Animal models and experimental scheme
All animal experiments in this study were performed under protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). The double transgenic APPSWE/PS1dE9 (APP/PS1) mice expressing Mo/HuAPP695swe (chimeric mouse and human amyloid precursor protein) and PS1-dE9 (mutant human presenilin 1), and the littermate control C57Bl/6J (WT; wild type) were purchased from Mutant Mouse Resource and Research Center (MMRRC). A total of 91 mice including 41 APP/PS1 (5 mo.: 6 female and 9 male; 10 mo.: 4 female and 4 male; 16 mo.: 6 female and 12 male) and 50 WT (5 mo.: 9 female and 9 male; 10 mo.: 6 female and 6 male; 16 mo.: 10 female and 10 male) were used in this study. The general experimental scheme of this study is shown in Additional file 1: Fig. S1. Briefly, animals selected from each age group received 7.4-11.1 MBq of [18F]ROStrace via tail vein injection. After a 20 mins (40-60 min post-injection) or a one hour (0-60 min post-injection) dynamic PET scan, a dose of 5 mg/kg DHE (Sigma-Aldrich, St. Louis, MO, USA) was injected to the animals. The animals were sacrificed 30 mins after DHE injection and brains were extracted for subsequent experiments.
Preparation of [18F]ROStrace
The radiosynthesis of [18F]ROStrace was prepared as previously described [34]. Briefly, [18F]ROStrace was accomplished on all-in-One module (Trasis, Belgium) with full automation. The final product was diluted with 0.6 mL of ethanol, 0.1% ascorbic acid and 6 mL normal saline, and filtered by 0.2 µM nylon filter. The radiochemical yield was 4~20%, and the specific activity was 74 GBq/mmol.
Micro-PET imaging
PET imaging was performed on the b-Cube PET scanner (Molecubes, Ghent, Belgium). The body weight of each animal was measured before the PET scan (Fig. 1a). Animals were anesthetized with 1-2% isoflurane, a tail vein catheter was placed for PET tracer administration, and the animal was placed on the scanner bed. Dynamic scans of 0-60 min or 40-60 min post-injection were acquired after injection of 7.4-11.1 MBq of [18F]ROStrace. After completion of the PET scan, the animal was transferred to the X-Cube CT scanner (Molecubes, Ghent, Belgium) and a general-purpose CT scan was acquired for anatomical reference and attenuation correction. PET images were reconstructed with a matrix size of 192 x 192 x 384, and a voxel size of 0.4 x 0.4 x 0.4 mm with frame lengths of 6 x 10 sec, 9 x 60 sec, and 10 x 300 sec for 60 minutes dynamic scans or 4 x 300 sec for 20 minutes scans. All corrections were applied using a manufacturer supplied reconstruction program. CT images were reconstructed with a matrix size of 200 x 200 x 550, and a voxel size of 0.2 x 0.2 x 0.2 mm with a manufacturer supplied reconstruction program.
Micro-PET image analysis
All the [18F]ROStrace micro-PET/CT imaging data were processed and analyzed by using Pmod software (version 3.7, PMOD Technologies Ltd., Zurich, Switzerland). Rigid body matching was manually performed on individual micro-CT images to co-register to the Mirrione mouse MR-T2 weighted brain template [73]. Then, the resulting transformation parameters were applied to the corresponding micro-PET image. Ten volumes of interest (VOIs) including cortex, thalamus, cerebellum, hypothalamus, brain stem, periaqueductal gray, striatum, hippocampus, amygdala, and midbrain were selected from the Mirrione atlas [73]. Due to the lack of the blood samples to perform absolute quantitation and a true reference region to semi-quantitate neuroinflammation images, a pseudo-reference region [44] is needed for [18F]ROStrace neuroinflammation imaging evaluation. Therefore, the periaqueductal gray, with no significant difference in standardized uptake values (SUVs; Fig 1b) among the different animal groups, was selected as the pseudo-reference region for calculating the SUV ratio (SUVR) for each VOI. The 1-3 min SUVR and the 40-60 min SUVR (SUVR40-60) were extracted from all the VOIs for the perfusion and the late phase comparison from the nineteen 16 mo. old animals (5 WT female, 5 WT male, 4 APP/PS1 female, and 5 APP/PS1 male) with 1 hour full dynamic [18F]ROStrace imaging.
Voxel-wise analyses were performed by using Statistical Parametric Mapping 12 (SPM12, Welcome Department of Cognitive Neurology, Institute of Neurology, London, UK; https://www.fil.ion.ucl.ac.uk/spm) and carried out with the SPMMouse (http://www.spmmouse.org/) [74] animal brain toolbox implemented in MATLAB R2017b (MathWorks Inc., Natick, MA). A voxel-wise two-sample t test was used to compare the [18F]ROStrace SUVR40-60 parametric images between WT and APP/PS1 in each age group (5, 10, and 16 mo.) for both female and male. Due to the small sample size of the studies, the SPMMouse analyses were evaluated by using a relative stringent threshold of a p value < 0.005 with uncorrected statistic and a voxel extent of 50.
Ex vivo autoradiography (ARG)
Mouse brains were collected immediately after [18F]ROStrace micro-PET scan completion. The mouse brains were frozen and sectioned sagittally with a thickness of 30 µm on a cryostat (Leica Biosystems, Germany), then the tissue slides were air dried for next step. The slides were exposed to phosphor plates (BAS 2040, GE Healthcare, Chicago, IL, USA) for 1 day and the ARG images were digitized by the Typhoon FLA 7000 (West Avenue, Stamford, USA). The ARG images were analyzed by using Pmod software. The ROI of periaqueductal gray was manually delineated for further calculating the ratio of images relative to it.
Immunohistochemical (IHC) analysis
Collected brain tissues were embedded with Tissue-Tek optimal cutting temperature (Sakura, Japan) and frozen, then cut into 12 µm sagittal sections. The brain sections were fixed with 4% paraformaldehyde (PFA) at room temperature before staining, and then rinsed with phosphate buffered saline (PBS). Then, 15 mins of 0.1% Triton X-100 in PBS was applied to permeabilized tissues. After rinse with PBS, brain sections were blocked with 10% bovine serum albumin (BSA) for 1 hour at room temperature. Selected slides were incubated with primary antibodies (Additional file 1: Table S2) overnight at 4˚C. The following day, after wash with 1% BSA in PBS, slides were incubated with corresponding secondary antibodies (Additional file 1: Table S2) for 1 h at room temperature. All slides were counterstained with Hoechst (1:2000, PBS, Thermo Fisher Scientific, H3570) and mounted with VectaShield Antifade Mounting Media (Vector Laboratories, 101098-042). Images were acquired by using Zeiss Axio Observer Microscope and Zeiss 710 Confocal Microscope (Germany). Panoramic brain tissue images were acquired by Keyence BZ-X800 Microscope (Osaka, Japan). For cortex amyloid burden and 3NT-positive cells quantification, images were uploaded onto Fiji software, amyloid plaques and 3NT-positive cells within the ROI (1.73mm x 0.65 mm) were counted manually by using the cell counter plugin, and quantification results were scaled to n/mm2. The average plaque sizes of each Aβ plaque in brain was measured by Zen software (Zeiss, Germany).
Statistical analysis
All the statistical analyses were performed on GraphPad Prism software, version 7.02 (GraphPad Inc., San Diego, CA), and the results were presented as mean ± standard error (SEM) or mean ± standard deviation (SD) as noted. The statistical significance between different animal groups was determined either with one-way or two-way ANOVA following Sidak’s or Turkey post hoc t test. A p value < 0.05 was considered as the threshold of statistically significant.