The study was divided in a behavioural evaluation to assess differences in responses to painful stimuli and real time reverse transcription polymerase chain reaction (RT-PCR) assays to evaluate the expression levels of Scn9a (NaV1.7) and Scn10a (NaV1.8). The rats were randomly assigned to Flutamide (F) and Control (C) groups.
Prior to each stage of the study, the animals were exposed to flutamide preparation administered by a blinded researcher other than the one who performed the behavioural tests. Group F rats received flutamide (Sigma-Aldrich, Brazil) at a dosage of 0.050 µg/g of animal weight/day, injected into the animals’ dorsal region subcutaneously for the 7 days prior to the experiment. The drug was diluted in a solution of propylene glycol/10% ethanol because the solubility of this vehicle is higher than that of vegetable oils. Flutamide is a non-hormonal medication that produces an anti-androgenic effect through its binding to androgen receptors, preventing the coupling of androgens to their receptors, and reduces the uptake of testosterone by peripheral tissues (Affaitati et al. [27]). Group C animals received only the propylene glycol solution/10% ethanol by the same administration route. After the preparation phase, the animals were submitted to the plantar incision pain model of acute postoperative pain for behavioural and gene expression experiments. The researcher in charge of this investigation was blinded to the animal group allocation (Fig. 1).
The behavioural tests were performed at the following times: Pre-incision (PRE); 2 h after incision (2 h); and on the 1st (D1), 2nd (D2), 3rd (D3) and 7th (D7) postoperative days. Gene expression was studied at PRE, D3 and D7.
Sample size
Sample size was calculated using G*Power software [17]. For the behavioral study, the number of animals was calculated using t-student test to obtain a 30% difference between groups for the paw withdrawal threshold test, with 80% power and 0.05 alfa error, obtaining 9 animals per group.
For the gene expression experiments, DRG samples were obtained from seven animals for each time point and for each group (total = 42). Sample size was calculated using ANOVA test with 80% power and 0.05 alfa error, obtaining 7 animals per group.
Plantar incision postoperative acute pain model
The animals were submitted to plantar incision of the right hind paw under general anaesthesia with 2–3% isoflurane. The procedure was performed aseptically as described by Brennan et al. [18].
Behavioral tests
Paw withdrawal threshold test [18]: The rats were placed in individual boxes with enough space to move around and kept on a metal screen with openings of 1 cm2. After a period of adaptation to the site, paw withdrawal threshold measurements were performed after mechanical stimulation with von Frey’s digital algesimeter (Insight Ltda, Ribeirão Preto, SP, Brazil). The mechanical stimulus was limited to 60 g to prevent worsening of the lesion and was applied to the right hind paw plantar region, close to the incision scar. Five measurements were obtained at intervals of 2 min (min) and the mean calculated, representing the test final value.
Guarding Pain Test [18]: A cumulative score was used to assess non-evoked pain behaviour. It was always performed prior to the evoked pain test to avoid confounders. Similarly, to the previous test, the animals were placed on a metal screen with openings of 1 cm2.
Incised and non-incised hind paws were carefully examined during a period of 1 min. This procedure was repeated every 5 min for 1 h (h). During the evaluation, scores ranging from 0 to 2 were assigned depending on the positioning most often adopted by each paw of the animal during the observation period.
The scores were assigned as follows: 0 = The surgical wound of the animal’s paw is totally in contact with or even distorted by the net or appears whitish. 1 = The rat paw gently touches the net, but without distortion of the surgical wound or a whitish appearance. 2 = The paw does not contact the net.
The sum of the 12 scores (0–24) obtained during 1 h of observation was recorded. The difference between the scores obtained for the healthy and incised paws represents the cumulative pain score.
At the end of the experiment, the animals were euthanized via cardiac puncture exsanguination under general anaesthesia with isoflurane.
Real-time quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was used to evaluate mRNA expression to determine whether surgical trauma-related hyperalgesia derives from change in expression of voltage-dependent sodium channels in the DRG, and whether this expression can be modulated by blocking testosterone receptors.
To obtain the DRG samples, the animals were anaesthetized according to protocol. To reduce bleeding in the operative field and thus the time to obtain the samples, the rats were submitted to thoracotomy and sacrificed similarly to the previously described method. Laminectomy was then performed to obtain the dorsal root ganglia of L4 and L5 ipsilateral to the plantar incision. The samples were frozen in liquid nitrogen and stored at − 80 °C.
Total RNA was extracted using the ReliaPrep™ RNA Miniprep Systems Kit (Promega, Madison, WI) according to the manufacturer’s instructions. The obtained RNA was subjected to spectrophotometric quantification with Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA) and only samples with an A260/A280 ratio higher than 1.8 were used. RNA integrity was analyzed by automated electrophoresis in the Agilent 2100 TapeStation Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples with RIN above 7.2 were included in this study. Synthesis of cDNA by reverse transcriptase was carried out using the IMProm-II Reverse Transcription System (Promega, Madison, WI). Gene expression analysis was performed using TaqMan specific assays for Scn9a (Rn00591020_ml), Scn10A (Rn00568398_ml) and TATA box protein/TBP (Rn01455646_ml, Thermo Fisher Scientific, Waltham, MA) as control. Results were obtained with the Ct methodology and expressed as arbitrary units.
Statistical analysis
All behavioural analyses were performed using Prism 4.0 software (GraphPad Software, Inc., USA). Data normality was assessed using the Kolmogorov–Smirnov test. The graphs present the data presented as median, lower and upper quartile. The variance in the results obtained in the behavioural tests and in gene expression analyses as a function of time was evaluated by the Kruskal–Wallis test, while the differences between the groups were determined by the Mann–Whitney test for each time point. A p value < 0.05 was considered statistically significant.