Animals and study design
All experiments were carried out in compliance with the German national guidelines for animal protection (TierSchG, Germany) and with approval of the local animal care committee (Regierung von Oberbayern), under supervision by a veterinarian. Experiments, analyses, and reporting were performed in accordance with the ARRIVE guidelines [15]. Animals were housed in a temperature- and humidity-controlled environment with a 12-h light–dark cycle, with free access to food (Ssniff, Soest, Germany) and water. Sample size calculation (G*Power, V3.1.9.2, University of Kiel, Germany) was based on earlier FDG-PET estimates in mice and assuming a type I error α = 0.05, a power of 0.8, and a dropout rate of 15% during follow-up. Assuming a relevant treatment effect of 5%, we calculated a group size of six, including one mouse for dropout compensation.
Twelve hTau mice and ten controls (all female) were purchased from Jackson Laboratories (JAX®) at 3 months of age and housed in our pathogen-free animal facility until attaining 14.5 months of age. We then undertook baseline FDG-PET imaging and randomized the mice into Anle138b treatment and vehicle treatment groups (n = 6/5 each). hTau mice had been designed to express all six isoforms of human tau in similar ratios to that in the diseased human brain, but do not express significant amount of mouse tau [14]. The model was generated by crossing 8c mice that express a tau transgene derived from a human P1-derived artificial chromosome, H1 haplotype [16] together with tau knock-out mice that have a targeted disruption of exon one of the tau gene [17].
The hTau mice that we used are homozygous for Mapt <tm1(EGFP)Klt> and heterozygous for Tg (NAPT)8cPdav, whereas controls only contain the homozygous Mapt <tm1(EGFP)Klt> mutation. Anle138b treatment was administered for 3 months, and follow-up PET imaging was performed in the last week of treatment. After a visual quality check of PET images, we performed transcardial perfusion with fixation in 4% paraformaldehyde and brain extraction for immunohistochemical analyses.
Anle138b treatment
We used the oligomer modulator Anle138b ([3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole]) [6] as a therapeutic against tau deposition. The compound was formulated in food pellets (Ssniff, Soest, Germany) at a concentration of 2 g/kg pellets [7], which were administered ad libitum over a period of 3 months. The food composition was based on regular maintenance diet (16.2 MJ/kg; 9% fat, 24% protein, 67% carbohydrates). Detailed specifications of the Anle138b formulation can be found in supplement #2 of [6]. In brief, (1-(1,3-benzodioxol-5-yl)-3-(3-bromophenyl)propane-1,3-dione), a previously reported pyrazole title compound [18], was used as a precursor for the chemical synthesis of Anle138b in several steps. Control mice were fed with unmodified food pellets.
PET imaging
PET data acquisition and analyses
All PET procedures followed standardized, established protocols [19]. In brief, mice were anesthetized with isoflurane (1.5%, delivered at 3.5 l/min) and placed in the aperture of the Siemens Inveon DPET [20] as described previously [21]. Mice had been fasted for at least 3 hours prior to tracer administration. Static FDG-PET imaging from 30 to 60 min p.i. was performed after administration of 13.2 ± 2.1 MBq 18F-FDG as previously established [19]. The emission recording was followed by a 15-min transmission scan using rotating 57Co point sources. The image was reconstructed as a single 30-min frame in 4 OSEM3D and 32 MAP 3D iterations, giving a target resolution of 1.0 mm and a zoom factor of 1.0, with scatter-, attenuation-, and decay-correction, resulting in a final voxel dimension of 0.78 × 0.78 × 0.80 mm. Following recovery from anesthesia, mice were returned to their home cages.
PET post-processing
FDG-PET images (30–60 min) were co-registered to an MRI mouse brain atlas [22] by a manual rigid-body transformation (TXrigid) using the PMOD fusion tool (V3.5, PMOD Technologies Ltd.). A reader who was blind to the type of mouse confirmed the initial registration. Then, we applied a reader-independent co-registration by generating treatment group-specific standard FDG-PET templates in the MRI atlas space. Non-linear brain normalization was performed with the PMOD fusion tool for each individual co-registered image to obtain a transformation matrix (TXBrainNorm) for each mouse brain to the template. The manual (TXrigid) and automatic (TXBrainNorm) transformations were concatenated and applied to the native space μPET data to guarantee a minimum of interpolation. As the μPET templates had been initially aligned to the atlas, all final fused μPET images had the same spatial orientation and voxel dimensions as the MRI mouse brain atlas, i.e., 0.064 × 0.064 × 0.064 mm.
PET analysis
An oval-shaped frontal cortical volume of interest (VOI; 28 mm3) and a bilateral circular-shaped hippocampal VOI (11 mm3) defined in the MRI atlas were placed on the resampled image to calculate the mean radioactivity concentrations in standardized uptake value (SUV) units. Absence of tau pathology in the brainstem of hTau mice has been shown earlier [14], justifying its use as a reference tissue in this mouse model. We calculated the SUV ratios (SUVRBST) as the ratio of the radioactivity concentrations in target and brainstem reference VOIs of the Mirrione atlas implemented in PMOD [23]. Longitudinal changes were computed for each mouse as the absolute difference of SUVR values between baseline and follow-up scans (ΔSUVR).
Immunohistochemical analyses
After removal from the skull, brains were bisected at the midline and one cerebral hemisphere randomly selected for immunohistochemical analysis after fixation by immersion in 4% paraformaldehyde at 4 °C for 24 h. A mean of three representative 50-μm-thick slices per animal was then cut in the sagittal plane between 1.5 and 2.0 mm from the midline using a vibratome (VT 1000 S, Leica, Wetzlar, Germany). Free-floating sections were permeabilized with 2% Triton X-100 overnight and then blocked with I-Block™ Protein-Based Blocking Reagent (Thermo Fischer Scientific). We obtained immunohistochemical labelling of hTau using the CP13 primary antibody (dilution; 1:25, 48 h, RT), which binds specifically to phosphorylated serine 202 (pS202) on tau, followed by incubation with the A-21244 secondary antibody, which contains Alexa Fluor 647 dye (Invitrogen, 1:500) [16, 24]. The unbound dye was removed by three washing steps with PBS, and the slices were then mounted on microscope slides with fluorescent mounting medium (Dako, Germany). Images were acquired with an inverted confocal Laser-Scanning Microscope LSM510=NLO (Zeiss). We imaged the frontal cortex and hippocampus of each slice three-dimensionally in tile scan mode, which allows automatic stitching of an array of fields of view. Tau load (%) in maximum intensity projected image stacks was calculated as the summed area of all tau-positive cells identified using an automated intensity threshold relative to the total inspected area in ImageJ software (Wayne Rasband, (NIH)). Second, we calculated the number of CP13-positive neurons per area (N/mm2) in the cortex and the hippocampus. The operator was blind to the PET results.
Statistics
Statistics were performed using SPSS (V25, IBM Cooperation) and GraphPad Prism (V5.03). FDG-PET measures of baseline, follow-up assessment, and longitudinal changes, as well as tau burden (%), the number of tau-positive neurons per area and body weight, were compared between the three groups of treated hTau mice, vehicle hTau mice, and non-carrier controls by a one-way ANOVA including Tukey post hoc correction for multiple comparisons. Longitudinal FDG-PET measures in the treatment group were also tested voxel-wise (paired t test) by a statistical parametric mapping approach [25] to allow a region-independent evaluation of metabolic rescue. Body weight was compared by an unpaired Student’s t test. For inter-modality correlation analyses, Pearson coefficients of correlation (R) were calculated between region-specific tau area (%) values and PET read-outs (SUVR, ΔSUVR). A Shapiro–Wilk test was performed to verify normal distribution of sample values (results are provided in Additional file 1). In all tests, a threshold of p < 0.05 was considered to be significant for rejection of the null hypothesis.