Assessment of brain beta-amyloid deposition in transgenic mouse models of Alzheimer’s disease with PET imaging agents 18F-flutemetamol and 18F-florbetaben

Background Although amyloid beta (Aβ) imaging is widely used for diagnosing and monitoring Alzheimer’s disease in clinical fields, paralleling comparison between 18F-flutemetamol and 18F-florbetaben was rarely attempted in AD mouse model. We performed a comparison of Aβ PET images between 18F-flutemetamol and 18F-florbetaben in a recently developed APPswe mouse model, C57BL/6-Tg (NSE-hAPPsw) Korl. Results After an injection (0.23 mCi) of 18F-flutemetamol and 18F-florbetaben at a time interval of 2–3 days, we compared group difference of SUVR and kinetic parameters between the AD (n = 7) and control (n = 7) mice, as well as between 18F-flutemetamol and 18F-florbetaben image. In addition, bio-distribution and histopathology were conducted. With visual image and VOI-based SUVR analysis, the AD group presented more prominent uptake than did the control group in both the 18F-florbetaben and 18F-flutemetamol images. With kinetic analysis, the 18F-florbetaben images showed differences in K1 and k4 between the AD and control groups, although 18F-flutemetamol images did not show significant difference. 18F-florbetaben images showed more prominent cortical uptake and matched well to the thioflavin S staining images than did the 18F-flutemetamol image. In contrast, 18F-flutemetamol images presented higher K1, k4, K1/k2 values than those of 18F-florbetaben images. Also, 18F-flutemetamol images presented prominent uptake in the bowel and bladder, consistent with higher bio-distribution in kidney, lung, blood and heart. Conclusions Compared with 18F-flutemetamol images, 18F-florbetaben images showed prominent visual uptake intensity, SUVR, and higher correlations with the pathology. In contrast, 18F-flutemetamol was more actively metabolized than was 18F-florbetaben (Son et al. in J Nucl Med 58(Suppl 1):S278, 2017]. Electronic supplementary material The online version of this article (10.1186/s12868-018-0447-7) contains supplementary material, which is available to authorized users.


Background
Recently, Aβ imaging with 18 F labeled radiotracers has been widely used for patients with Alzheimer's disease (AD). 18 F-flutemetamol is 18 F labeled analogue of 11 C-PiB produced by GE Healthcare (Buckinghamshire, UK) [2]. It has been useful in differentiating between patients with AD and healthy subjects with high specificity (96%) and sensitivity (93%) in the detection of AD, as well as high test-retest reliability [3,4]. 18 F-florbetaben is an 18 F labeled polyethylene glycol stilbene derivative showing high in vitro affinity and specificity for β-amyloid plaques [3]. 18 F-florbetaben and 18 F-flutemetamol are widely used for the diagnosis and monitoring of AD in a routine medical field. However, there are many unknown issues regarding the difference in tracer dynamics and biodistribution between 18 F-flutemetamol and 18 F-florbetaben. Because of the difficulty conducting the direct comparison between two tracers for humans due to the weighted exposure to radiation, a preclinical animal study is a good alternative option for a baseline study.
Recently, several imaging studies using newly developed 18 F labeled Aβ PET tracers were reported in AD mouse models. In a previous study, the in vivo 18 F-flutemetamol binding of Aβ deposits was tested in various AD mouse models [5]. In old APP23 mice, significant 18 F-flutemetamol retention was observed in the brain. But, 18 F-flutemetabmol did not show a outstanding advantage in APPswe-PS1dE9 and Tg2576 mice.
However, transgenic mice with various genetic backgrounds have been related with different pathologies, which make it difficult to interpret the overlapping study results [6]. Therefore, comparisons between β-amyloid imaging regarding AD mouse have to be accomplished with some caution as brain sizes and anatomic landmarks of target VOIs greatly affect accurate PET signal quantification. Until now, there has been no antecedent report comparing between 18 F-flutemetamol and 18 F-florbetaben images in an AD mouse model, so comparative conclusions draw special interest.
Herein, we tested a recently developed APPsw mouse model (C57BL/6-Tg(NSE-hAPPsw)Korl) enhancing expressing Swedish double mutation form of human APP (K670 N, M671L) under regulation of the neuron specific enolase (NSE) promoter. For this mouse model, there has been no attempt regarding its application for the evaluation of new Aβ imaging ligands. Hence, we performed a small animal study conducting direct comparisons between two 18 F labeled Aβ PET tracers, 18 F-flutemetamol and 18 F-florbetaben in (C57BL/6-Tg(NSE-hAPPsw) Korl) mouse model in terms of following aspects: the ability to discriminate a transgenic from a control mouse, intensity of uptake and distribution pattern in visual images, difference of static ratio and kinetic parameters, bio-distribution and correlation with neuropathologic findings.

Animals
Experiments were conducted with 7 APPsw transgenic mice (genetic background C57BL/6-Tg(NSE-hAPPsw) Korl) augmenting human APP with the Swedish double mutation (K670N, M671L) under regulation of the NSE promoter. As controls, 7 littermates with the corresponding genetic background, C57BL/6J, were used. Age and sex were matched between the two groups (mean age and mean weight: 18 weeks and 24.84 ± 1.01 g for APPsw mice and 18 weeks and 29.20 ± 3.49 g for C57BL/6 J control mice). The mice used in the study were donated from the Division of Laboratory Animal Resources, Korea FDA (Food and Drug safety administration, National Institute of Toxicological Research, registration number: KNL-HYD-TG0615). Details on number of animals per study group, sex, mean age and mean body weight are summarized in Table 1. Two mice from each AD and control group were sacrificed for pathology at 18 weeks

PET/CT imaging
Seven transgenic and 7 control mice underwent sequential PET imaging for direct comparison of the two tracers (total 28 scans). The time interval between 18 F-florbetaben and 18 F-flutemetamol PET imaging was 2-3 days. Inhalation anesthesia was maintained by 3.5 L/min oxygen and 0.6-2% isoflurane, 15 min prior to scanning. The body temperature was kept at 37 °C with a temperaturecontrolled heating pad, and the respiratory rate stayed at 80-100/min. Small animal PET data was acquired with a nanoscan PET scanner (Mediso Medical Imaging Systems, USA). After the induction of anesthesia, the animals were positioned with their heads in the center of the field of view and were fixed in the PET scanner in the prone head first position (HFP). At the beginning of the PET scanning procedure, computed tomography (CT) scans were acquired for attenuation correction and anatomical reference (50 kVp, 250 mA). Next, simultaneous with an i.v. injection of 8.51 MBq (0.23 mCi) of 18 F-flutemetamol or 18 F-florbetaben, a 90-min dynamic emission scan was started. Dynamic acquisition was performed in the 3D list mode for 90 min. The emission data were normalized and corrected for decay and dead time. The sinograms were reconstructed with FBP (filtered back-projection using a ramp filter with a cut-off at the Nyquist frequency). Static images and dynamic images with 20 imaging frames were generated.

Radiosynthesis
The radiosynthesis of 18 F-florbetaben (4-ethoxy)phenyl] vinyl}-N-methylaniline, commercial name: Neuraceq) was performed using an auto-synthesizer according to the protocol of Piramal Enterprises Ltd. The radiochemical purity was > 99%, as determined by analytical HPLC. The radiochemical yield averaged 45% (decay-corrected) at the end of synthesis (EOS) based on 18 F-fluorine. The specific activity averaged 774 GBq/umol at the EOS. The commercial products were purchased from the company (Duchem Bio, South Korea). The radiosynthesis of 18 F-flutemetamol (6-Benzothiazolol, 2-[3-( 18 F) fluoro-4-(methylamino) phenyl], commercial name: Visamyl) was performed to using an auto-synthesizer according to the protocol of GE Healthcare. The radiochemical purity was > 96% as determined by analytical HPLC. The radiochemical yield averaged 27% (decay-corrected) at the end of synthesis (EOS) based on 18 F-fluorine. The specific activity averaged 1862 GBq/umol at the EOS. The commercial products were purchased from the company (Carecamp Co., Ltd., South Korea).

Analysis of PET data
PET data was analyzed with the fusion toolbox embedded in PMOD version 3.7.0 software (PMOD Technologies, Zurich, Switzerland). The CT image was thresholded at 2/3 of the maximal value (approximately 1340 Hounsfield units), and the skull image was obtained. For the shape of an atlas to properly fit with the skull CT, the thresholded CT image was manually fused with the magnetic resonance brain template, called M.Mirrione. Then, the transformation information was saved in a MAT-file format. Using the Initialize/Match function of the fusion toolbox, the PET image was re-sliced to match M.Mirrione template [7]. Then, the transformation information between the thresholded CT and the mouse magnetic resonance template was loaded on re-sliced PET image. Then, the re-sliced PET image was co-registered manually using the shift, rotate and scale functions and normalized to the mouse MR brain template (M. Mirrione) [7,8]. The final co-registered PET image was masked with the M. Mirrione brain mask. The corresponding template and mask files can be found in the resources/usertemplates directory embedded in PMOD version 3.7.0 software (Pmod Technologies, Zurich, Switzerland). The same step was applied to all frames of dynamic data.
Volumes of interest (VOIs) of embedded mouse brains are presented in Fig. 1. The areas of the VOIs are the cortex (Cor), right hippocampus (Rhip), left hippocampus (Lhip), thalamus (Thal), right striatum (Rstr), left striatum (Lstr) and the cerebellum (Crbl). To investigate the difference between 18 F-florbetaben and 18 F-flutemetamol images, different images using the image algebra option embedded in PMOD fusion tool (version 3.7.0; Pmod Technologies, Zurich, Switzerland) were created. For the analysis of static PET image, the standardized uptake value (SUV) and the standardized uptake value ratio (SUVR) between the cortex and cerebellum was calculated with a VOI based method.
To determine the optimal compartment model for amyloid specific tracers, the 2 tissue compartment model from previous studies was used [9][10][11]. For the 2 tissue compartment model analysis with the Image Derived Input Function (IDIF) method, 1 mm 3 volumes of interest (VOIs) were drawn on the center of the left ventricle on the initial time frame image.

Sample preparation
The animals were deeply anesthetized with zoletil and xylazine and were sacrificed by intra-cardiac perfusion with 4% paraformaldehyde (pH 7.4). The brains were embedded in paraffin wax for 48 h. The tissue samples were serially sectioned at a thickness of 10 µm on a rotary microtome for immuno-histochemical analysis.

Thioflavin S staining
The sections were deparaffinated and rehydrated before staining. The sections were incubated in a 1% (1 g per 100 ml water) thioflavin S (TfS, T1892, Sigma Aldrich, St. Louis, MI, USA) solution for 30 min. The sections were washed with water three times for 2 min, 80% ethanol for 6 min, washed again with water and cover slip mounted with VectaShield as the mounting medium. The slides were stored for 4 °C. The sections were washed with water three times for 2 min and with 80% ethanol for fluorescence microscopy using filter sets for DAPI and GFP. The DAPI (contained in the mounting medium) fluorescence was used by the scanner to set the optical focus, and the GFP contained the specific signal of thioflavin S.

Immunohistochemistry for amyloid beta 40
Non-specific reactions were blocked with 3% fetal bovine serum in phosphate buffered saline (PBS) for 1 h. Slides were incubated with mouse monoclonal primary amyloid beta 40 antibody (diluted 1:150; Millipore, USA). The secondary antibody was Streptavidin Alexa fluor 594 conjugated anti-mouse IgG (1:400, Invitrogen, USA). The fluorescence was observed using Nikon-80i fluorescence microscopy using filter sets for Fig. 1 a Volume of interest (VOI) of mouse brain. VOI was drawn under guidance of the PMOD embedded mouse brain atlas (Mouse (M. Mirrione)-T2 MRI atlas) to cover the cortex (Cor: blue), right hippocampus (Rhip: dark green), left hippocampus (Lhip: dark green), thalamus (Thal: light green), right striatum (Rstr: red), left striatum (Lstr: red) and the cerebellum (Crbl: yellow), b volume of interest in blood input area, c time activity curve of blood input area DAPI and RFP. The DAPI (contained in the mounting medium) fluorescence was used by the scanner to set the optical focus, and the RFP contained the specific signal of amyloid beta 40.

Bio-distribution
The 18 F-florbetaben and 18 F-flutemetamol binding to different brain regions and peripheral organs in AD transgenic (N = 1) and control mice (N = 1) using ex vivo gamma counting. Mice were anaesthetized with isoflurane and injected with 0.23 mCi of 18 F-florbetaben and 18 F-flutemetamol. The tracer was allowed to distribute for 90 min. Mice were sacrificed by cervical dislocation and the brain was rapidly removed. Then, the blood, heart, lung, liver, kidney, medulla, cerebellum, right cortex, left cortex, olfactory bulb were dissected. 18 F-radioactivity was measured with a gamma counter.

Statistics
For the analysis of static PET data, group comparison of SUVR and kinetic parameters were conducted with the Mann-Whitney U. A threshold of P less than 0.05 was considered significant. All statistical analyses were performed using IBM SPSS Statistics (version 20.0; SPSS) and Medcalc 16.8.4.

Comparative overview of representative visual brain PET image
A comparative overview of the representative brain PET images is presented in Fig. 2. On the 18 F-florbetaben PET image, the AD transgenic mice showed significantly higher tracer retention in the cortical regions than did the control mice. On the 18 F-flutemetamol PET image, the transgenic mice showed mild, focal uptake in cortical brain regions; however, higher uptake was shown in the transgenic mice than in the control mice. Overall, regardless of the AD transgenic and control group, 18 F-florbetaben imaging showed much higher retention than did 18 F-flutemetamol imaging. Both the AD transgenic and control groups showed high tracer retention in the cerebellum and pons than did the cortical regions.

Difference image obtained from image algebra calculation: ( 18 F-florbetaben-18 F-flutemetamol)
A visual representation of comparisons of the difference between 18 F-florbetaben and 18 F-flutemetamol is presented in Fig. 3. In the AD transgenic group, 18 F-florbetaben showed higher and more extensive cortical uptakes compared with 18 F-flutemetamol. Figure 4 provides a comparative overview of the representative whole body PET images. The color bar of the PET image was adjusted to (0-30% ID/g) to optimize for visualization of the peripheral organ uptakes. 18 F-flutemetamol PET imaging showed much more intense uptake in the bowel and bladder than did 18 F-florbetaben.

Bio-distribution
Bio-distribution data of both 18 F-florbetaben and 18 F-flutemetamol are presented in Fig. 5. The highest radioactivity in the brain was measured in the cortex, followed by the medulla and cerebellum. 18 F-florbetaben (Rt. cortex: 1.39 ID/g (%), Lt. cortex: 1.209 ID/g (%)) showed higher absolute differences between AD transgenic mice and control mice than did 18 F-flutemetamol (Rt. cortex: 0.619 ID/g (%), Lt. cortex: 0.608 ID/g (%)). In AD transgenic mouse, 18 F-florbetaben showed higher uptake in the cortex than did 18 F-flutemetamol. In addition, for both 18 F-florbetaben and 18 F-flutemetamol, the right cortex in the AD mouse showed higher uptake, showing right side laterality. In terms of the visceral distribution of 18 F-florbetaben, the highest radioactivity was measured in the liver, followed by the kidney, lung, blood and heart in both transgenic and control mice. In terms of visceral distribution of 18 F-flutemetamol, the highest radioactivity was measured in the kidney, followed by the liver, lung, blood and heart in transgenic mice. In control mice, the highest radioactivity was measured in the kidney, followed by the lung, liver, blood and heart. Comparative analysis of 18 F-florbetaben and 18 F-flutemetamol biodistribution revealed that 18 F-florbetaben imaging showed higher radioactivity in the cortex than did 18 F-flutemetamol. In contrast, 18 F-flutemetamol showed higher radioactivity in the kidney, lung, blood and heart, although the liver showed higher radioactivity with 18 F-florbetaben. In contrast with the imaging findings, the bio-distribution data showed higher uptake in the cortex than in the cerebellum.

SUVmean and SUVR based analysis of static PET image
The SUVmean and SUVR values of PET images in both the AD transgenic and control groups are presented in Tables 2, 3. The mean SUVmean values of the 18 F-florbetaben images in the AD and control mice were 0.804 and 0.699, respectively. In contrast, the mean SUVmean values of the 18 F-flutemetamol images in the AD and control mice were 0.332 and 0.297, respectively. The mean SUVR values of the 18 F-florbetaben images in the AD and control mice were 0.926 and 0.829, respectively. In contrast, the mean SUVR values of the 18 F-flutemetamol images in the AD and control mice were 0.854 and 0.687, respectively. On both the 18 F-florbetaben and 18 F-flutemetamol scans, the mean SUVmean and SUVR values of the AD transgenic group showed higher values than those of the control group. The mean SUVmean and SUVR of 18 F-florbetaben showed higher values than those of 18 F-flutemetamol in the AD transgenic and control groups, respectively. The mean of the differences in the SUVmean between the AD and control group was 0.106 for 18 F-florbetaben and 0.03 for 18 F-flutemetamol.

Statistical analysis of static PET data: AD vs. control
The quantitative parameters of the static PET images (SUVR) in the AD transgenic and control groups were tested. On the 18 F-florbetaben images, the AD transgenic group showed significantly higher SUVR values (p = 0.011) than did the control group. On the 18 F-flutemetamol images, the AD group showed significantly higher SUVR values (p = 0.001) than did the control group. Moreover, on the 18 F-flutemetamol images, the AD group showed significantly higher SUVR values than did the control group in all brain areas. However, on the 18 F-florbetaben images, the AD group showed significantly higher SUVR values than did the control group only in the cortex.

F-flutemetamol
The quantitative parameters of the static PET images (SUVR) between the 18 F-florbetaben and 18 F-flutemetamol groups are presented in Table 4. The significant differences of the SUVR (cortex/cerebellum) between the scans of the two tracers in each AD and control group were compared. 18 F-florbetaben presented a higher   SUVR value in the cortex than did 18 F-flutemetamol in both the AD (p = 0.049) and control groups (p = 0.017).

Quantitative compartment model dynamic analysis of 18 F-florbetaben and 18 F-flutemetamol image Statistical analysis of dynamic PET data: AD versus. control
In the 18 F-florbetaben group, there was a significant difference in the K1 (p = 0.011) and k4 (p = 0.017) parameters between the AD transgenic and control groups. However, in the 18 F-flutemetamol group, there was no significant difference in K1, k2, k3, k4, K1/k2, or k3/k4 between the AD transgenic and control groups.

Difference in the time-activity curve between 18 F-florbetaben and 18 F-flutemetamol
Dynamic PET time activity curves of the cortex-VOI and cerebellum-VOI for the two tracer images in representative AD and control mice are illustrated in Fig. 6. Visual inspection of the time-activity curves revealed that 18 F-florbetaben showed higher initial uptake and later retention than did 18 F-flutemetamol. In contrast, 18 F-flutemetamol showed lower initial upstroke and faster washout than did 18 F-florbetaben.

Neuropathologic findings (at 18 weeks) Hematoxylin and eosin (H & E) staining
In Fig. 7, the AD mice show a more immature pattern as a result of disarrangement of hippocampal cell migration  (pathogenic sign of AD) along the dentate gyrus of the hippocampus compared with the wild type.

Thioflavin S staining image
On thioflavin S staining images, Aβ deposits were found broadly in various brain regions including the cortex, hippocampus and thalamus in AD mice. Thioflavin S positive plaque areas predominantly diffuse in a morphologic characteristic nature rather than in a compact nature (Fig. 8).

Immunohistochemistry for Aβ 40 staining
The results of immunohistochemistry for Aβ 40 staining in AD transgenic and wild type were represented in Figs. 9, 10. In wild type mouse, there was no Aβ 40 expression in the cortex and hippocampus. In contrast, the Aa 40 expression of AD transgenic mouse significantly increased, correlating our H & E staining findings.

Correlation with neuropathologic findings and visual PET image
Finally, as shown in Fig. 11, the 18 F-florbetaben PET images more closely correlated with the thioflavin S staining image in terms of spatial distribution pattern. However, the 18 F-flutemetamol images revealed less prominent signal intensity and poor correlation of spatial distribution with neuropathologic plaque distribution shown in thioflavin S staining images.

Follow-up neuropathologic findings (at 48 weeks)
The results of the follow-up immunohistochemistry for Ai 40

Discussion
In this study, 18 F-florbetaben and 18 F-flutemetamol images could differentiate AD and control group on visual and SUVR analysis. The 18 F-florbetaben group presented differences in K1 and k4 kinetic parameters between AD and control groups, although 18 F-flutemetamol did not show difference. Several differences emerged between 18 F-florbetaben and 18 F-flutemetamol. 18 F-florbetaben images showed more prominent visual uptake intensity and higher SUVR than the 18 F-flutemetamol images did. Moreover, 18 F-florbetaben PET images more correlated well with the thioflavin S staining. However, according  to bio-distribution and kinetic results, 18 F-flutemetamol is more actively metabolized than is 18 F-florbetaben, suggesting that 18 F-flutemetamol has faster transport from arterial plasma into the first tissue compartment and faster dissociation from the amyloid tracer complex.
In the static analysis data, the results were grossly consistent with a previous study [12]. In another 18 F-florbetaben PET study, the SUVR in APPswe/PS2 at 5 months was 0.95 ± 0.04, and the SUVR in APPswe/PS1G384A mice at 5 months was 0.93 [12]. The traditional SUVR method measures the radioactivity ratio of brain target   8 Visual overview of the thioflavin S staining images of a wild type mouse and b AD transgenic mouse. Left column shows DAPI (blue channel), middle column shows thioflavin S (green channel with specific staining signal) and right column shows merged image. Aβ deposits were found broadly in various brain regions including the cortex, hippocampus and thalamus in AD mice regions to reference tissue during a fixed time interval after injection of the tracer [11]. This relative quantitative approach for static PET data is practical for routine clinical setting. However, due to the kinetic compartment model for reversible binding radiotracers such as 18 F-florbetaben or 18 F-flutemetamol, the kinetic model reflects the available binding site density and also the perfusion signal and tracer clearance to and from brain tissue [11]. In this study, the 2 tissue compartment model with IDIF method was used, and the IDIF appears to be an attractive non-invasive alternative option obviating the need for arterial cannulation, blood handling and analysis [13][14][15]. Furthermore, to avoid the effects of non-specific binding, we prolonged the uptake time, resulting in a longer wash-out of non-specifically bound tracer. A clinical protocol for 18 F-florbetaben involves a 90 min uptake periods [14]. A similar protocol was used in the previous APPPS1-21 mouse cohort study, allowing a 90-min uptake time [15].
The reasons for the disparity in imaging characteristics between 18 F-florbetaben and 18 F-flutemetamol are related to their chemico-physiological properties.   40 (RFP red channel with specific staining signal) and Right column shows merged image. In wild type mouse, there was no Aβ 40 expression in the cortex. In contrast, the Aa40 expression of AD transgenic mouse significantly increased F-florbetaben and 18 F-flutemetamol belong to different families of imaging probes. 18 F-flutemetamol is a member of the thioflavin derivatives imaging probe family [16], and 18 F-florbetaben belongs to a different branch of imaging probe family, the trans-stilbene derivatives [16]. Because these two tracers belong to distinct chemical families, they showed differences in binding affinity. In the bio-distribution data, 18 F-flutemetamol showed lower brain and higher peripheral organ uptake responsible for metabolite excretion compared with 18 F-florbetaben. These findings and kinetic parameter results suggest that 18 F-flutemetamol is more actively metabolized than is 18 F-florbetaben. The tracer metabolites were more polar than were the parent molecules and therefore less able to enter the brain [17]. In a preclinical study comparing the pharmacokinetic characteristics of 18 F-flutemetamol with that of 11 C-PiB, the metabolism of 18 F-flutemetamol was faster than that of 11 C-PiB [18]. This finding can be explained by the higher lipophilicity of 18 F-flutemetamol (logPC18 = 1.7) than that of 11 C-PiB (logPC18 = 1.2) [19]. In another study, the lipophilicity of 18 F-florbetaben (Log Doct/PBS = 1.58) was higher than that of 11 C-PiB (Log Doct/PBS = 1.50) [20]. These results indirectly demonstrate that the rapid metabolism of 18 F-flutemetamol could be explained by the higher lipophilicity of Previous studies reported that various transgenic animal models showed differences in binding affinity with imaging tracers and this phenomenon was thought to be related with variations in plaques configurations. In this study, 18-week-old AD transgenic mice carrying NSE-controlled APPswe, C57BL/6-Tg (NSE-hAPPsw) Korl were selected due to their rapid and robust amyloid plaque development at that early age [21]. In contrast, Tg2576 mice showed late onset and slower accumulation [5]. In another previous report using APPPS1 mice coexpressing L166P mutated Presenilin 1 under the control of a neuron-specific Thy1 promoter and KM670/671NL mutated amyloid precursor protein, cortical amyloidosis was reported at the age of 6-8 weeks [22]. In APPPS1-21 mice, amyloid was known to accumulate in a 4-week and cortical microglia increased threefold from 1 to 8 months of age [22]. Hence, APPPS1 mice are good for investigating the mechanism of amyloidosis and treatment strategies because of their early onset of amyloid deposition and convenient cross-breeding with other genetically engineered mouse models.
In humans, the APPswe gene caused early presentation of familial AD [23]. In the NSE-controlled APPswe mouse model, the Swedish double mutation at the 670/671 codon in the human APP gene under the control of the NSE promoter caused increased cleavage by the beta secretase and accelerated amyloid accumulation at young age [24]. Amyloid deposition in the NSEcontrolled APPswe mouse induces subsequent neuronal apoptosis through the mechanisms of the mitogenactivated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathway or caspase-3 pathway [25,26]. Although the mouse model in our study was relatively young, compelling evidence from previous studies regarding the dynamics of cerebral amyloidosis using a young APP mouse model indicates that NSE-augmented APPswe mice are suitable for the neuropathological phenotype of AD [27]. Moreover, we selected only male mice to control the effect of sex. Several studies reported the effect of sex on β-amyloid accumulation and AD phenotype. Latest studies have investigated the effects of sex on hippocampal atrophy in normal aging, MCI and AD [28]. Sex could regulate the relation of amyloid positivity and cognition [28]. Also, significant sex differences in pathology of 3xTg-AD mice suggested these differences may be due to organizational actions of sex hormones during development [29].
The results of this study are in contrary to those of antecedent 18 F-FDDNP study that presented affinity for both amyloid and neurofibrillary tangles [30]. There was no increase in cortical uptake even in 13-15-month-old Tg2576 mice, even if technical issues, such as low spatial resolution, were regarded as the reasons for this negative PET finding [30]. However, in another 18 F-FDDNP study in triple-transgenic rats, previous partial volume effects were overcome and contrasting results were observed; prominent uptake was presented in the frontal cortex and hippocampus [31]. In an 11 C-PiB study, old Tg2576 mice showed prominent cortical binding than did control mice [9]. Those paradoxical results were explained by the confounding effects of cortical perfusion and the low distribution of 11 C-PiB binding sites per plaque [32]. However, following the 11 C-PiB study with high specific activity overcame such confounding effects, so significant cortical uptake and excellent correlation between PET uptake and a pathologic amyloid burden were observed in APP23 mice compared with age-matched healthy controls [33]. Additionally, in a recent 11 C-PiB PET study of APP/PS1 mice, an outstanding correlation can be found between imaging results and the plaque burden measure obtained ex vivo and in vitro in the same animals [34]. In a previous preclinical imaging study comparing 18 F-florbetaben and 11 C-PiB, which is of the same thioflavin T derivative family with 18 F-flutemetamol, two aged AD mouse models with contrasting levels of amyloid deposition to high (APPPS 1-21) and low (BRI 1-42) target state were investigated [15]. Compared with control mice, APPPS 1-21 mice (high target state) presented prominent Fig. 13 Comparison of the Aβ 40 staining images of AD mouse in dentate gyrus of hippocampus between a 18 week and b 48 weeks. AD mice showed extensive and significantly increased Aβ40 expression in the hippocampus and cortex compared with the images at 18 weeks fibrillary amyloid accumulation in both 11 C-PiB and 18 F-florbetaben, but the difference of uptake between AD and control mice was higher for 11 C-PiB than for 18 F-florbetaben [15]. However, BRI1-42 mice (low target state) did not show enhanced tracer uptake [15]. Taking into consideration the difference in the mouse ages, our results broadly resemble their findings. Another 18 F-florbetaben study using the same mouse cohort reported only a 14.5% difference between control and transgenic mice (5XFAD) found with 18 F-florbetapir in comparison to a 21% difference found with 11 C-PiB [35].
Before the interpretation of 18 F-florbetaben or 18 F-flutemetamol images in clinical settings, preclinical approaches can provide baseline information regarding differences in the kinetic and metabolic properties of two tracers. The visual image and SUVR of 18 F-florbetaben showed extensive cortical uptake in the same cohorts compared with 18 F-flutemetamol images in both the AD and control groups. On the 18 F-flutemetamol images, high lipophilicity and fast metabolism might complicate the analysis of PET data. In this study, the metabolism and kinetics of the tracer also have a great influence on the visual uptake of the amyloid tracers. Using these points, the human amyloid image should be read in consideration of the pharmacokinetic and metabolic properties of the tracer. Therefore, preclinical imaging might provide valuable information about the possibilities and limits of a given approach in humans by helping to better understand the in vivo binding characteristics of an imaging agent. The results of this study suggest that appropriate outcome measures are important in monitoring disease progression and response to therapeutic approaches in human settings. In this study, both tracers for VOI-based ratio analysis could discriminate the AD transgenic and control groups. However, on kinetic parameters from dynamic data, 18 F-flutemetamol images could not be used as an indicator to distinguish between AD transgenic and control groups. Moreover, the detection of amyloid PET signal in this early aged mouse model used in this study suggests the sensitivity of the PET imaging bio-marker, suggesting the possibility of early detection of amyloid pathology before the manifestation of behavioral abnormalities.
There are several limitations that should be mentioned. First, the distribution patterns between 18 F-florbetaben and 18 F-flutemetamol were compared at a single time point. Therefore, the current data are insufficient to judge the superiority between the two tracers based. In a follow-up study, the scope of the analysis should be extended to cover the comparison of serial and chronological accumulation pattern between 18 F-florbetaben and 18 F-flutemetamol.
Second, there are some issues regarding methodological perspectives. Herein, for the shape of the merged atlas to match well with the skull CT, the thresholded CT image was manually fused with the magnetic resonance template. However, limitations could exist regarding the method of manual registration. More accurate, automatic algorithm is required in the further study. Additionally, partial volume correction was not conducted when defining the VOI in the blood input area, because the VOI size of the blood input area was larger than the volumetric PET spatial resolution (0.343 mm 3 ). Therefore, the effect of the partial-volume correction should be investigated in a further study.
Moreover, in further studies, the different amyloid isoform structures and the range of fibrillarity influencing PET imaging results should be investigated. Amyloid plaques can be sub-classified according to the presence of dystrophic neuritis or reactive astrocytes and the morphological features as either diffuse, fibrillary or dense core types [36,37]. In the thioflavin S image in our study, the thioflavin S positive plaque areas were predominantly diffuse rather than compact in terms of morphologic characteristic nature. Diffuse plaques are known to occur early in the disease course and to progress towards typical cored plaques [27,38]. Dense-core plaques are often observed in AD mouse models, at an advanced age [39]. Morphological and biochemical compositional differences of plaques can influence the affinity binding sites for amyloid imaging tracers. Between 18 F-florbetaben and 18 F-flutemetamol, which tracer has higher binding affinity to diffuse type plaques? The answer to this question should be investigated in a further study including an in vitro binding assay. In addition, as plaques are amorphous three-dimensional configurations, further three-dimensional analysis of plaque structures with more precise detection stringency should be required.
Moreover, we have not performed Aβ 1-42 staining along with the Aβ 1-40 staining, in the follow-up of immunohistochemistry, because we simply wanted to demonstrate the establishment of AD mouse model and regarded Aβ 1-40 was better choice. Aβ 1-40 presents the most prominent Aβ isoform in the AD brain, while the Aβ 1-42 shows a substantial increase with specific forms of AD [40,41]. Moreover, extraordinary expressions in AD mice carrying NSE-controlled APPsw presented that Aβ 1-40 was more prominent than Aβ 1-42 in the APPsw mice [42]. 18 F-florbetaben and 18 F-flutemetamol images could distinguish between the AD and control group by both visual and SUVR-based analysis. The 18 F-florbetaben and 18 F-flutemetamol images showed disparate character in aspects of visual uptake intensity, quantitative parameters, bio-distribution and relations with neuropathological finding. 18 F-flutemetamol was more actively metabolized than was 18 F-florbetaben, although 18 F-florbetaben presented higher visual uptake intensity, SUVR and close correlation with the pathology.