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  • All procedures with animals were approved by Douglas Institute Facility Animal Care Committee and McGill University Animal Care Committee, in line with the guidelines of the Canadian Council on Animal Care. Adult male Sprague-Dawley rats (2–3 months old, weighing 250–300 g at the time of experiment; Charles River, Saint Constant, QC, Canada) were used in the experiments. The animals were housed individually in a controlled environment with a regular 12 h:12 h LD cycle (∼200 lux in the light periods) in an ambient temperature of 21±2°C. Regular chow and water were available ad libitum. In all experiments, animals were treated with either 0.1 mL of purified turpentine oil (TURP) (Riedel-deHaen, Sleeze, Germany) or 0.1 mL of sterile physiological saline by intramuscular (I.M.) injection into the right gastrocnemius muscle (hind-limb). In one experiment, animals were treated with human recombinant (hr) IL-1Ra (provided by Dr. Stephen Poole, National Institute for Biological Standards and Control, Hertfordshire, UK). Animals were treated intraperitoneally (I.P.) at a dose of 1 mg/kg at 0, 4, 8, and 12 h after TURP treatment. The hrIL-1Ra was dissolved in physiological saline to a concentration of 1 mg/mL, and control animals were treated similarly with an equal volume of physiological saline. The times of treatment and sacrifice varied depending on the experiment. Animals were sacrificed by decapitation, blood was collected by trunk bleed into sterile tubes and serum was isolated by immediate centrifugation at 4000 rpm and 4°C for 10 min. Aliquots were made and samples frozen at −80°C until use. Livers, hearts, kidneys and spleens were collected upon sacrifice, frozen on dry ice and stored at −80°C until use. Analgesics were not used in our study, primarily because of the potential impact on some of our endpoint measures: Analgesics were shown to alter inflammatory responses and to affect the levels of pro-inflammatory cytokines [30], [31]. Moreover, analgesic treatment has impacts on circadian rhythms and on clock gene expression [32], [33]. Additionally, the pain experienced by the rats upon injection of the dose of TURP used in our study (0.1 mL) is relatively minor, compared to the effects of the higher dose of 0.6 mL of TURP used in some of our previous studies [34], and the animals did not exhibit any outward signs of distress nor any pain-induced vocalization. All injections during the dark phase (ZT12-ZT0) were performed under dim red light. When animals were to be sacrificed during the dark phase, they were taken out of the room in a lightproof box and were not exposed to light until less than a minute before sacrifice. Temperature was monitored using remote radio-biotelemetry (Data Science International, St Paul, MN, USA) as previously described [35]. Briefly, two weeks before the experiment, animals were abdominally implanted with pre-calibrated temperature-sensitive radio transmitters (TA10TA-F40, Data Sciences International) under isoflurane anaesthesia. Two days before the experiment, animals were placed on individual receiver boards containing an antenna to monitor the temperature output frequency (Hz) every 10 min until sacrifice. The temperature frequency was converted into degrees centigrade with Dataquest software (Data Sciences International). The baseline 24 h temperature fluctuation was established two days prior to treatment in each animal to ensure a normal diurnal profile. Before each of the following three experiments, animals were habituated for 2 weeks in a 12 h:12 h LD cycle with daily handling at least one week before the experiment. This involved taking the animals out of their cages and manipulating them in the same way they would be at the time of injection (i.e., for handling prior to I.M. injection, animals held at the side of the experimenter and their rear gastrocnemious muscle revealed; prior to I.P. injections, animals wrapped in the same towel as for the injections and positioned such as to receive an injection). Experiment 1: The effect of a morning TURP injection on clock gene expression: Animals were treated with either TURP or saline at ZT2 (2 h after lights on given that Zeitgeber Time 0 [ZT0] corresponds to lights on and ZT12 to lights off). Animals were then sacrificed 2, 6, 10, 14, 18, and 22 h after treatment (i.e. ZT4, ZT8, ZT12, ZT16, ZT20 and ZT0 time of sacrifice (TOS), respectively). Each group included 4 animals. All measurements for clock genes and cytokines were performed at the TOS. Experiment 2: The effect of injection time on TURP-induced changes of clock gene expression: Animals were treated with either TURP or saline at 4 time points over 24 h: ZT2, ZT8, ZT14 and ZT20 time of injection (TOI). Animals were then sacrificed exactly 10 h after the TOI (i.e., ZT12, ZT18, ZT0 and ZT6 TOS, respectively) as this time corresponds with the maximal fever response in response to TURP [27]. There were 5 animals per time point each for TURP and saline. Measurements for clock genes and cytokines were performed at the TOS. Experiment 3: The effects of anti-inflammatory IL-1Ra treatment on TURP-induced changes of clock gene expression: Animals were treated with either TURP or saline at ZT2 and sacrificed at ZT12 or ZT16 (10 h and 14 h after TOI, respectively). In each of the injection groups, animals were treated with either hrIL-1Ra or saline making a total of 4 groups per time of sacrifice (saline-saline, saline-hrIL-1Ra, TURP-saline, and TURP-IL-1Ra). The ZT12 group had a total of 3 hrIL-1Ra treatments at 0, 4 and 8 h after TOI (i.e., ZT2, ZT6, and ZT10, respectively) and the ZT16 group had 4 hrIL-1Ra treatments at 0, 4, 8 and 12 h after TOI (i.e., ZT2, ZT6, ZT10 and ZT14, respectively). Given the short half-life of IL-1Ra, multiple injections were required for a full effect on fever and IL-6 [36]. Further, IL-1Ra blocks the upregulation of IL-6 and to completely attenuate its response, IL-1Ra must be administered early in the TURP time-course when IL-6 begins to be produced [36]. The human carcinoma HepG2 cells were provided by Dr. Cindy Goodyer (McGill University, Montreal). Cells were seeded in 6-well plates at a density of 0.3×106 cells per plate in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Burlington, ON) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), 2 mM L-glutamine (Life Technologies), and 100 mg/mL penicillin-streptomycin and grown to confluency at 37°C in 5% CO2. One day (24 h) before the first time point, growth media was replaced with a starvation media containing 0.5% FBS. On the day of the experiment, cells were transferred to a serum-free media containing the various concentrations of human recombinant IL-6 (CedarLane, Burlington, ON). Cells were then washed with cold PBS, lysed in Trizol (Life Technologies), and frozen at −80°C until use. Serum concentrations of IL-6, IL-1Ra and TNFα were obtained in duplicate using an in-house-developed sandwich enzyme-linked immunosorbent assay (ELISA) (secondary enzymes supplied by NIBSC, Potters Bar, UK) as previously described [35]. The detection limit was between 30 and 50 pg/mL for IL-6, TNFα and IL-1Ra, as indicated in figure legends. The inter-assay variability was below 4% and the intra-assay variability was below 9% for all experiments. RNA was extracted using Trizol (Life Technologies) according to the manufacturer’s protocol. cDNA was synthesized using the MultiScribe reverse-transcription kit (Life Technologies) according to manufacturer’s instructions. In all rat tissues, clock gene expression was assessed using SYBR Green quantitative PCR (qPCR) (Life Technologies, 7500 Real-Time PCR System). Primers for clock genes Per1, Per2 and Rev-erbα, were designed using Primer3 software (sequences in Table S1). The control primers tested were Histone H1, HPRT (hypoxanthine-guanine phosphoribosyl transferase), GAPDH (glyceraldehyde-3-phosphate), Ubi (ubiquitin), and Tbp (TATA box binding protein) (sequences in Table S1) [37]. In order to determine the most stable control genes over time and condition the GeNorm v3.3 software was used [38]. For each experiment conducted, two control genes were selected (Table S2). In the human HepG2 cell line, mRNA expression was assessed with quantitative PCR using TaqMan probes (Life Technologies) as outlined by the manufacturer. From a set of 5 genes (B2M; beta-2-microglobin, PPiA; peptidylprolyl isomerase A, TBP; TATA-binding protein, RPLPO; large ribosomal protein, GAPDH; glyceraldehyde-3-phosphate dehydrogenase, ACTB; beta-actin), the most stable control genes over both time and treatment were determined using GeNorm as described above: B2M (H200984230_m1) and PPiA (Hs04194521_s1). For clock gene expression, the following probe sets were used: Per1 (H200242988_m1), Per2 (Hs00256143_m1), Rev-erbα (Hs00253876_m1), Serum amyloid A2 (Hs00605928_g1) and Haptoglobin (Hs01667582_m1). The relative mRNA expression of each clock gene was assessed by normalizing to the geometric mean of the two control genes, and then to a reference sample, using the 2−ΔΔCT method [39]. In Experiments 1 and 2, the temperature curve differences, cytokine induction over time and changes in clock gene RNA levels were analyzed by two-way ANOVAs with factors Time and Condition. In Experiment 3, the variation in temperature over time in all the groups was analyzed with a three-way ANOVA with factors Time and Condition for TURP and Condition for IL-1Ra; and group differences in cytokine induction and clock gene expression were analyzed with a one-way ANOVA. Finally, clock gene expression in HepG2 genes was analyzed using two-way ANOVAs with factors Time and Condition. For ANOVAs, significant effects were decomposed using Tukey’s post hoc analysis for pair-wise comparisons when applicable. Induction of SAA2 and HP gene expression in HepG2 cells was analyzed with Student t-tests. Statistical significance was set to p<0.05 and results are reported as mean ± SEM.
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