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Introduction
During the rapid development of in vivo
amyloid imaging agents for the early diagnosis of Alzheimer's disease
(AD)[1], some efforts were made to use high-resolution
micro-positron emission tomography (PET) imaging in the
transgenic mouse model of central nervous system
amyloid deposition to further study Pittsburgh compound-B
([11C]PIB), a PET tracer that has been shown to be increased in
amyloid-aggregate areas of the AD brain. However, some types of
transgenic mice, including PS1/APP and Tg2576, showed no
significant retention of PIB in these models, even at 12 or 22
months of age when amyloid deposition
exceeded that distinctly seen in
AD[2,3]. Klunk et al studied the reason and
found that PS1/APP mouse brain tissues contained less than
1 high-affinity PIB binding site per 1000 molecules of
β-amyloid, whereas the AD brain contained >500 PIB binding sites per
1000 molecules of β-amyloid. The tiny amount
(nmol/L) of the PET tracer, the small amount of high-binding sites, and the
low detection efficiency (2%) of the micro-PET might be the
main reasons for the absence of tracer retention in the
transgenic mice. Cai et al reported their initial efforts to evaluate
[11C]PIB for imaging brains of Tg2576 mice. They also did not
find any specific binding in the cerebrum, only non-specific
binding comparable to that in the cerebellum. Interestingly,
when they used much older transgenic Tg2576 mice (28
months' old), they observed a ratio of radioactivity
in the cerebrum to that in the cerebellum of approximately
1.8 at 25 min after an injection of
[11C]PIB[4]. Their case was
successful in exhibiting micro-PET imaging of β-amyloid deposits in
rodent brains.
Shimadzu et al found a hot spot at 180 min after the
intravenous administration of [18F]BF-168 (see the structure in
ref 5) on the same site of the rat brain where β-amyloid (1_42)
and a buffer only on the opposite site had been injected 6 d
before with an autoradiographic
image[5]. Previous studies showed the
compound O-FEt-PIB had a high affinity to β-amyloid and high
lipophilicity[6_8]. In the present study, we
first observed the increased uptake of
[18F]O-FEt-PIB in the right hemicerebrum of an injected AD rat where
β-amyloid (1_40) and a saline only on the opposite site were injected
using micro-PET imaging.
Materials and methods
General equipment and reagents HPLC was carried out
on a Dionex system equipped with a P680 pump, a PDA-100
photodiode array detector (Dionex Corporation, Sunnyvale,
CA, USA), and a NaI (Tl) scintillation detector (Bioscan,
Washington, DC, USA). The columns used for the
identification and purification were LiChrosorb C18 (5
μm, 300 mm×3.9 mm and 10 μm, 300 mm×7.8 mm (Waters, Milford, MA, USA),
respectively;. The column effluent passed through an UV
(365 nm) detector and scintillation radioactivity detector in
series. The HPLC solvents consisted of
CH3OH (solvent A) and 0.1%
CF3COOH/H2O (solvent B). HPLC gradient was
set as follows: 0_1 min 70% B, 1_25 min 20%, 25_40 min 20%.
The flow rates of purification and analysis were 3 mL/min
(condition 1, with column 300 mm×7.8mm) and 1 mL/min
(condition 2, with column 300 mm×3.9 mm), respectively.
Radio-TLC (Thin Layer Chromatography, Merck, Darmstadt, Germany) analyses were performed using silica
gel 60 GF-254 plates (Merck, Darmstadt, Germany) on a
Bioscan system AR-2000 (Bioscan, Washington, DC, USA)
with Winscan software version 3.09 (Bioscan, Washington,
DC, USA). The radioactivity measurements were done
using an automatic gamma counter (3 inch NaI[Tl] well crystal;
Jiading, Shanghai, China) coupled to a multichannel
analyzer (SN682B; Jiading, Shanghai, China)
[9].
Synthetic human β-amyloid protein (1_40) was
purchased from Sigma-Aldrich Synthesis (Steinheim, Germany).
Other commercial reagents were purchased from Shanghai
Reagent (Shanghai, China), and used without further
purification unless otherwise specified.
Synthesis of [18F]O-FEt-PIB
The cold reference compound O-FEt-PIB was synthesized as described in our
previous work[6]. The radiosynthesis procedure of
[18F]O-FEt-PIB and its primary characters were described in
Zheng et al[10]. [18F]Fluoride, produced by a cyclotron using
18O(p, n)18F reaction, was passed through a Sep-Pak light QMA cartridge
(C18, Waters, Milford, MA, USA) as an aqueous solution
and then dried azeotropically with CH3CN (3×400 µL) at 110
°C under a stream of nitrogen. Ethylene glycol bistosylate (6
mg) in anhydrous CH3CN (400 µL) was added to the reaction
vial and heated at 110 °C for 10 min to generate
2-18F-fluoroethyl tosylate
([18F]FEtOTs). The products were purified through a pre-saturated C18 Sep-Pak cartridge. In total,
3 mg PIB (dissolved in 200 µL DMSO, with 20 µL of 1 mol/L
K2CO3 solution, and 400 µL anhydrous
CH3CN) was added to the purified
[18F]FEtOTs in a vessel and heated for 20 min
at 110 °C. The reaction products were then purified using a
semi-preparative HPLC column (condition 1). The elution
was collected from 13 to 15 min and dried at 60 °C under a
nitrogen stream. Phosphate-buffered saline (pH 7.20) was
added to dilute the product to a 37 MBq/mL solution. The
analytical HPLC condition (condition 2) and radio-TLC were
used for quality control (Chemical Purity and Radio
Chemical Purity) >95%, Retention time
(tR) =18.2 min [reference
tR=17.4 min]; Rf=0.66 in
CHCl3 [reference Rf=0.62]). Specific
activity estimated by comparing the UV peak intensity of
purified [18F]O-FEt_PIB with a known concentrations was
more than 740 GBq/µmol.
Animal preparation The model and control rats were
prepared as described in Wang et al and Nitta
et al[11,12]. Male Sprague_Dawley rats, weighing 200_220 g at the
beginning of the experiment, were housed individually in a room
maintained at 23 °C with a 12 h light-dark cycle for the
duration of the experiment. In total, 6 μg aggregated
β-amyloid (1_40) with 6 μL dissolved in sterile, distilled water (prepared
by incubating soluble β-amyloid [1_40] at 37 °C for at least 7
d) was stereotaxically injected into the hippocampus (_3.0
mm AP, _2.2 mm ML, and _3 mm DV) in rats under barbital
anesthesia using a microsyringe. One week was allowed for
recovery from the surgery. The control rats were infused with
the same quantity of sterile, distilled water. One month after
the surgery, 3 randomly-selected model rats were killed by
decapitation after heart perfusion. The brains were rapidly
removed and placed on ice. The brain tissues were fixed for
immunohistochemical evaluation. Other normal and
postoperative rats were used for micro-PET imaging or
other in vivo/in vitro conventional experiments. All of the
experiments complied with the current laws of China and met
ethical approval.
Brain biodistribution of
[18F]O-FEt_PIB in normal rats
Studies were performed in 5 male Sprague-Dawley rats,
weighing 400-500 g (~3 months old). Under anesthesia, each
animal was weighed and received approximately 7.4 MBq (200
µCi) of [18F]O-FEt_PIB
in 100 μL saline through the lateral tail vein. The animals were killed by cardiac excision
following cardiac puncture to obtain arterial blood samples and
decapitation to obtain brains and crania at 2, 30, 60, and 120
min postinjection. The brains were rapidly excised and
divided into cerebellum, cortex, and cerebrum. All of the samples
were weighed and counted, and the counts were corrected
for decay. The radioactivity of tissues was determined as
the percentage of injected dose per gram of tissue
normalized to body weight (in kg) or (%ID-kg)/g.
High-resolution micro-PET scanning PET imaging was
performed using a micro-PET R4 rodent model scanner (CTI
Concorde Microsystems, Knoxville, TN, USA), which was
equipped with micro-PET manager for data acquisition in list
mode and ASIPro for preparing sinograms and image
reconstruction[13]. The scanner had a computer-controlled bed
and 10.8 cm transaxial and 7.8 cm axial field of view (FOV)
with an image resolution of <1.8 mm.
[18F]FDG (0.5 mCi each rat) was prepared with a specific activity of 500 Ci/mmol,
which was provided by the Department of Nuclear Medicine,
Zhejiang University School of Medicine (Hangzhou, China).
A total of 4 AD and 3 control male Sprague-Dawley rats
were imaged by micro-PET using
[18F]O-FEt-PIB. To determine the appropriate times for data acquisition, 37 MBq (1
mCi; the amount was 2 times than that in
[18F]FDG imaging) of
[18F]O-FEt_PIB was injected via the tail vein while the rat
was placed at the center of the FOV (field of view) of the
scanner and scanned just after the injection. The dynamic
PET data were acquired on an AD rat for 40 min (10 s×6
frames, 30 s×6 frames, 60 s×5 frames, 300 s×4 frames, and 600
s×1 frame). The other animals were anesthetized first,
receiving an injection of PET tracers, and were then placed at
the center of the FOV of the micro-PET R4 scanner and
underwent a 15 min static scan. Three AD rats were scanned
for 15 min at 5 min postinjection. The control rats
(n=3) were scanned based on the same methods as the model rats.
Images were reconstructed by a maximum posteriori
probability algorithm. Corrections for dead time, random
scattering, and attenuation were performed for all scans.
PET data analysis Regions of interest (ROI) were
manually placed over the cerebellum, hemicerebrum, and cortex. A
3-D digital map of the rat was used for the identification of
the anatomical structures[14] and the
[18F]FDG images of the normal rat brains. Both summed images of the dynamic scan
and 3-D images (or single slice) of the static scan were
analyzed. Adjacent slices containing hippocampi were picked
out from both the model and control rats. The activity
difference value (ADV) between 2 hemicerebrums was calculated
as follows:
ADV=(|AL_AR|/([
AL+AR]/2)×100%.
AL represented the radioactivity in the left hemicerebrum, and
AR represented the radioactivity in the right hemicerebrum. This value could
evaluate the extent of radioactivity in the 2 parts of the brain.
Results
Brain distribution of [18F]O-FEt_PIB in normal
rats Figure 1 shows the brain distribution
of [18F]O-FEt-PIB in normal Sprague-Dawley rats in terms of (%ID-kg)/g. The brain
entry of this tracer was rapid, and peaked at approximately 2
min with the average (%ID-kg)/g value at approximately 0.5.
This was a good character for further developing it as a
potential neuroreceptor radioligands. The low uptake and
radioactivity in the bone indicated that little
18F_ ion was generated in the metabolism. This result was confirmed by
the dynamic scan with micro-PET. Even at 40 min, there was
no distinct increased radioactivity in the crania. However,
the clearance rate of radioactivity from the brain tissues was
slow. The 2_60 min brain radioactivity ratio was
approximately 6, and this characteristic was not as good as that of
PIB (2_30 min ratio, 12[15]).
Considering the disadvantage of AD transgenic mouse
models in micro-PET imaging, we prepared a commonly-used
AD rat model, in which the amyloid protein (1_40) was
administered to the rats in vivo to evaluate the AD PET tracer
using micro-PET. This model has successfully evaluated
the effects of a Chinese traditional drug, huperzine
A[11]. To understand the position and pervasion of the injected
amyloid-β (1_40), we performed an immunohistochemical
evaluation. The results are shown in Figure 2. Figure 2
shows the amyloid deposits in the right hippocampus. The
deposits were diffused in the area of hippocampus, and a
spot on it was caused by an air bubble (Figure 2B); there
were no detected amyloid deposits in the left hippocampus
(Figure 2A).
Micro-PET results Figure 3 shows the time-activity
curves (TAC) of the regional distribution of radioactivity in
the rat brain after the injection of the tracer. The maximum
radioactivity obtained from the data was defined as 100, and
other values were calculated as a ratio of the maximum
radioactivity. This result correlated with the conventional
method (Figure 1) and showed a quick brain uptake and slow
washout.
The distribution of [18F]O-FEt-PIB in the rat
brains in vivo using micro-PET is also shown (Figures 4_7), and the
results are interesting. Figure 4 shows the representative
summed image of the dynamic scanning, which contained 23
frames of images. The fusional picture exhibited an
unsymmetrical distribution of the radiotracer in the 2 hemicerebrums.
The distribution of [18F]O-FEt-PIB in the single images, which
were neither from early time (recorded at 0_5 min) or at late
time (recorded at 30_40 min) showed no distinct difference
in 2 hemicerebrums because the images were blurry or
radioactivity was low (Figure 3). Figure 5 shows the increased
aggregates of radioactivity in the right hemicerebrum in the
AD model rats infused with 6 µg β-amyloid (1_40). Figure 5
shows a similar uptake of the radio tracer between the 2
hemicerebrums. This result indicated that the surgery could
not induce an increased aggregate of the tracer after
recovery. Figure 6 shows the adjacent transverse slices (each
0.12 mm thick), which confirmed the special distribution of
the tracer in the special regions (the areas adjacent to the
hippocampus) of the rat brains. ROI were defined on the
3-D digital map, and the mean activities were auto-given by
the microPET ASIPro (Figure 7). The
ADV(HT) was approximately 14.6% in the AD model rats, which was approximately
4 times great than that of the control rats (3.9%). The ADV in
the cortex was 2.9% in the model rats compared to 5.6% in
the control rats.
Discussion
Animal models of AD play an important role in
understanding the pathology of the disease, the diagnosis, or
therapeutic drugs. Although no current model has
developed the full pathological spectrum of the disease, the
transgenic rodent model was an ideal choice. However,
recent studies have shown that although a large amount of
amyloid plaques exist in the transgenic mouse brain, not
enough high-binding sites could "capture" enough amounts
of the [11C]PIB to form an aimed images using micro-PET
imaging. Nitta et al and Wang et al have reported an
injected rat model, which shows that injected β-amyloid could
impair memory and elicit a degree of
Alzheimer-type neurodegeneration. In the present study, we tried to apply
this rat model for the in vivo imaging of β-amyloid deposits
in the right hippocampus with micro-PET using a new
synthesized PET tracer, [18F]O-FEt-PIB instead of
[11C]PIB. In the present study, the brain, blood, and bone uptake and
micro-PET imaging demonstrated excellent brain uptake of
radioactivity in the rat brains. To our knowledge, this study
is the first to show an increased aggregate of a small
molecular PET probe for the aggregates of β-amyloid
using in vivo micro-PET imaging with an AD-injected rat model. Further
studies about new tracers and new AD rodent models are
currently being undertaken in our laboratory.
Acknowledgements
The authors will thank the Amersham Kexing
Pharmaceuticals (Jiading, Shanghai, China) for supplying the
non-carrier-added [18F]F_ solution. The authors also thank Prof
Jiang-ning ZHOU from Hefei National Laboratory for
Physical Sciences at Microscale and the Department of
Neurobiology, School of Life Sciences, University of
Science and Technology of China (Hefei, Anhui, China) for his
help in the analyses of the micro-PET images.
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