[18F]RoSMA-18-d6 was synthesized by nucleophilic substitution of the tosylate precursor with [18F]KF/Kryptofix222 in acetonitrile . The crude product was purified by reverse phase semi-preparative high-performance liquid chromatography and formulated with 5 % ethanol in water for intravenous injection and for biological evaluations. In a typical experiment, a moderate radiochemical yield of ~ 12 % (decay corrected) was achieved with a radiochemical purity > 99 %. The molar activities ranged from 156 to 194 GBq/μmol at the end of synthesis. The identity of the final product was confirmed by comparison with the HPLC retention time of the non-radioactive reference compound by co-injection. [18F]FDG was obtained from a routine clinical production from the University Hospital Zurich, Switzerland.
Twenty-four male C57BL/6J mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France). The mice were scanned at 8–10 weeks of age (20–25 g body weight). Mice were randomly allocated to sham-operation (n = 10) or tMCAO (n = 14). Mice underwent MRI, µPET/ computed tomography (CT), and 2,3,5-Triphenyltetrazolium chloride (TTC) histology staining for validation 24 h or 48 h after reperfusion. Animals were housed in ventilated cages inside a temperature-controlled room, under a 12-hour dark/light cycle. Pelleted food (3437PXL15, CARGILL) and water were provided ad-libitum. Paper tissue and red Tecniplast mouse house® (Tecniplast, Milan, Italy) shelters were placed in cages as environmental enrichments. All experiments were performed in accordance with the Swiss Federal Act on Animal Protection and were approved by the Cantonal Veterinary Office Zurich (permit number: ZH018/14 and ZH264/16).
Surgeries for tMCAO and sham-operation were performed using standard-operating procedures as described before [67, 68]. Anaesthesia was initiated by using 3 % isoflurane (Abbott, Cham, Switzerland) in a 1:4 oxygen/air mixture, and maintained at 2 %. Before the surgical procedure, a local analgesic (Lidocaine, 0.5 %, 7 mg/kg, Sintectica S.A., Switzerland) was administered subcutaneously (s.c.). Temperature was kept constant at 36.5 ± 0.5 °C with a feedback controlled warming pad system. All surgical procedures were performed in 15-30 min. After surgery, buprenorphine was administered as s.c. injection (Temgesic, 0.1 mg/kg b.w.), and at 4 h after reperfusion and supplied thereafter via drinking water (1 mL/32 mL of drinking water) until 24 h or 48 h. Animals received softened chow in a weighing boat on the cage floor to encourage eating. tMCAO animals were excluded from the study if they met one of the following criteria: Bederson testing was performed 2h post-reperfusion. Bederson score of 0, no reflow after filament removal, and premature death.
mRNA isolation, reverse-transcription reaction and real-time polymerase chain reaction
Brain hemispheres of C57BL/6 mouse, tMCAO mice at 24 h and 48 h post reperfusion were used for total mRNA isolation according to the protocols of the Isol-RNA Lysis Reagent (5 PRIME, Gaithersburg, USA) and the bead-milling TissueLyser system (Qiagen, Hilden, Germany). QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany) was used to generate cDNA. The primers (Microsynth, Balgach, Switzerland) used for the quantitative polymerase chain reaction (qPCR) are summarized in Supplementary Table 1. Quantitation of CNR2, Iba1, TNF-a, MMP9, GFAP and MAP-2 mRNA expression was performed with the DyNAmo™ Flash SYBR® Green qPCR Kit (Thermo Scientific, Runcorn, UK) using a 7900 HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, USA). The amplification signals were detected in real-time, which permitted accurate quantification of the amounts of the initial RNA template during 40 cycles according to the manufacturer's protocol. All reactions were performed in duplicates and in two independent runs. Quantitative analysis was performed using the SDS Software (v2.4) and a previously described 2−ΔΔCt quantification method . The specificity of the PCR products of each run was determined and verified with the SDS dissociation curve analysis feature.
In vivo MRI
Data were acquired at 24 h after reperfusion on a 7 T Bruker Pharmascan (Bruker BioSpin GmbH, Germany), equipped with a volume resonator operating in quadrature mode for excitation and a four element phased-array surface coil for signal reception and operated by Paravision 6.0 (Bruker BioSpin) [67, 70-72]. Mice were anesthetized with an initial dose of 4 % isoflurane in oxygen/air (200:800 ml/min) and maintained at 1.5 % isoflurane in oxygen/air (100:400 ml/min). Body temperature was monitored with a rectal temperature probe (MLT415, ADInstruments) and kept at 36.5 °C ± 0.5 °C using a warm water circuit integrated into the animal support (Bruker BioSpin GmbH, Germany). T2-weighted MR images were obtained using a spin echo sequence (TurboRARE) with an echo time 3 ms, repetition time 6 ms, 100 averages, slice thickness 1 mm, field-of-view 2.56 cm × 1.28 cm, matrix size 256 × 128, giving an in-plane resolution of 100 μm × 100 μm. For DWI, a four-shot spin echo–echo planar imaging sequence with an echo time = 28 ms, repetition time = 3000 [70, 71] acquired with a field-of-view of 3.3 cm × 2 cm and a matrix size of 128 × 128, resulting in a nominal voxel size of 258 μm × 156 μm. Diffusion-encoding was applied in the x-, y-, and z-directions with b-values of 100, 200, 400, 600, 800, and 1000 s/mm2, respectively, acquisition time 3 min 48 s. The ischemic lesion was determined as an area of significant reduction of the apparent diffusion coefficient (ADC) value compared with the unaffected contralateral side . On T2-weighted images, the lesion was determined as an area of hyperintensities compared with the contralateral side.
In vivo microPET studies
MicroPET/CT scans were performed at 24 h after reperfusion with a calibrated SuperArgus µPET/CT scanner (Sedecal, Madrid, Spain) with an axial field-of-view of 4.8 cm and a spatial resolution of 1.6–1.7 mm (full width at half maximum). tMCAO and the sham-operated C57BL/6J mice were anesthetized with ca. 2.5 % isoflurane in oxygen/air (1:1) during tracer injection and the whole scan time period. The formulated radioligand solution ([18F]FDG: 9.9-11 MBq or [18F]RoSMA-18-d6: 7.2-13 MBq) was administered via tail vein injection, and mice were dynamically scanned for 60 min. For blocking experiments, 1.5 mg/kg GW405833 was dissolved in a vehicle of 2 % Cremophor (v/v), 10 % ethanol (v/v), and 88 % water for injection (v/v) and injected together with [18F]RoSMA-18-d6. Body temperature was monitored by a rectal probe and kept at 37 °C by a heated air stream (37 °C). The anesthesia depth was measured by the respiratory frequency (SA Instruments, Inc., Stony Brook, USA). µPET acquisitions were combined with CT for anatomical orientation and attenuation correction. The obtained data were reconstructed in user-defined time frames with a voxel size of 0.3875 × 0.3875 × 0.775 mm3 as previously described .
Triphenyltetrazolium chloride (TTC) staining
To assess the ischemic lesion severity in the brain of tMCAO mice and to validate the absence of lesion in the sham-operated mice, staining with TTC staining was performed. After measurements mice were euthanized, their brains were removed and 1-mm thick brain slices were obtained with a brain matrix. Slices were incubated in a 2.5 % TTC solution (Sigma-Aldrich, Switzerland) in PBS at 37 °C for 3 min. Photographs of the brain sections were taken. Edema-corrected lesion volumes were quantified as described .
Biodistribution studies in the mouse brain
After PET/CT scanning of tMCAO mice at 24 h after reperfusion with [18F]RoSMA-18-d6, animals were sacrificed at 70 min post injection by decapitation. The spleen and brain regions of ischemic ipsilateral area and contralateral hemisphere were collected for analysis with a gamma counter. The accumulated radioactivities in the different tissues were expressed as percent normalized injected dose per gram of tissue normalized to 20 g body weight of the animals (norm. percentage injected dose per gram tissue (% ID/g tissue)).
Data analysis and Statistics
Images were processed and analyzed using PMOD 4.2 software (PMOD Technologies Ltd., Zurich, Switzerland). The time−activity curves were deduced from specific volume-of-interest that were defined based on a mouse MRI T2-weighted image template . Radioactivity is presented as standardized uptake value (SUV) (decay-corrected radioactivity per cm3 divided by the injected dose per gram body weight). [18F]RoSMA-18-d6 SUVR was calculated by using the midbrain in the corresponding hemisphere as reference brain region. For [18F]FDG PET, regional SUV was calculated. Two-way ANOVA with Sidak post-hoc analysis was used for comparison between groups (Graphpad Prism 9.0, CA, U.S.A).