first trapped on a
preconditioned Sep-Pak® Accell Plus QMA Light cartridge
(Waters, Milford, MA, USA) and subsequently eluted from
the cartridge with a solution of Kryptofix K2.2.2/K2CO3 in
a mixture of water and acetonitrile. After azeotropic drying
of the solvent, a solution containing the appropriate
tosylate precursor (4.0 mg) in dimethylsulfoxide (0.7 mL)
was added, and the mixture was heated at 165 °C for
5 min. The reactor was then cooled at room temperature;
the reaction crude was diluted with a mixture of
acetonitrile and water (2/1, 3 mL) and purified by HPLC using a
Nucleosil 100-7 C18 column (Macherey-Nagel, Düren,
Germany) as stationary phase and 0.1 M aqueous
ammonium formate solution (pH = 3.9)/acetonitrile (30/70) as the
mobile phase at a flow rate of 7 mL/min. The desired
fraction (10–11 min for [18F] DPA-714, 12–13 min for [18F]
VUIIS1008) was collected, diluted with water (20 mL), and
the radiotracer was retained on a C-18 cartridge (Sep-Pak®
Light, Waters, Milford, MA, USA) and further eluted with
ethanol (1 mL). The ethanol solution was finally
reconstituted with saline solution (9 mL). Filtration through
a 0.22 μm filter yielded the final solution, ready for
injection. Radiochemical yields (non-decay corrected)
were in the range of 8–13% for both [18F] DPA-714 and
[18F] VUIIS1008. Radiochemical purity was consistently
higher than 98% at the time of injection. The specific
activity for both tracers at injection time was in the range
of 175–800 GBq/μmol. Considering the amount of
radioactivity injected (ca. 70 MBq/animal), this results in a mass
amount injected in the range of 0.04–0.17 μg/animal.
Positron emission tomography scans and data acquisition
PET scans were repeatedly performed before (day 0) and at
1, 3, 7, 14, 21, and 28 days after reperfusion using a
General Electric eXplore Vista CT camera (GE Healthcare).
Scans were performed in rats anesthetized with 4%
isoflurane and maintained by 2–2.5% of isoflurane in 100% O2.
The tail vein was catheterized with a 24-gauge catheter for
intravenous administration of the radiotracer. Animals
were placed into a rat holder compatible with the PET
acquisition system and maintained normothermia using
a water-based heating blanket. Two groups of animals
were subjected to PET scans to assess TSPO expression
with [18F] VUIIS1008 and [18F] DPA-714 at each time
point before and after ischemia onset. The radiotracers
([18F] VUIIS1008 or [18F] DPA-714, ~ 70 MBq) were
injected concomitantly with the start of the PET
acquisition, and dynamic brain images were acquired (31 frames:
3 × 5, 3 × 10, 3 × 15, 3 × 30, 4 × 60, 4 × 120, 5 × 180, 6 ×
300 seconds) in the 400–700 keV energetic window, with
a total acquisition time of 60 min. For the displacement
studies, unlabeled compounds (VUIIS1008 and DPA-714,
1 mg/Kg) were injected 20 min after the injection of [18F]
VUIIS1008. After each PET scan, CT acquisitions were
also performed (140 μA intensity, 40 kV voltage),
providing anatomical information of each animal as well
as the attenuation map for the later image reconstruction.
Dynamic acquisitions were reconstructed (decay and
CT-based attenuation corrected) with filtered back
projection (FBP) using a ramp filter with a cutoff frequency
of 0.5 mm−1.
Positron emission tomography image analysis
PET images were analyzed using PMOD image analysis
software (PMOD Technologies Ltd., Zürich, Switzerland).
To verify the anatomical location of the signal, PET
images were co-registered to the anatomical data of a MRI
rat brain template. Two types of volumes of interest
(VOIs) were established as follows: (i) A first set of VOIs
was defined to study the whole brain PET signal over time.
Whole brain VOIs were manually drawn in both the
entire ipsilateral and contralateral hemispheres containing
the territory irrigated by the middle cerebral artery on
slices of a MRI (T2W) rat brain template from the PMOD
software. (ii) A second set of VOIs was automatically
generated in the cortex, striatum, hippocampus, thalamus,
and cerebellum by using the regions proposed by the
PMOD rat brain template. The last three time frames of
the time-activity curve in a steady state were used to
calculate the summed PET binding uptake during the last
15 min of acquisition for both radiotracers. PET signal
uptake was averaged in each VOI and expressed as a
percentage of injected dose per cubic centimeter (%ID/cm3).
Immunohistochemistry staining was performed at day 7
after reperfusion. The animal was terminally anesthetized
and sacrificed by decapitation. The brain was removed,
frozen, and cut into 5-μm-thick sections in a cryostat.
Sections were fixed in acetone (− 20 °C) during 2 min,
washed with phosphate-buffered saline (PBS) and saturated
with a solution of bovine serum albumine (BSA) 5%/Tween
0.5% in PBS during 15 min at room temperature, and
incubated during 1 h at room temperature with primary
antibodies in a solution of BSA (5%)/Tween (0.5%) in PBS.
The section was stained for CD11b with mouse anti-rat
CD11b (1:300; Serotec, Raleigh, NC, USA) and for TSPO
with a rabbit anti-rat TSPO (NP155, 1:1000). Sections were
washed (3 × 10 min) in PBS and incubated for 1 h at room
temperature with secondary antibodies Alexa Fluor 350
goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse
IgG (Molecular Probes, Life Technologies, Madrid, Spain,
1:1000) in BSA 5%/Tween 0.5% in PBS, washed again (3 ×
10 min) in PBS, and mounted with a prolong antifade kit
in slices (Molecular Probes Life Technologies, Madrid).
Standardized images acquisition was performed with an
Axio Observer Z1 (Zeiss, Le Pecq, France) equipped with a
For PET signal values, the statistical analysis was
performed as follows: The percentage of injected dose per
cubic centimeter (%ID/cm3) for each animal, the brain
hemisphere (ipsilateral and contralateral), and the region
(cortex, striatum, hippocampus, thalamus, and cerebellum)
were calculated at each time point after cerebral ischemia.
Values of %ID/cm3 within each region and time point
following cerebral ischemia were averaged and compared
with the averaged baseline control values at days 0 and 7
using a two-way ANOVA followed by Tukey’s multiple
comparison tests for post hoc analysis. The ratio of the
lesion to the contralateral brain hemispheres of
nondisplaced and displacement experiments was compared
using a one-way ANOVA followed by Tukey’s multiple
comparison tests for post hoc analysis. The level of
significance was regularly set at P < 0.05. Statistical analyses were
performed with GraphPad Prism version 6 software.
Coronal and horizontal T2W-MRI images showed the
extension and evolution of the cerebral infarction by means
of the brain edema at days 0 (control), 1, 3, 7, 14, 21, and
28 after ischemic stroke in rats (Figs. 1 and 3). MRI-T2W
images were used to co-register PET signal data and to
measure the infarct volume at 24 h after the ischemia
onset. All rats included in the PET binding time course
evaluation for both [18F] VUIIS1008 and [18F] DPA-714
presented similar infarct volume values that affected
the cortical and striatal regions ([18F] VUIIS1008
287.43 ± 84.32 mm3, n = 8; [18F] DPA-714 290.58 ±
94.47 mm3, n = 8) (Fig. 2a). [18F] VUIIS1008 and [18F]
DPA-714 PET signals showed higher uptake in hyperintense
lesions on T2W-MRI images evidencing the expression of
TSPO following cerebral ischemia.
[18F] VUIIS1008 and [18F] DPA-714 PET after cerebral
The coronal and horizontal brain images shown in Figs. 1
and 3, illustrate the evolution of the [18F] VUIIS1008
and [18F] DPA-714-PET signals in ischemic animals at
day 0 (control) and at 1, 3, 7, 14, 21, and 28 days after
reperfusion. Quantification of the images provided
information related to the time-course activity of [18F] VUIIS1008
and [18F] DPA-714 in both the ipsilateral and contralateral
brain whole hemispheres, cerebellum (Fig. 2, n = 8), and
particularly in the ipsilateral cortex, striatum, hippocampus,
and thalamus (Fig. 4, n = 8) at the different time points
following MCAO. In the ipsilateral whole brain, the PET
signal for [18F] VUIIS1008 and [18F] DPA-714 showed
similar low signal intensity before (day 0) and at day 1, followed
by a PET signal increase from days 3 to 14 after ischemia
(P < 0.001, Fig. 2b). In fact, the highest PET uptake signal
for both [18F] VUIIS1008 and [18F] DPA-714 was observed
at day 7 compared to days 3, 14, 21, and 28 after MCAO
(P < 0.01 and P < 0.001, Fig. 2b). Subsequently, the PET
signal showed a progressive decline from days 14 to 28.
Moreover, the [18F] DPA-714 uptake signal displayed a mild
increase from days 3 to 21 after ischemia compared to the
[18F] VUIIS1008 PET signal in the ipsilateral hemisphere. In
the contralateral whole brain, [18F] VUIIS1008 and [18F]
DPA-714 PET uptake at day 1 showed similar value
controls followed by an increase at days 3 and 7 after ischemia
(P < 0.05; P < 0.01, Fig. 2c). In addition, [18F] DPA-714
signal showed a higher increase at day 7 compared to days
21 and 28 (P < 0.05; P < 0.01, Fig. 2c). The cerebellum also
showed a PET signal increase at day 7 for both radiotracers
compared to different days following ischemia (P < 0.05;
P < 0.001, Fig. 2d). In addition, this increase was followed
by a slight decline from days 14 to onwards (P < 0.01;
P < 0.001, Fig. 2d).
The main brain areas affected in this animal model of
cerebral ischemia (MCAO) are the cortex and striatum.
PET signal uptake for both radiotracers displayed similar
distribution pattern over time in the cortex and striatum;
nevertheless, [18F] DPA-714 showed a mild uptake
increase in comparison to [18F] VUIIS1008 (Fig. 3a, b).
The ipsilateral cortex and striatum displayed a PET signal
increase from days 3 to 14 (P < 0.001, Fig. 4a, b).
Moreover, the PET signal for both radiotracers peaked
at day 7 in comparison to different days after MCAO
(P < 0.05; P < 0.01; P < 0.001, Fig. 4a, b).
Neighboring regions of the brain lesion such as the
hippocampus and the thalamus exhibited PET signal
uptake increase for both [18F] VUIIS1008 and [18F]
DPA-714. In fact, the hippocampus showed a higher
PET signal uptake compared to the thalamus due to its
closer location to the cerebral infarction. The
hippocampus and thalamus showed a progressive increase
during the first week after reperfusion (P < 0.05; P < 0.01;
P < 0.001, Fig. 4c, d), followed by a decline after day 7
(P < 0.05; P < 0.01; P < 0.001, Fig. 4c, d).
[18F] VUIIS1008 PET displacement studies after MCAO
The time-activity curve (TAC) generated in the ischemic
cerebral hemisphere at day 7 after brain ischemia showed
that [18F] VUIIS1008 uptake reached a peak of radioactivity
a few minutes after bolus injection and remained constant
from 10 to 60 min. In the contralateral hemisphere, the
uptake showed a peak of radioactivity during the first
minutes followed by a fast washout (Fig. 5a). Displacement
studies were performed by injecting an excess (1 mg/Kg)
of DPA-714 or VUIIS1008 20 min after the radiotracer
injection. Ten to 15 min after the injection of the cold
compound, the radioactivity concentration in the ischemic
area decreased to the concentration levels of the
contralateral area (Fig. 5b, c). Likewise, the ratios of the lesion to the
contralateral brain hemisphere showed a significant decrease
in the PET uptake signal after displacement by DPA-714
and VUIIS1008 7 days after ischemia (P < 0.01; P < 0.001,
Fig. 5d). In addition, displacement by VUIIS1008 showed a
mild decrease compared to the displacement achieved by
DPA-714 (Fig. 5d). [18F] VUIIS1008 PET images showed the
PET signal in the lesion before the displacement (0–20 min)
and the [18F] VUIIS1008 uptake decrease after displacement
with VUIIS1008 and DPA-714 (40–60 min) (Fig. 6).
Microglial/macrophage expression of TSPO after ischemia
The evaluation of TSPO expression was assessed with
immunohistochemistry at different magnifications in the
contralateral hemisphere and the core of the infarction, as
represented in the co-registered [18F] VUIIS1008 PET-MRI
T2W image at day 7 after cerebral ischemia (20×*, 20×**
and 100×**, Fig. 7a). In the contralateral hemisphere,
immunofluorescence staining showed the low expression
of TSPO in few activated microglial cells (Fig. 7b); unlike
the core of the lesion, TSPO showed an overexpression of
TSPO in activated microglia/infiltrated macrophages at
different magnifications (Merged, Fig. 7e). Cells with the
morphology of amoeboid reactive microglia/macrophage
showed intense CD11b immunoreactivity in the ischemic
lesion (in red; Fig. 7f ). Subsequently, the overreactivity of
microglia co-localized with the expression of TSPO at day
7 after ischemia (in green, Fig. 7g).
PET imaging of TSPO has widely grown over the last two
decades to evaluate the role of neuroinflammation in the
central nervous system diseases and to assess novel
antiinflammatory therapeutic strategies [
several PET radioligands have been evaluated as markers
of microglial neuroinflammation after stroke in both
human and animal models . Among them, [18F]
DPA714 has been considered as an excellent candidate for
imaging neuroinflammation after cerebral ischemia in
3, 24, 33, 34
]. Alternatively, [18F] VUIIS1008, a
novel radiotracer [18F] VUIIS1008 with an optimized
(See figure on previous page.)
Fig. 4 Time course of the progression of the [18F] VUIIS1008 and [18F] DPA-714 PET signals before and after cerebral ischemia. %ID/cm3 (mean ± SD)
of [18F] VUIIS1008 and [18F] DPA-714 was quantified in the ipsilateral cortex (a), striatum (b), hippocampus (c), and thalamus (d). The upper right panels
of each figure show the selected brain ROIs for the quantification defined on a slice of a MRI (T2W) template. Rats (n = 8 per group) were repeatedly
examined by PET before (day 0) and at 1, 3, 7, 14, 21, and 28 after ischemia. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control (day 0) and day 1;
&p < 0.05, &&p < 0.01, and &&&p < 0.001 compared with day 7
pyrazolopyrimidine scaffold and approximately 36-fold
enhancement in affinity compared to its analog [18F]
DPA-714, has been recently developed . Because of
this, we have assessed the evaluation of both PET
radiotracers during the first month following cerebral ischemia
in rats, in combination with magnetic resonance imaging
(MRI) and immunohistochemistry.
[18F] VUIIS1008 and [18F] DPA-714 PET after cerebral ischemia
[18F] VUIIS1008 has been previously evaluated as a novel
radiotracer for PET imaging of glioma in rats [
these studies, the authors showed that [18F] VUIIS1008
exhibited a rapid uptake in TSPO-rich tissues and
suggested that this radiotracer might improve tumor
detection, particularly for those tumors expressing modest levels
of TSPO . Likewise, [18F] VUIIS1008 displayed a higher
tumor-to-background ratio and binding potential
compared to [18F] DPA-714 suggesting its promising properties
for cancer imaging [
]. Nevertheless, the suitability of this
radiotracer still needed to be confirmed in other animal
models of neuroinflammation such as cerebral ischemia.
Thus, the present study has tackled the unprecedented
evaluation of [18F] VUIIS1008 as a potential biomarker for
in vivo PET imaging of neuroinflammation, particularly in
a rat model of transient ischemia.
Cerebral ischemia leads to an overexpression of TSPO
which has been considered as a sensitive marker related
to the size of the brain lesion and clinical outcome [
For this reason and to avoid bias, rats with similar brain
infarct volumes measured with MRI-T2W at 24 h after
reperfusion were included in the PET studies with [18F]
VUIIS1008 and [18F] DPA-714.
Previous in vivo PET imaging of TSPO with [18F]
DPA-714 showed low levels of the PET signal before the
ischemia onset evidencing the low expression of TSPO
in the healthy cerebral tissue [
]. In the present
study, both [18F] VUIIS1008 and [18F] DPA-714
radiotracers showed non-significant binding differences in the
healthy brain parenchyma before the induction of the
MCAO (0. 204%ID/cm3 vs. 0.215%ID/cm3, respectively).
These results stand in agreement with those observed by
Tang and collaborators, who described similar tracer
uptake values in the healthy brain at 90 min after injection
of [18F] VUIIS1008 and [18F] DPA-714. Despite this, by
applying compartmental modeling, the same authors
observed a lower influx-to-efflux parameter ratio (k1/k2)
and volume of distribution (Vt) for [18F] VUIIS1008
compared to [18F] DPA-714, but similar binding
potential values (k3/k4) [
Here, we have observed that following cerebral ischemia,
both radiotracers displayed significant accumulation in the
injured brain regions at different days after reperfusion
(Figs. 1 and 3). In the ischemic hemisphere and particularly
in the specific brain regions evaluated (cortex, striatum,
hippocampus, and thalamus), PET signal uptake showed
the same pattern observed recently by Domercq and
colleagues, a progressive [18F] DPA-714 uptake increase
during the first week followed by a binding gradual
decrease afterwards [
] (Figs. 2 and 4). Moreover, we
have observed that [18F] DPA-714 displayed a mild
signal increase from day 3 to day 21 after ischemia in
relation to [18F] VUIIS1008. In fact, the [18F] DPA-714
uptake increase observed at these time points was
concomitant with the overexpression of TSPO, particularly
at day 7 after ischemia as shown using
immunohistochemistry for TSPO (Fig. 7). On the contrary, both
radiotracers displayed similar PET uptake values in the
ischemic tissue at days 1 and 28 and in non-ischemic
brain regions (contralateral hemisphere and cerebellum),
scenarios with poor TSPO expression. Therefore, these
findings might suggest that [18F] VUIIS1008 did not exhibit
a greater performance than [18F] DPA-714 for imaging high
levels of TSPO expression after experimental stroke.
[18F] VUIIS1008 PET displacement studies after cerebral
At day 7 after ischemia, the time-activity curve for [18F]
VUIIS1008 showed a fast increased uptake in the ischemic
hemisphere that reached a peak 10 min after tracer injection
and was maintained at a plateau level during the following
50 min. These findings are in agreement with those
displayed by [18F] DPA-714 at day 7 after MCAO in
]. In contrast, the contralateral hemisphere
showed lower [18F] VUIIS1008 uptake because of the
low presence of TSPO in healthy tissue. Actually, the
contralateral binding was displaced by cold VUIIS1008
and DPA-714 supporting the view that the modest and
transient increase of [18F] VUIIS1008 binding is TSPO
specific (Figs. 5 and 6). Likewise, [18F] VUIIS1008 binding
was rapidly displaced from the injured hemisphere by an
excess of the corresponding unlabeled compound, where,
after 10 min, its uptake was close to that in the
nonischemic area. The target-to-background ratio after the
displacement by DPA-714 and VUIIS1008 was 1.4 and
1.2, respectively, showing a slight displacement increase
performed by VUIIS1008 in relation to DPA-714. In fact,
this situation is mainly caused by a more effective
displacement of both specific and non-specific TSPO binding
of [18F] VUIIS1008 by VUIIS1008 in relation to DPA-714.
In addition, the fact that [18F] DPA-714 could successfully
displace [18F] VUIIS1008 supports the specificity of this
latter to bind to TSPO in vivo following cerebral ischemia.
Finally, the radioactivity remaining after displacement
studies observed in the lesion compared to the
contralateral hemisphere for both radiotracers might be due to the
increase of the radiotracer influx/efflux ratio (k1/k2) as a
consequence of the blood-brain barrier disruption
following cerebral ischemia.
We report here the PET imaging of TSPO with both [18F]
VUIIS1008 and [18F] DPA-714 in a rat model of cerebral
ischemia. Our results confirmed the progressive binding
increase of [18F] VUIIS1008 in the ischemic hemisphere
during the first week after cerebral ischemia, followed by a
decline later on. These findings are consistent with the
PET signal uptake pattern of [18F] DPA-714, a well-known
radiotracer for TSPO, and with the expression dynamics
of this transporter after cerebral ischemia. Therefore, these
results provide novel information about the feasibility of
[18F] VUIIS1008 to monitor neuroinflammation following
neurological diseases such as ischemic stroke.
The authors would like to thank A Leukona, L Morales, V Salinas, A Arrieta,
and A Cano for the technical support in the radiosynthesis and technical
assistance in PET studies and the Spanish Ministry of Economy and
Competitiveness through the grant SAF2014-54070-JIN for financial support.
This study was funded by Spanish Ministry of Economy and Competitiveness
(SAF2014-54070-JIN)Ministry of Economy and Competitiveness
(SAF201454070-JIN) and the project FATENANO (PCIN-2015-116 and ERA-NET SIINN –
KRP carried out the synthesis of the radiotracer and radiosynthesis. LC carried
out PET studies, imaging analysis, and immunohistochemistry. DP carried out
MRI studies and analysis. SP-G participated in MRI studies, VG-V performed
radiosynthesis, MH helped to draft the manuscript and supplied the NP155
antibody, and JLL participated in the design and coordination of the study.
AM conceived the study, participated in its design and coordination, and
wrote the manuscript. All authors read and approved the final manuscript.
All applicable international, national, and/or institutional guidelines for the
care and use of animals were followed. This article does not contain any
studies with human participants performed by any of the authors.
The authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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