Influence of androgenic blockade with flutamide on pain behaviour and expression of the genes that encode the NaV1.7 and NaV1.8 voltage-dependent sodium channels in a rat model of postoperative pain

The phenomenon of pain receives the influence of several systems in the body, including the limbic, adrenergic, endogenous opioid and hormonal systems. Notably, the latter has been receiving more attention in the last 20 years. Some studies have focused on the potential relationships between gonadal hormones and pain behaviour, having revealed that it may be intensified by female gonadal hormones [1‐5]. Data on male hormones, however, are scarce. The observation that patients with temporomandibular chronic pain and low testosterone levels display higher pain expression indicates that this hormone may modulate pain [6, 7]. In male rats, testosterone presented an antinociceptive effect in models of inflammatory pain (carrageenan, formalin and Freud’s adjuvant) [7‐10]. The mechanisms underlying such effects of sex hormones, however, are not fully understood.

Gonadal hormones appear to increase expression of voltage-dependent sodium channels (NaV channels) in the trigeminal ganglion after inflammatory stimulation with formalin [1, 11]. NaV channels are crucial in neuronal excitation and signalling and are expressed at various sites of the nervous system, including primary afferents and the dorsal root ganglion (DRG).

Studies have demonstrated increased expression of NaV1.7 channels after inflammation [1, 11], an increase that contributes to exacerbation of pain behaviour [12‐14]. Likewise, the role of NaV1.8 was established in inflammatory pain, where knockout animals for its encoding gene had reduced response to thermal and chemical stimuli [15, 16]. In studies that assessed the influence of estradiol on the modulation of such channel’s expression, increased NaV1.7 expression was observed. Whether testosterone has the same potential to influence their expression, however, remains unknown [1].

To expand the knowledge on the relationship between pain and testosterone, the current study evaluated the effect of peripheral blockade of testosterone on pain behavior and quantified Scn9a and Scn10a expression (rat orthologs of human SCN9A and SCN10A, the genes that encode NaV1.7 and NaV1.8 sodium channels, respectively) in DRG using a rat model of acute postoperative pain.

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.

Our data revealed that androgenic blockade promoted exacerbation of nociception in a classical rat pain model, supporting the concept that androgens are associated with antinociception. This conclusion was based on a lower threshold for paw withdrawal and more evident guarding behaviour of the incised hind paw developed by flutamide-treated animals. Interestingly, gene expression analyses showed increased Scn9a expression in DRG post-incision whereas Scn10a expression did not significantly vary after this trauma. Flutamide did not modulate, however, the expression of Scn9a.

The plantar incision model has been used to better reproduce the mechanisms observed in acute postoperative pain, facilitating the translation of results to humans and to clinical practice [18]. Plantar incision causes a state of hyperalgesia by inducing the production of local inflammatory cytokines, neural growth factor (NGF) [19] and brain-derived growth factor in DRG [20], in addition to an increase in the spontaneous activity of DRG neurons [18].

The scores based on the guarding pain test were similar in the two groups, peaking at 2 h and then decreasing progressively over time. The same trend was observed for the paw withdrawal threshold, which reached the lowest value at 2 h and gradually increased until the seventh postoperative day in both groups. This pattern is similar to the ones reported in other studies using the same experimental model, where the peak of hyperalgesia occurred in the first postoperative hours and was followed by spontaneous resolution around the tenth postoperative day [18, 19, 21].

The animals treated with flutamide presented a lower paw withdrawal threshold compared to the controls, as well as higher scores in the spontaneous pain test. The results associated with the behavioural tests suggest a greater degree of hyperalgesia in animals submitted to testosterone blockade by flutamide. The antinociceptive action of testosterone has already been demonstrated in acute inflammatory pain. Orchiectomized animals submitted to carrageenan injection in the paw presented more pronounced exacerbation of hyperalgesia than controls, whereas testosterone replacement reversed this difference [22]. Additionally, after formalin injection orchiectomized males exhibited potentiated pain behaviour, while females receiving testosterone exhibited less pain [2, 23]. The plantar incision model was also used to compare postoperative pain between males and females, but no difference was observed in pain behaviour or response to opioid analgesics [24].

Although several studies have used surgical castration as means of inducing hypogonadism, this method has the disadvantage of impairing the control of physiological mechanisms related to sex hormones, leading to inadequate maintenance of elevated or depleted hormonal levels [25]. Wistar and Sprague–Dawley rats presented high plasma oestradiol concentrations as a result of orchiectomy, reaching levels equivalent to those found in females of these strains [26]. Flutamide is used to achieve androgenic blockade without the complications of surgical castration [27]. A recent study demonstrated that the analgesic effect of testosterone is dependent on activation of the testosterone receptor and that flutamide reversed this protective effect after formalin injection into the temporomandibular joint (TMJ) [28].

The evidence accumulated over the last decade corroborates the results obtained in the present study, suggesting a protective role of testosterone against pain, but the underlying mechanisms are still unclear [8, 22, 23, 28, 29]. The increase in the expression of voltage-dependent sodium channels, particularly NaV1.7 and NaV1.8, has already been demonstrated in previous studies using inflammatory pain models [15, 30, 31]. It was suggested that oestradiol might modulate the expression of these channels. This hypothesis was in fact confirmed by a study where animals were treated with the oestrogen receptor blocker ICI 182,780. In this case, NaV1.7 expression was increased in the trigeminal ganglion in a model of inflammation of the TMJ, with concomitant increase in mechanical hyperalgesia [1, 11].

Scn9a expression increased in the present study following the established surgical trauma, a pattern that did not occur for Scn10a. This lack of increase in NaV1.8 expression corroborates data reported by previous studies evaluating the role of sodium channels in postoperative pain [12, 15]. Despite the increase in Scn9a expression, it was apparently not related to testosterone effects, therefore not participating in mechanisms of testosterone-induced analgesia. Our data thus support that androgenic blockade increases pain behaviour in rats, suggesting an antinociceptive role for testosterone, not related to modulation of NaV1.7 channel expression.

The choice of using L4 and L5 DRGs was based on the fact that the hindpaw innervation of these dermatomes are made by these nerve roots. We believe that if L3 or L6 DRGs had been added, it would have been more difficult to obtain a more precise change in NaV channel expression. One possibility that was not explored in this paper is the study of dorsal horns along with the DRGs, that might bring relevant additional information to the field.

Recently, a possible role of the central opioid system in the protective effect of testosterone was described. After treatment with naloxone, gonadectomized male rats on testosterone replacement that received formalin TMJ injection presented increased behavioral response, while the opioid antagonist had no effect on gonadectomized animals without hormonal replacement [7]. Additional evidence of a relationship between androgens and opioid system supports this hypothesis. An androgen receptor (AR) binding site was found in the promoter region of the mu opioid receptor (MOR) gene Oprm1. In the presence of testosterone, AR can transcriptionally activate Oprm1 and amplify the opioid receptor synthesis. This effect was not observed after AR blockade [32].

Some limitations may be considered in our study. The use of subcutaneous route facilitates the injection of the study drug; however, it carries the risk of erratic absorption. Moreover, the chosen time points to evaluate the expression levels of Scn9a and Scn10a may not have been sufficient to obtain accurate time expression curves. Also, the stimuli provided by the plantar incision may not have been intense or long enough to induce amplification of genes that encode NaV channels.