Animals and peripheral pseudoinfection generation
Forty-six male Sprague-Dawley rats (6 weeks old) were purchased from Japan SLC (Hamamatsu, Japan). The rats were housed in a temperature- (23 ± 1°C), humidity- (60 ± 5%), and light- (lights on at 8:00 and off at 20:00) controlled environment. A standard laboratory diet and tap water were available ad libitum. For acclimation, rats were housed in the experimental room for at least 1 week before the week-long pre-level measurement of spontaneous activity, and randomly divided into saline- (control) and poly I:C-treated groups. A pseudo-viral infection in rats (8 weeks old) was induced by intraperitoneal injection of poly I:C (GE Healthcare Life Science, Buckinghamshire, UK), a synthetic double-stranded RNA, dissolved in saline at a dose of 10 mg/kg body weight [21, 22]. In the control group, rats were injected with saline at analogous procedure. The injection was performed between 10:00 and 11:00 in the morning. The experimental procedures in the present study were approved by the Institutional Animal Care and Use Committee of RIKEN, Kobe Branch, and were performed in accordance with the Guide for the care and use of laboratory animals (NIH publication No. 85-23, revised 2011).
Measurement of spontaneous activity
To quantitatively evaluate fatigue state, the spontaneous activity of each rat was recorded with an infrared beam sensor (NS-AS01; Neuroscience Inc., Tokyo, Japan) prior to and following a poly I:C injection. The infrared beam sensor was placed 15 cm above the center of each cage, and the activities of rats housed in individual cages were measured. The level of night-time spontaneous activity was normalized by the mean value of the 3 days prior to poly I:C injection. The fatigue of rats was calculated by assessing night-time spontaneous activity, which was added up every 60 min and analyzed in Clock Lab (Neuroscience Inc.). In addition, the spontaneous activity in all rats used in [18F]DPA-714 PET scan was also examined separately throughout the entire experimental period for the correlation analysis.
Body temperature measurement
Body temperature of rats was monitored using an implantable programmable temperature transponder (IPTT-300, Bio Medic Data Systems, Inc., Seaford, USA), which was implanted gently into the subcutaneous tissue between the scapulae of each rat under anesthesia (with a mixture of 1.5% isoflurane and nitrous oxide/oxygen 7:3) with a syringe-like action 7 days before intraperitoneal injection of poly I:C or saline. Temporal changes in the body temperature of the rats were measured wirelessly using an IPTT reader from 0 h (before injection) to 48 h following the poly I:C or saline injection.
Besides the pre-injection levels, at 2 h, 4 h, 8 h, 24 h, and 48 h after poly I:C injection, rats were shortly anesthetized with a mixture of 1.5% isoflurane and nitrous oxide/oxygen (7:3), and blood samples were collected from an indwelling catheter in the tail vein implanted prior to sampling. Venous blood was centrifuged at 12,000 rpm for 10 min at 4°C and cytokine levels were measured on the resulting plasma. The cytokines IL-1β, IL-6 and TNF-α were simultaneously assessed using the Bio-Plex Pro Rat Cytokines Assay (Bio-Rad Laboratories Inc., California, USA) according to the manufacturer’s instructions .
In the present study, [18F]DPA-714 was synthesized as reported by Sydney group . The product was identified and purified using high-performance liquid chromatography on a COSMOSIL C18-AR-II column (10 × 250 mm, Nacalai, Kyoto, Japan). Molar activity ranged from 33 to 160 GBq/µmol. Radiochemical purity analyzed using HPLC exceeded 99%.
All PET scans were performed using a microPET Focus220 (Siemens, Knoxville, USA) designed for small laboratory animals. Rats were anesthetized with 1.5% isoflurane and nitrous oxide/oxygen (7:3) and placed in a prone position in the PET scanner gantry. During the PET scan, the body temperature was maintained at 37°C using a small animal warmer connected to a thermometer (BWT-100A; Bio Research Center, Nagoya, Japan). A 45-min emission scan was performed immediately after the bolus injection of [18F]DPA-714 (≈75 MBq per animal) via a cannula inserted into the tail vein; the energy window was 400‒650 keV and the coincidence time window was 6 ns. Emission data were collected in list mode and sorted into dynamic sonograms (6 × 10 s, 6 × 30 s, 11 × 60 s, and 10 × 180 s, for a total of 33 frames). The acquired data were reconstructed by standard 2D-filtered back projection (FBP) (ramp filter, cutoff frequency at 0.5 cycles per pixel) for quantification, and by a statistical maximum a posteriori probability (MAP) algorithm (12 iterations with point spread function effect) for image registration.
PET images were co-registered to a magnetic resonance imaging (MRI) template which was placed in a Paxinos and Watson stereotactic space using the PMOD imaging processing software (version 3.6, PMOD Technologies, Ltd, Zürich, Switzerland). Each FBP image was spatially smoothed using an isotropic Gaussian kernel (0.6-mm FWHM) for enhancement of the statistical power. The radioactivity was normalized with cylinder phantom data and expressed as standardized uptake values (SUVs).
A voxel-based statistical analysis was performed using SPM 8 software (Welcome Department of Imaging Neuroscience, London, UK). A two sample t-test was used for estimating the statistical differences between groups. The statistical threshold was set to be P < 0.005 (FWE) with an extent threshold of 200 contiguous voxels.
All results are expressed as the mean ± SEM. All data were analyzed in SPSS (version 24.0, IBM, Armonk, USA). A one-way analysis of variance (ANOVA) with Bonferroni’s multiple-comparison procedure was used to assess changes in body temperature, cytokines, and spontaneous activity prior to and following the poly I:C injection. A two-way repeated measures ANOVA with a Bonferroni’s multiple-comparison procedure was used to assess differences in body temperature and spontaneous activity between the two groups of rats. A Pearson’s test was used for correlation analysis of accumulation of [18F]DPA-714 in each brain region and fatigue-like behavior. Differences were considered statistically significant for P < 0.05.
The data that support the findings of this study are available on request from the corresponding author.