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DHEA administration modulates stress-induced analgesia in rats
Physiology & Behavior 157 (2016) 231–236
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DHEA administration modulates stress-induced analgesia in rats Ana Lúcia Cecconello a,c,⁎, Iraci L.S. Torres b,c, Carla Oliveira b, Priscila Zanini a, Gabriela Niches a, Maria Flávia Marques Ribeiro a,c a Laboratório de Interação Neuro-Humoral, Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Av. Sarmento Leite, 500, Porto Alegre, Rio Grande do Sul CEP 90050-170, Brazil b Laboratório de Farmacologia da Dor e Neuromodulação, Investigações Pré-Clinicas, Departamento de Farmacologia, ICBS, UFRGS, Av. Sarmento Leite, 500, Porto Alegre, Rio Grande do Sul CEP 90050-170, Brazil c Programa de Pos-Graduação em Ciências Biológicas, Fisiologia, ICBS, UFRGS, Av. Sarmento Leite, 500, Porto Alegre, Rio Grande do Sul CEP 90050-170, Brazil
H I G H L I G H T S • We evaluated the effect of DHEA on stress-induced analgesia in rats. • Acute treatment with DHEA had the same effect as the exposure to acute stress. • The treatment with DHEA prolongs acute stress-induced analgesia.
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Article history: Received 14 September 2015 Received in revised form 27 January 2016 Accepted 3 February 2016 Available online 4 February 2016 Keywords: Steroids Pain neuropathic Responses of fight or flight
a b s t r a c t An important aspect of adaptive stress response is the pain response suppression that occurs during or following stress exposure, which is often referred to as acute stress-induced analgesia. Dehydroepiandrosterone (DHEA) participates in the modulation of adaptive stress response, changing the HPA axis activity. The effect of DHEA on the HPA axis activity is dependent on the state and uses the same systems that participate in the regulation of acute stress-induced analgesia. The impact of DHEA on nociception has been studied; however, the effect of DHEA on stress-induced analgesia is not known. Thus, the aim of the present study was to evaluate the effect of DHEA on stress-induced analgesia and determine the best time for hormone administration in relation to exposure to stressor stimulus. The animals were stressed by restraint for 1 h in a single exposure and received treatment with DHEA by a single injection before the stress or a single injection after the stress. Nociception was assessed with a tail-flick apparatus. Serum corticosterone levels were measured. DHEA administered before exposure to stress prolonged the acute stress-induced analgesia. This effect was not observed when the DHEA was administered after the stress. DHEA treatment in non-stressed rats did not alter the nociceptive threshold, suggesting that the DHEA effect on nociception is state-dependent. The injection of DHEA had the same effect as exposure to acute stress, with both increasing the levels of corticosterone. In conclusion, acute treatment with DHEA mimics the response to acute stress indexed by an increase in activity of the HPA axis. The treatment with DHEA before stress exposure may facilitate adaptive stress response, prolonging acute stress-induced analgesia, which may be a therapeutic strategy of interest to clinics. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Individuals exposed to threatening situations (stressor) present physical and behavioral changes (stress response) with the objective of maintaining homeostasis [1, 2, 3]. Stress responses mainly include activation of the hypothalamic-pituitary-adrenocortical (HPA) axis, and induction of the corticotropin-releasing hormone (CRH) and
⁎ Corresponding author at: Departamento de Fisiologia, ICBS, Universidade Federal do Rio Grande do Sul, R. Sarmento Leite 500, Porto Alegre RS 90170-050, Brazil. E-mail address: [email protected] (A.L. Cecconello).
http://dx.doi.org/10.1016/j.physbeh.2016.02.004 0031-9384/© 2016 Elsevier Inc. All rights reserved.
vasopressin (VP) by parvocellular neurons of the paraventricular nucleus of the hypothalamus (PVN) [2]. CRH acts on the anterior pituitary gland, releasing adrenocorticotrophic hormone (ACTH), which in turn stimulates the release of glucocorticoids by the adrenal cortex. During the stress response, the PVN receives information from brainstem afferents, among them the locus coeruleus, an ascending noradrenergic pathway with an important role in HPA activation [4], inducing an increase in the synthesis of CRH and VP [5, 6]. The PVN is a central integrator of endocrine, vegetative and behavioral responses [2]. An important aspect of the adaptive stress response is the pain response suppression that occurs during or following stress exposure. It contributes to the expression of appropriate behaviors to face stressors,
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facilitating the responses of fight or flight [7, 8]. This phenomenon is often referred to as stress-induced analgesia (SIA), and occurs in both laboratory animals and humans [7, 8]. SIA involves activation of the descending inhibitory pain pathway, originating in the neurons of the cortex, hypothalamus and amygdala, and project into the periaqueductal gray and rostroventral medulla and finally to the dorsal horn of the spinal cord. Activation of this pathway results in an inhibitory effect at the level of the dorsal horn by inhibiting the ascending transmission of nociceptive information [7]. It is known that acute stress-induced analgesia involves the modulation of opioid receptors [9] and nonopioid receptors (e.g. NMDA and GABA receptors) [10]. In addition, corticosterone can facilitate analgesia induced by stress, since this phenomenon may be blocked by adrenalectomy and reestablished by treatment with the administration of corticosterone [7]. Another hormone that participates in the modulation of adaptive stress response is the adrenal hormone dehydroepiandrosterone (DHEA). DHEA is considered a neuroactive steroid, and a crucial endogenous modulator of numerous physiological functions [11, 12]. The main biological functions of DHEA on the nervous system are neuroprotection, catecholamine synthesis and secretion, as well as antioxidant and anti-inflammatory activities [12]. DHEA has been shown to modulate various receptors, such as GABA(A), N-methyl-D-aspartate (NMDA), kainate, ionotropic glutamate, nicotinic acetylcholine, muscarinic, glycine, σ1 and neurotrophin receptors and ionic channels such as calcium, sodium and potassium channels [13, 14]. In humans, elevated levels of DHEA have been observed in response to stress exposure [13]. DHEA may be secreted in response to ACTH in humans at least [15], and it modulates HPA axis activity [16, 17]. In an in vitro study, ACTH-induced corticosterone release by rat adrenal zona fasciculatereticularis cells was attenuated by DHEA [18]. During repeated stress exposure, a single injection of DHEA can reduce the serum corticosterone levels [17]. On the other hand, it was observed that a single injection of DHEA stimulates the secretion of hypothalamic CRH, ACTH by the pituitary and corticosterone by the adrenal cortex in nonstressed rats [16]. In addition, chronic DHEA treatment increases CRH mRNA levels in hypothalamic PVN independent of age and sex in nonstressed rats [19], suggesting that the effect of DHEA on the HPA axis activity is dependent on state. The impact of DHEA on nociception has been studied [20, 21], although it is still not well understood. DHEA effects on nociceptive mechanisms are complex. Acute DHEA treatment exerts a biphasic effect on nociception (a rapid pro-nociceptive action and a delayed antinociceptive effect). On the other hand, chronic treatment with DHEA elevates the nociceptive threshold [21, 22]. However, the effect of DHEA on stress-induced analgesia is not known. SIA may be thought of as an important component of the fight or flight response [7]. The understanding of the fundamental mechanism of pain suppression that occurs during or following exposure to stress and how this effect can be modulated becomes a potential new therapeutic target for pain and stress-related disorders. Thus, the aim of the present study was to evaluate the effect of DHEA on stress-induced analgesia and determine the best time for hormone administration in relation to exposure to stressor stimulus. 2. Material and methods 2.1. Animals Male adult Wistar rats (60–70 days, mean weight 300 g) were obtained from the Center for Laboratory Animal Reproduction and Research (CREAL) at the Universidade Federal do Rio Grande do Sul (UFRGS). The number of animals was four rats per cage with food and water available ad libitum and they were maintained in a 12 h light/ dark cycle (lights on at 7:00 a.m. and lights off at 7.00 p.m.) in a humidity- and temperature-controlled environment (22 ± 2 °C).
Initially, the rats were divided into four animals per cage and acclimated to the vivarium for one week before beginning treatment. After the acclimation period, the animals were randomly selected by weight and subsequently divided into a control group (rats were housed only with other control rats) and stress group (rats that underwent restraint stress were housed only with other rats that underwent restraint stress). They were handled for 14 days prior to the experiments. All experiments and procedures were approved by the Institutional Animal Care and Use Committee (CEUA-UFRGS protocol No. 19788) and were compliant with Brazilian guidelines involving the use of animals in research (Law No. 11.794). Additionally, all efforts were made to minimize the suffering, pain and discomfort of the animals, as well as to reduce the number of animals. The animals were euthanized 15 min after the last measure of TFL and the trunk blood collected. Death by decapitation was carried out by a trained professional and in a separate room to where they were experiencing stress and treatment. 2.2. Stress procedures The animals were stressed by restraint for 1 h in a single exposure [23]. Restraint stress was carried out by placing each animal in a 25 × 7 cm plastic tube, and adjusting it with plaster tape on the outside so that the animal was unable to move. There was a 1 cm hole in the far end for breathing. Control animals were manipulated, but not submitted to restraint. Stressed animals were submitted to a single exposure to the restraint. The immobilization procedure was carried out between 1000 and 1200 h a.m. 2.3. Treatment Each group (stressed and control) was subdivided into three treatment groups (n = 7–8 per group): 1) injection of DHEA 30 min before the stress and injection of vehicle 30 min after the stress; 2) injection of vehicle 30 min before the stress and injection of DHEA 30 min after the stress; and 3) injection of vehicle 30 min before the stress and injection of vehicle 30 min after the stress (Fig. 1). Animals received DHEA (Calbiochem) as a single i.p. dose of 25 mg/kg diluted in 20% cyclodextrin. 2.4. Tail-flick measures Nociception was assessed with a tail-flick apparatus. Rats were wrapped in a towel and placed on the apparatus. A photo beam with adjustable sensitivity detects the tail flick and the latency is automatically presented on a digital display on the control unit. The light source positioned below the tail was focused on a point 2.3 cm rostral to the tip of the tail. Deflection of the tail activated a photocell and automatically terminated the trial. Light intensity was adjusted to obtain the baseline tail-flick latency (TFL) of 4–6 s as described in the manufacturer's protocol (LE7106 Tail-flick Meter/Harvard Apparatus). Measurements of reaction time are given with a 0.1 second precision. Once this intensity had been established, rats with baseline latency N 7 s and b3 s were excluded from the experiment. A cutoff time of 10 s was used to avoid tissue damage. The groove system for the tail and the adjustment of response sensitivity ensure optimum repeatability and reliability of results. The general procedure was as follows [23, 24]: Day 1, the animals were familiarized with the apparatus; day 2, the baseline TFL value was obtained; day 3, animals were subjected to three TFL measurements for the analgesia test. The first TFL measurement was taken 15 min after the first injection, the second immediately after stress and the third 15 min after the second injection (Fig. 1). 2.5. Corticosterone serum concentration The rats were killed and trunk blood samples were collected in tubes containing clot activator gel (BD, Vacutainer®). After centrifugation at
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Fig. 1. Representative diagram of the experimental design.
1000 ×g for 15 min, the serum was separated and stored at −20 °C until analysis. Serum corticosterone levels were measured by enzyme-linked immunosorbent assay (ELISA) (Corticosterone EIA Kit 500655, Cayman Chemical). 2.6. Data analysis Levene's test was used to check for homogeneity of variance. When the data showed homogenous variance, repeated measures analysis of variance (ANOVA) (Tail-flick measures) or two-way analysis of variance (Corticosterone serum concentration: treatment × stress) or one-way analysis of variance (Corticosterone serum concentration: treatment) followed by the post hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). p-Values b0.05 were considered significant.
levels immediately after the stress, and reduced levels after 30 min of recovery [13]. The impact of DHEA on nociception has been studied [20, 21]; however, the effect of DHEA on stress-induced analgesia is not known. In this pre-clinical model, we propose that DHEA administration before exposure to stress such as invasive procedures may facilitate adaptive stress response, prolonging acute stress-induced analgesia. The most important is that experimental models can help to elucidate the fundamental mechanisms of nociceptive threshold increase that occurs in situations of acute stress. Studies of stressinduced analgesia have enhanced our understanding of the fundamental physiology of pain and stress, and can be a useful approach for uncovering new therapeutic targets for the treatment of pain and
3. Results 3.1. Effects of DHEA administration on stress-induced analgesia (SIA) Repeated measures ANOVA showed an effect of stress. As can be observed, the animals presented increased tail-flick latency immediately after being stressed, regardless of treatment with DHEA (F = 8.88; p b 0.05 — DHEA before the stress; F = 5.57; p b 0.05 — DHEA after the stress) (Fig. 2A and B). Moreover, a significant interaction between stress and DHEA treatment was observed only when the injection of DHEA was administered before the stress (F = 6.37; p b 0.05) (Fig. 2A). When the DHEA was administered before the stress exposure, we observed stress-induced analgesia (SIA), which was maintained for a longer time (until 45 min after stress) (Fig. 2A). On the other hand, when the DHEA was administered after the stress, this effect was not observed (Fig. 2B). Moreover, the DHEA treatment did not alter the TFL latency in non-stressed rats (Fig. 2). 3.2. Effects of exposure to restraint stress and DHEA treatment on serum corticosterone levels Two-way ANOVA showed a significant interaction between stress and DHEA treatment both when the DHEA was administered before (F = 7.63; p b 0.05) and after (F = 13.28; p b 0.05) the stress (Fig. 3A and B). In the animals that received vehicle the stress produced an increase in serum corticosterone levels. However, in the animals that received DHEA the stress produced no effect on serum corticosterone levels. Nonstressed animals showed an increase in serum corticosterone levels when treated with DHEA. One-way ANOVA showed that nonstressed animals that received DHEA 150 min before blood was collected presented concentrations of corticosterone that were lower than those observed in animals that received DHEA 30 min before blood was collected (F = 21.97; p b 0.05) (Fig. 4). 4. Discussion As a positive aspect and from an evolutionary perspective, SIA may be thought of as a component of the fight or flight response [7]. Interestingly, DHEA is involved in the modulation of adaptive stress response, changing the HPA axis activity [7, 8]. In humans, significantly elevated levels of DHEA have been observed in response to acute stress. The predominant pattern for DHEA levels during the acute stress was increased
Fig. 2. Effect of acute stress upon TFLs on DHEA-treated rats. The animals were restrained for 1 h and subjected to three TFL measurements. (A) Injection of DHEA administered before the stress: basal = 24 h before; TFL 1 = 15 min after injection of DHEA — 25 mg/ kg; TFL 2 = immediately after stress; TFL 3 = 45 min after stress (15 min after injection of vehicle). (B) Injection of DHEA administered after the stress: basal = 24 h before; TFL 1 = 15 min after injection of vehicle; TFL 2 = immediately after stress; TFL 3 = 45 min after stress (15 min after injection of DHEA — 25 mg/kg). Repeated measures ANOVA followed by the post-hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). *p b 0.05 stress vs. control; #p b 0.05 stress-DHEA vs. all others.
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Fig. 3. Effect of acute stress upon serum corticosterone levels on DHEA-treated rats. (A) Injection of DHEA (25 mg/kg) administered before the stress. (B) Injection of DHEA administered after the stress: Two-way ANOVA followed by the post-hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). Different letters represent p b 0.05.
stress-related disorders. In this way, we can propose that DHEA administration may be a therapeutic tool in pain management. In this study, we showed stress-induced analgesia (Fig. 2), corroborating previous literature reports [7, 23], and this effect was potentiated by DHEA treatment. Given that the effect of treatment with DHEA on SIA depends on the time at which the intervention with DHEA is made in relation to the exposure to stressor stimulus (Fig. 2A and B), we suggest that the DHEA effect on nociception is state-dependent like the DHEA effect on the HPA axis activity [19]. It is interesting to note that the effect of prolonging SIA occurs only when the intervention with DHEA is made at the same time as the HPA axis is in the basal state (Fig. 2A). It has been observed that a single injection of DHEA stimulates the secretion of CRH by the hypothalamus, ACTH by the pituitary and corticosterone by the adrenal cortex in nonstressed rats [16]. In the present study, it was observed that the treatment with DHEA in nonstressed rats was able to increase the secretion of corticosterone (Fig. 3). It is known that both stress and treatment with DHEA stimulate the secretion of corticosterone [13, 16]. Based on the fact that corticosterone facilitates SIA, since this phenomenon may be blocked by adrenalectomy and reestablished by treatment with corticosterone [7], the effect of prolonging the SIA could have been caused by a more durable increase of serum corticosterone levels induced by the action of DHEA followed by stressor exposure. It was observed that the stressed animals that received vehicle showed an increase in serum corticosterone levels (Fig. 3A and B),
corroborating previous literature reports [2, 3]. However, in the stressed animals, DHEA showed no significant difference in the serum corticosterone levels, irrespective of the time at which the intervention with DHEA was made. It is possible that when the HPA axis is in a state of maximum activation induced by acute stress, intervention with DHEA is not able to stimulate the axis activity further. As can be seen in Fig. 3, the injection of DHEA had the same effect as the exposure to acute stress, with both of them increasing the levels of corticosterone. In addition, the animals that received DHEA 150 min before blood was collected presented concentrations of corticosterone that were lower than those observed in animals that received DHEA 30 min before blood was collected (Fig. 4). This result is in agreement with a previous study [16] that showed that a single injection of DHEA, in the same dose as that used in the present model, increases the concentration of corticosterone. It reached a maximal concentration at 60 min, and although this was the lowest concentration, it was still significantly elevated 300 min after DHEA injection. It is interesting to note that the pattern of secretion of DHEA-induced corticosterone [16] is similar to that of corticosterone induced by acute stress [3]. This suggests that acute treatment with DHEA can mimic a situation of acute stress. This rapid effect of DHEA on the activity of the HPA axis is in accordance with the model of action of neurosteroids on CNS functions, which rapidly alter neuron excitability, in seconds to minutes, by binding to membrane-bound receptors for inhibitory and excitatory neurotransmitters [12, 14, 25]. DHEA generally acts as a noncompetitive antagonist at GABAA receptors and as a positive allosteric modulator of the NMDA receptor [12]. Moreover, DHEA can potentiate NMDA receptor function by its actions as a sigma receptor agonist [12, 25]. HPA axis activity is under GABA, opioid and glutamate control, since microinjection of GABA antagonists, such as bicuculline, into the PVN activates the HPA axis [26]; also, intracerebroventricular (i.c.v.) administration of morphine stimulates the HPA axis [27]. Moreover, exposing a rat to a single stressor increases the activity of NMDA receptors in the paraventricular nucleus of the hypothalamus [28]. This indicates that the rapid DHEA effects on the HPA axis could be mediated through negative modulation of the GABAA receptor and positive modulation of NMDA and opioid receptors. Interestingly, DHEA modulates the activity of the HPA axis using the same systems that participate in the regulation of SIA [7, 10]. SIA may be opioid or nonopioid in nature [7], and depend on the stress severity, the pain test employed, and the sex and genotype of the animal used [7, 10]. It has been hypothesized that a stressor of mild severity produces a naloxone-sensitive SIA (opioid), whereas a more severe stressor induces a naloxone-insensitive SIA (nonopioid)
Fig. 4. Effect of injections of DHEA upon serum corticosterone levels on rats. DHEA 30 min = injection of DHEA (25 mg/kg) 30 min before blood collect. DHEA 150 min = injection of DHEA (25 mg/kg) 150 min before blood collect. One-way ANOVA followed by the post-hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). Different letters represent p b 0.05.
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that can be blocked by MK-801, a noncompetitive NMDA receptor antagonist [10, 29]. Studies employing GABA receptor agonists (muscimol) and antagonists (bicuculline) have established a role for GABA receptor in some forms of SIA [7]. In a published study of the effect of an inflammatory pain model and stress on the antinociceptive effect in mice it was shown that immobilization stress activated the hypothalamic proopiomelanocortin (POMC) gene and β-endorphin expression [30]. It is well established that the POMC is the precursor of βendorphin, but also of ACTH, which activates the release of corticosterone [3]. However, during the expression of SIA, a reduction in the expression of the POMC gene is observed [30], which possibly occurs due to negative feedback of corticosterone in the hypothalamus diminishing the expression of the POMC gene. These findings suggest that a single immobilization stress activates the descending pain modulatory system, which is mainly mediated through endorphinergic activation. Interestingly, POMC-containing neurons project reciprocally to the PVN (neurons of the paraventricular nuclei), CRH (corticotropin-releasing hormone) and AVP (arginine-vasopressina) neurons, innervate LC/NE-sympathetic neurons (locus ceruleus/norepinephrinesympathetic system) of the central stress system in the brainstem and terminate pain control neurons of the hind brain and spinal cord. Consequently, activation of the stress system, via CRH and catecholamines, stimulates the hypothalamic β-endorphin and other POMC-peptide secretion, which reciprocally inhibits the activity of the stress system, and produces the stress-induced analgesia [31]. It is interesting to mention that the POMC promotes SIA through the action of β-endorphin and by being the precursor of ACTH, which stimulates the release of corticosterone. Furthermore, stress induces analgesia through mechanisms within and outside the brain. Another study demonstrated that sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue [32]. In this case, the antinociception induced by acute stress was significantly attenuated by the adrenergic antagonists propranolol and phentolamine, indicating that sympathetic neurotransmitters (e.g. noradrenaline) mediate stress-induced peripheral opioid analgesia [32], thereby acting in parallel with the activation of the hypothalamic-pituitary-adrenocortical axis to promote SIA. Interestingly, DHEA can induce an increase in POMC mRNA levels [33] and sympathetic activation [34]. Given that DHEA modulates HPA axis activity, GABA, NMDA and opioid receptors and sympathetic nervous system activity, we can suggest that DHEA may prolong SIA by modulating one or more pathways that control SIA. This is possibly, by effect of higher corticosterone serum levels by more time, increased modulation of the GABAergic, glutamatergic and/or opioid pathways and/or sympathetic activation, or even due to the complex involvement of all of these neurotransmission systems. However, the exact mechanism by which DHEA prolongs the effect of SIA is still unknown. Since DHEA is a natural steroid and precursor hormone produced by the adrenals in humans, its adverse effects are gender specific [35]. It is important to note that studies using oral or percutaneous DHEA administration with doses of up to 1600 mg taken by mouth daily for one month have been well tolerated [36, 37]. An interesting issue is that a dose of 50 mg of DHEA taken by mouth daily has been shown to be safe for up to six months [37]. Most importantly, in women, only minimal adverse effects have been described, such as mild acne, seborrhea, facial hair growth and ankle swelling [37]. It has also been proposed that in animal models of chronic DHEA administration (10 mg/kg/ week for 5 weeks), it exerts a dual effect [38]. These effects can be antioxidant or pro-oxidant, depending on dose and on the tissue specificity [38]. After DHEA treatment, SOD activity (antioxidant enzyme) was significantly decreased on heart tissue [38]. On the other hand, lipid peroxidation in rat erythrocytes was significantly increased [38]. In another study, Jahn et al. [39] report that DHEA administration reduced the levels of proteins essential for cell survival (p-AKT) and also thioredoxin (the most important mechanism for the regulation of the redox balance) in muscle tissue, showing an environment conducive to redox
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imbalance. Moreover, in a study conducted in diabetic rats, chronic DHEA treatment was detrimental to renal tissue, since it reduced the glomerular filtration rate and renal medulla metabolism [40]. However, it is important to emphasize that these effects are observed in chronic DHEA administration. Furthermore, as our model uses a single dose of DHEA, adverse effects are unlikely to be observed. In conclusion, acute treatment with DHEA mimics the response to acute stress indexed by increased activity of the HPA axis. Treatment with DHEA before stress exposure may facilitate an adaptive stress response, prolonging the SIA, which may be a therapeutic strategy of interest to clinics. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This study was supported by the following Brazilian funding agencies: National Council for Scientific and Technological Development — CNPq (Dr. Torres, I.L.S. (grant 14-0103)); Committee for the Development of Higher Education Personnel — CAPES (Dr. Ribeiro, M.F. (grant PNPD3149/2010)). References [1] G.P. Chrousos, P.W. Gold, The concepts of stress system disorders: overview of behavioral and physical homeostasis, J. Am. Med. Assoc. 267 (1992) 1244–1252. [2] G.P. Chrousos, Stress and disorders of the stress system, Nat. Rev. Endocrinol. 5 (2009) 374–381. [3] K. Pacák, M. Palkovits, Stressor specificity of central neuroendocrine responses: implications for stress-related disorders, Endocr. Rev. 224 (2001) 502–548. [4] J.P. Herman, H. Figueiredo, N.K. Mueller, Y. Ulrich-Lai, M.M. Ostrander, D.C. Choi, W.E. Cullinan, Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary adrenocortical responsiveness, Front. Neuroendocrinol. 24 (2003) 151–180. [5] S.L. Lightman, W.S. Young, Changes in hypothalamic preproenkephalin A mRNA following stress and opiate withdrawal, Nature 6131 (1987) 643–645. [6] S.L. Lightman, W.S. Young, Corticotrophin-releasing factor, vasopressin and proopiomelanocortin mRNA responses to stress and opiates in the rat, J. Physiol. 403 (1988) 511–523. [7] R.K. Butler, D.P. Finn, Stress-induced analgesia, Prog. Neurobiol. 88 (2009) 184–202. [8] M. al'absi, M. Nakajima, J. Grabowski, Stress response dysregulation and stressinduced analgesia in nicotine-dependent men and women, Biol. Psychol. 93 (2013) 1–8. [9] L.B. Hough, J.W. Nalwalk, W. Yang, X. Ding, Significance of neuronal cytochrome P450 activity in opioid-mediated stress-induced analgesia, Brain Res. 1578 (2014) 30–37. [10] L.F. Vendruscolo, F.A. Pamplona, R.N. Takahashi, Strain and sex differences in the expression of nociceptive behavior and stress-induced analgesia in rats, Brain Res. 1030 (2004) 277–283. [11] L. Stárka, M. Dusková, M. Hill, Dehydroepiandrosterone: a neuroactive steroid, J. Steroid Biochem. Mol. Biol. 145 (2015) 254–260. [12] N. Maninger, O.M. Wolkowitz, V.I. Reus, E.S. Epel, S.H. Mellon, Neurobiological and neuropsychiatric effects of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), Front. Neuroendocrinol. 30 (2009) 65–91. [13] A.K. Lennartsson, M.M. Kushnir, J. Bergquist, I.H. Jonsdottir, DHEA and DHEA-S response to acute psychosocial stress in healthy men and women, Biol. Psychol. 90 (2012) 143–149. [14] M. Hill, M. Dusková, L. Stárka, Dehydroepiandrosterone, its metabolites and ion channels, J. Steroid Biochem. Mol. Biol. 145 (2015) 293–314. [15] P.D. Kroboth, F.S. Salek, A.L. Pittenger, T.J. Fabian, R.F. Frye, DHEA and DHEA-S: a review, J. Clin. Pharmacol. 39 (1999) 327–348. [16] G. Naert, T. Maurice, L. Tapia-Arancibia, L. Givalois, Neuroactive steroids modulate HPA axis activity and cerebral brain-derived neurotrophic factor (BDNF) protein levels in adult male rats, Psychoneuroendocrinol 32 (2007) 1062–1078. [17] T.A. Obut, M.V. Ovsyukova, T.Y. Dementeva, O.P. Cherkasova, S.K. Saryg, Effects of dehydroepiandrosterone sulfate on the conversion of corticosterone into 1dehydrocorticosterone in stress: a regulatory scheme, Neurosci. Behav. Physiol. 39 (2009) 695–699. [18] L.L. Chang, W.S. Wun, L.L. Ho, P.S. Wang, Effects of dehydroepiandrosterone on corticosterone release in rat zona fasciculata–reticularis cells, Naunyn Schmiedeberg's Arch. Pharmacol. 368 (2003) 487–495. [19] L. Givalois, S. Li, G. Pelletier, Age-related decrease in the hypothalamic CRH mRNA expression is reduced by dehydroepiandrosterone (DHEA) treatment in male and female rats, Brain Res. Mol. Brain Res. 48 (1997) 107–114.
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DHEA administration modulates stress-induced analgesia in rats Ana Lúcia Cecconello a,c,⁎, Iraci L.S. Torres b,c, Carla Oliveira b, Priscila Zanini a, Gabriela Niches a, Maria Flávia Marques Ribeiro a,c a Laboratório de Interação Neuro-Humoral, Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Av. Sarmento Leite, 500, Porto Alegre, Rio Grande do Sul CEP 90050-170, Brazil b Laboratório de Farmacologia da Dor e Neuromodulação, Investigações Pré-Clinicas, Departamento de Farmacologia, ICBS, UFRGS, Av. Sarmento Leite, 500, Porto Alegre, Rio Grande do Sul CEP 90050-170, Brazil c Programa de Pos-Graduação em Ciências Biológicas, Fisiologia, ICBS, UFRGS, Av. Sarmento Leite, 500, Porto Alegre, Rio Grande do Sul CEP 90050-170, Brazil
H I G H L I G H T S • We evaluated the effect of DHEA on stress-induced analgesia in rats. • Acute treatment with DHEA had the same effect as the exposure to acute stress. • The treatment with DHEA prolongs acute stress-induced analgesia.
a r t i c l e
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Article history: Received 14 September 2015 Received in revised form 27 January 2016 Accepted 3 February 2016 Available online 4 February 2016 Keywords: Steroids Pain neuropathic Responses of fight or flight
a b s t r a c t An important aspect of adaptive stress response is the pain response suppression that occurs during or following stress exposure, which is often referred to as acute stress-induced analgesia. Dehydroepiandrosterone (DHEA) participates in the modulation of adaptive stress response, changing the HPA axis activity. The effect of DHEA on the HPA axis activity is dependent on the state and uses the same systems that participate in the regulation of acute stress-induced analgesia. The impact of DHEA on nociception has been studied; however, the effect of DHEA on stress-induced analgesia is not known. Thus, the aim of the present study was to evaluate the effect of DHEA on stress-induced analgesia and determine the best time for hormone administration in relation to exposure to stressor stimulus. The animals were stressed by restraint for 1 h in a single exposure and received treatment with DHEA by a single injection before the stress or a single injection after the stress. Nociception was assessed with a tail-flick apparatus. Serum corticosterone levels were measured. DHEA administered before exposure to stress prolonged the acute stress-induced analgesia. This effect was not observed when the DHEA was administered after the stress. DHEA treatment in non-stressed rats did not alter the nociceptive threshold, suggesting that the DHEA effect on nociception is state-dependent. The injection of DHEA had the same effect as exposure to acute stress, with both increasing the levels of corticosterone. In conclusion, acute treatment with DHEA mimics the response to acute stress indexed by an increase in activity of the HPA axis. The treatment with DHEA before stress exposure may facilitate adaptive stress response, prolonging acute stress-induced analgesia, which may be a therapeutic strategy of interest to clinics. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Individuals exposed to threatening situations (stressor) present physical and behavioral changes (stress response) with the objective of maintaining homeostasis [1, 2, 3]. Stress responses mainly include activation of the hypothalamic-pituitary-adrenocortical (HPA) axis, and induction of the corticotropin-releasing hormone (CRH) and
⁎ Corresponding author at: Departamento de Fisiologia, ICBS, Universidade Federal do Rio Grande do Sul, R. Sarmento Leite 500, Porto Alegre RS 90170-050, Brazil. E-mail address: [email protected] (A.L. Cecconello).
http://dx.doi.org/10.1016/j.physbeh.2016.02.004 0031-9384/© 2016 Elsevier Inc. All rights reserved.
vasopressin (VP) by parvocellular neurons of the paraventricular nucleus of the hypothalamus (PVN) [2]. CRH acts on the anterior pituitary gland, releasing adrenocorticotrophic hormone (ACTH), which in turn stimulates the release of glucocorticoids by the adrenal cortex. During the stress response, the PVN receives information from brainstem afferents, among them the locus coeruleus, an ascending noradrenergic pathway with an important role in HPA activation [4], inducing an increase in the synthesis of CRH and VP [5, 6]. The PVN is a central integrator of endocrine, vegetative and behavioral responses [2]. An important aspect of the adaptive stress response is the pain response suppression that occurs during or following stress exposure. It contributes to the expression of appropriate behaviors to face stressors,
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facilitating the responses of fight or flight [7, 8]. This phenomenon is often referred to as stress-induced analgesia (SIA), and occurs in both laboratory animals and humans [7, 8]. SIA involves activation of the descending inhibitory pain pathway, originating in the neurons of the cortex, hypothalamus and amygdala, and project into the periaqueductal gray and rostroventral medulla and finally to the dorsal horn of the spinal cord. Activation of this pathway results in an inhibitory effect at the level of the dorsal horn by inhibiting the ascending transmission of nociceptive information [7]. It is known that acute stress-induced analgesia involves the modulation of opioid receptors [9] and nonopioid receptors (e.g. NMDA and GABA receptors) [10]. In addition, corticosterone can facilitate analgesia induced by stress, since this phenomenon may be blocked by adrenalectomy and reestablished by treatment with the administration of corticosterone [7]. Another hormone that participates in the modulation of adaptive stress response is the adrenal hormone dehydroepiandrosterone (DHEA). DHEA is considered a neuroactive steroid, and a crucial endogenous modulator of numerous physiological functions [11, 12]. The main biological functions of DHEA on the nervous system are neuroprotection, catecholamine synthesis and secretion, as well as antioxidant and anti-inflammatory activities [12]. DHEA has been shown to modulate various receptors, such as GABA(A), N-methyl-D-aspartate (NMDA), kainate, ionotropic glutamate, nicotinic acetylcholine, muscarinic, glycine, σ1 and neurotrophin receptors and ionic channels such as calcium, sodium and potassium channels [13, 14]. In humans, elevated levels of DHEA have been observed in response to stress exposure [13]. DHEA may be secreted in response to ACTH in humans at least [15], and it modulates HPA axis activity [16, 17]. In an in vitro study, ACTH-induced corticosterone release by rat adrenal zona fasciculatereticularis cells was attenuated by DHEA [18]. During repeated stress exposure, a single injection of DHEA can reduce the serum corticosterone levels [17]. On the other hand, it was observed that a single injection of DHEA stimulates the secretion of hypothalamic CRH, ACTH by the pituitary and corticosterone by the adrenal cortex in nonstressed rats [16]. In addition, chronic DHEA treatment increases CRH mRNA levels in hypothalamic PVN independent of age and sex in nonstressed rats [19], suggesting that the effect of DHEA on the HPA axis activity is dependent on state. The impact of DHEA on nociception has been studied [20, 21], although it is still not well understood. DHEA effects on nociceptive mechanisms are complex. Acute DHEA treatment exerts a biphasic effect on nociception (a rapid pro-nociceptive action and a delayed antinociceptive effect). On the other hand, chronic treatment with DHEA elevates the nociceptive threshold [21, 22]. However, the effect of DHEA on stress-induced analgesia is not known. SIA may be thought of as an important component of the fight or flight response [7]. The understanding of the fundamental mechanism of pain suppression that occurs during or following exposure to stress and how this effect can be modulated becomes a potential new therapeutic target for pain and stress-related disorders. Thus, the aim of the present study was to evaluate the effect of DHEA on stress-induced analgesia and determine the best time for hormone administration in relation to exposure to stressor stimulus. 2. Material and methods 2.1. Animals Male adult Wistar rats (60–70 days, mean weight 300 g) were obtained from the Center for Laboratory Animal Reproduction and Research (CREAL) at the Universidade Federal do Rio Grande do Sul (UFRGS). The number of animals was four rats per cage with food and water available ad libitum and they were maintained in a 12 h light/ dark cycle (lights on at 7:00 a.m. and lights off at 7.00 p.m.) in a humidity- and temperature-controlled environment (22 ± 2 °C).
Initially, the rats were divided into four animals per cage and acclimated to the vivarium for one week before beginning treatment. After the acclimation period, the animals were randomly selected by weight and subsequently divided into a control group (rats were housed only with other control rats) and stress group (rats that underwent restraint stress were housed only with other rats that underwent restraint stress). They were handled for 14 days prior to the experiments. All experiments and procedures were approved by the Institutional Animal Care and Use Committee (CEUA-UFRGS protocol No. 19788) and were compliant with Brazilian guidelines involving the use of animals in research (Law No. 11.794). Additionally, all efforts were made to minimize the suffering, pain and discomfort of the animals, as well as to reduce the number of animals. The animals were euthanized 15 min after the last measure of TFL and the trunk blood collected. Death by decapitation was carried out by a trained professional and in a separate room to where they were experiencing stress and treatment. 2.2. Stress procedures The animals were stressed by restraint for 1 h in a single exposure [23]. Restraint stress was carried out by placing each animal in a 25 × 7 cm plastic tube, and adjusting it with plaster tape on the outside so that the animal was unable to move. There was a 1 cm hole in the far end for breathing. Control animals were manipulated, but not submitted to restraint. Stressed animals were submitted to a single exposure to the restraint. The immobilization procedure was carried out between 1000 and 1200 h a.m. 2.3. Treatment Each group (stressed and control) was subdivided into three treatment groups (n = 7–8 per group): 1) injection of DHEA 30 min before the stress and injection of vehicle 30 min after the stress; 2) injection of vehicle 30 min before the stress and injection of DHEA 30 min after the stress; and 3) injection of vehicle 30 min before the stress and injection of vehicle 30 min after the stress (Fig. 1). Animals received DHEA (Calbiochem) as a single i.p. dose of 25 mg/kg diluted in 20% cyclodextrin. 2.4. Tail-flick measures Nociception was assessed with a tail-flick apparatus. Rats were wrapped in a towel and placed on the apparatus. A photo beam with adjustable sensitivity detects the tail flick and the latency is automatically presented on a digital display on the control unit. The light source positioned below the tail was focused on a point 2.3 cm rostral to the tip of the tail. Deflection of the tail activated a photocell and automatically terminated the trial. Light intensity was adjusted to obtain the baseline tail-flick latency (TFL) of 4–6 s as described in the manufacturer's protocol (LE7106 Tail-flick Meter/Harvard Apparatus). Measurements of reaction time are given with a 0.1 second precision. Once this intensity had been established, rats with baseline latency N 7 s and b3 s were excluded from the experiment. A cutoff time of 10 s was used to avoid tissue damage. The groove system for the tail and the adjustment of response sensitivity ensure optimum repeatability and reliability of results. The general procedure was as follows [23, 24]: Day 1, the animals were familiarized with the apparatus; day 2, the baseline TFL value was obtained; day 3, animals were subjected to three TFL measurements for the analgesia test. The first TFL measurement was taken 15 min after the first injection, the second immediately after stress and the third 15 min after the second injection (Fig. 1). 2.5. Corticosterone serum concentration The rats were killed and trunk blood samples were collected in tubes containing clot activator gel (BD, Vacutainer®). After centrifugation at
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Fig. 1. Representative diagram of the experimental design.
1000 ×g for 15 min, the serum was separated and stored at −20 °C until analysis. Serum corticosterone levels were measured by enzyme-linked immunosorbent assay (ELISA) (Corticosterone EIA Kit 500655, Cayman Chemical). 2.6. Data analysis Levene's test was used to check for homogeneity of variance. When the data showed homogenous variance, repeated measures analysis of variance (ANOVA) (Tail-flick measures) or two-way analysis of variance (Corticosterone serum concentration: treatment × stress) or one-way analysis of variance (Corticosterone serum concentration: treatment) followed by the post hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). p-Values b0.05 were considered significant.
levels immediately after the stress, and reduced levels after 30 min of recovery [13]. The impact of DHEA on nociception has been studied [20, 21]; however, the effect of DHEA on stress-induced analgesia is not known. In this pre-clinical model, we propose that DHEA administration before exposure to stress such as invasive procedures may facilitate adaptive stress response, prolonging acute stress-induced analgesia. The most important is that experimental models can help to elucidate the fundamental mechanisms of nociceptive threshold increase that occurs in situations of acute stress. Studies of stressinduced analgesia have enhanced our understanding of the fundamental physiology of pain and stress, and can be a useful approach for uncovering new therapeutic targets for the treatment of pain and
3. Results 3.1. Effects of DHEA administration on stress-induced analgesia (SIA) Repeated measures ANOVA showed an effect of stress. As can be observed, the animals presented increased tail-flick latency immediately after being stressed, regardless of treatment with DHEA (F = 8.88; p b 0.05 — DHEA before the stress; F = 5.57; p b 0.05 — DHEA after the stress) (Fig. 2A and B). Moreover, a significant interaction between stress and DHEA treatment was observed only when the injection of DHEA was administered before the stress (F = 6.37; p b 0.05) (Fig. 2A). When the DHEA was administered before the stress exposure, we observed stress-induced analgesia (SIA), which was maintained for a longer time (until 45 min after stress) (Fig. 2A). On the other hand, when the DHEA was administered after the stress, this effect was not observed (Fig. 2B). Moreover, the DHEA treatment did not alter the TFL latency in non-stressed rats (Fig. 2). 3.2. Effects of exposure to restraint stress and DHEA treatment on serum corticosterone levels Two-way ANOVA showed a significant interaction between stress and DHEA treatment both when the DHEA was administered before (F = 7.63; p b 0.05) and after (F = 13.28; p b 0.05) the stress (Fig. 3A and B). In the animals that received vehicle the stress produced an increase in serum corticosterone levels. However, in the animals that received DHEA the stress produced no effect on serum corticosterone levels. Nonstressed animals showed an increase in serum corticosterone levels when treated with DHEA. One-way ANOVA showed that nonstressed animals that received DHEA 150 min before blood was collected presented concentrations of corticosterone that were lower than those observed in animals that received DHEA 30 min before blood was collected (F = 21.97; p b 0.05) (Fig. 4). 4. Discussion As a positive aspect and from an evolutionary perspective, SIA may be thought of as a component of the fight or flight response [7]. Interestingly, DHEA is involved in the modulation of adaptive stress response, changing the HPA axis activity [7, 8]. In humans, significantly elevated levels of DHEA have been observed in response to acute stress. The predominant pattern for DHEA levels during the acute stress was increased
Fig. 2. Effect of acute stress upon TFLs on DHEA-treated rats. The animals were restrained for 1 h and subjected to three TFL measurements. (A) Injection of DHEA administered before the stress: basal = 24 h before; TFL 1 = 15 min after injection of DHEA — 25 mg/ kg; TFL 2 = immediately after stress; TFL 3 = 45 min after stress (15 min after injection of vehicle). (B) Injection of DHEA administered after the stress: basal = 24 h before; TFL 1 = 15 min after injection of vehicle; TFL 2 = immediately after stress; TFL 3 = 45 min after stress (15 min after injection of DHEA — 25 mg/kg). Repeated measures ANOVA followed by the post-hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). *p b 0.05 stress vs. control; #p b 0.05 stress-DHEA vs. all others.
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Fig. 3. Effect of acute stress upon serum corticosterone levels on DHEA-treated rats. (A) Injection of DHEA (25 mg/kg) administered before the stress. (B) Injection of DHEA administered after the stress: Two-way ANOVA followed by the post-hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). Different letters represent p b 0.05.
stress-related disorders. In this way, we can propose that DHEA administration may be a therapeutic tool in pain management. In this study, we showed stress-induced analgesia (Fig. 2), corroborating previous literature reports [7, 23], and this effect was potentiated by DHEA treatment. Given that the effect of treatment with DHEA on SIA depends on the time at which the intervention with DHEA is made in relation to the exposure to stressor stimulus (Fig. 2A and B), we suggest that the DHEA effect on nociception is state-dependent like the DHEA effect on the HPA axis activity [19]. It is interesting to note that the effect of prolonging SIA occurs only when the intervention with DHEA is made at the same time as the HPA axis is in the basal state (Fig. 2A). It has been observed that a single injection of DHEA stimulates the secretion of CRH by the hypothalamus, ACTH by the pituitary and corticosterone by the adrenal cortex in nonstressed rats [16]. In the present study, it was observed that the treatment with DHEA in nonstressed rats was able to increase the secretion of corticosterone (Fig. 3). It is known that both stress and treatment with DHEA stimulate the secretion of corticosterone [13, 16]. Based on the fact that corticosterone facilitates SIA, since this phenomenon may be blocked by adrenalectomy and reestablished by treatment with corticosterone [7], the effect of prolonging the SIA could have been caused by a more durable increase of serum corticosterone levels induced by the action of DHEA followed by stressor exposure. It was observed that the stressed animals that received vehicle showed an increase in serum corticosterone levels (Fig. 3A and B),
corroborating previous literature reports [2, 3]. However, in the stressed animals, DHEA showed no significant difference in the serum corticosterone levels, irrespective of the time at which the intervention with DHEA was made. It is possible that when the HPA axis is in a state of maximum activation induced by acute stress, intervention with DHEA is not able to stimulate the axis activity further. As can be seen in Fig. 3, the injection of DHEA had the same effect as the exposure to acute stress, with both of them increasing the levels of corticosterone. In addition, the animals that received DHEA 150 min before blood was collected presented concentrations of corticosterone that were lower than those observed in animals that received DHEA 30 min before blood was collected (Fig. 4). This result is in agreement with a previous study [16] that showed that a single injection of DHEA, in the same dose as that used in the present model, increases the concentration of corticosterone. It reached a maximal concentration at 60 min, and although this was the lowest concentration, it was still significantly elevated 300 min after DHEA injection. It is interesting to note that the pattern of secretion of DHEA-induced corticosterone [16] is similar to that of corticosterone induced by acute stress [3]. This suggests that acute treatment with DHEA can mimic a situation of acute stress. This rapid effect of DHEA on the activity of the HPA axis is in accordance with the model of action of neurosteroids on CNS functions, which rapidly alter neuron excitability, in seconds to minutes, by binding to membrane-bound receptors for inhibitory and excitatory neurotransmitters [12, 14, 25]. DHEA generally acts as a noncompetitive antagonist at GABAA receptors and as a positive allosteric modulator of the NMDA receptor [12]. Moreover, DHEA can potentiate NMDA receptor function by its actions as a sigma receptor agonist [12, 25]. HPA axis activity is under GABA, opioid and glutamate control, since microinjection of GABA antagonists, such as bicuculline, into the PVN activates the HPA axis [26]; also, intracerebroventricular (i.c.v.) administration of morphine stimulates the HPA axis [27]. Moreover, exposing a rat to a single stressor increases the activity of NMDA receptors in the paraventricular nucleus of the hypothalamus [28]. This indicates that the rapid DHEA effects on the HPA axis could be mediated through negative modulation of the GABAA receptor and positive modulation of NMDA and opioid receptors. Interestingly, DHEA modulates the activity of the HPA axis using the same systems that participate in the regulation of SIA [7, 10]. SIA may be opioid or nonopioid in nature [7], and depend on the stress severity, the pain test employed, and the sex and genotype of the animal used [7, 10]. It has been hypothesized that a stressor of mild severity produces a naloxone-sensitive SIA (opioid), whereas a more severe stressor induces a naloxone-insensitive SIA (nonopioid)
Fig. 4. Effect of injections of DHEA upon serum corticosterone levels on rats. DHEA 30 min = injection of DHEA (25 mg/kg) 30 min before blood collect. DHEA 150 min = injection of DHEA (25 mg/kg) 150 min before blood collect. One-way ANOVA followed by the post-hoc Tukey-Kramer test was used for statistical analysis (presented as mean ± SEM). Different letters represent p b 0.05.
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that can be blocked by MK-801, a noncompetitive NMDA receptor antagonist [10, 29]. Studies employing GABA receptor agonists (muscimol) and antagonists (bicuculline) have established a role for GABA receptor in some forms of SIA [7]. In a published study of the effect of an inflammatory pain model and stress on the antinociceptive effect in mice it was shown that immobilization stress activated the hypothalamic proopiomelanocortin (POMC) gene and β-endorphin expression [30]. It is well established that the POMC is the precursor of βendorphin, but also of ACTH, which activates the release of corticosterone [3]. However, during the expression of SIA, a reduction in the expression of the POMC gene is observed [30], which possibly occurs due to negative feedback of corticosterone in the hypothalamus diminishing the expression of the POMC gene. These findings suggest that a single immobilization stress activates the descending pain modulatory system, which is mainly mediated through endorphinergic activation. Interestingly, POMC-containing neurons project reciprocally to the PVN (neurons of the paraventricular nuclei), CRH (corticotropin-releasing hormone) and AVP (arginine-vasopressina) neurons, innervate LC/NE-sympathetic neurons (locus ceruleus/norepinephrinesympathetic system) of the central stress system in the brainstem and terminate pain control neurons of the hind brain and spinal cord. Consequently, activation of the stress system, via CRH and catecholamines, stimulates the hypothalamic β-endorphin and other POMC-peptide secretion, which reciprocally inhibits the activity of the stress system, and produces the stress-induced analgesia [31]. It is interesting to mention that the POMC promotes SIA through the action of β-endorphin and by being the precursor of ACTH, which stimulates the release of corticosterone. Furthermore, stress induces analgesia through mechanisms within and outside the brain. Another study demonstrated that sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue [32]. In this case, the antinociception induced by acute stress was significantly attenuated by the adrenergic antagonists propranolol and phentolamine, indicating that sympathetic neurotransmitters (e.g. noradrenaline) mediate stress-induced peripheral opioid analgesia [32], thereby acting in parallel with the activation of the hypothalamic-pituitary-adrenocortical axis to promote SIA. Interestingly, DHEA can induce an increase in POMC mRNA levels [33] and sympathetic activation [34]. Given that DHEA modulates HPA axis activity, GABA, NMDA and opioid receptors and sympathetic nervous system activity, we can suggest that DHEA may prolong SIA by modulating one or more pathways that control SIA. This is possibly, by effect of higher corticosterone serum levels by more time, increased modulation of the GABAergic, glutamatergic and/or opioid pathways and/or sympathetic activation, or even due to the complex involvement of all of these neurotransmission systems. However, the exact mechanism by which DHEA prolongs the effect of SIA is still unknown. Since DHEA is a natural steroid and precursor hormone produced by the adrenals in humans, its adverse effects are gender specific [35]. It is important to note that studies using oral or percutaneous DHEA administration with doses of up to 1600 mg taken by mouth daily for one month have been well tolerated [36, 37]. An interesting issue is that a dose of 50 mg of DHEA taken by mouth daily has been shown to be safe for up to six months [37]. Most importantly, in women, only minimal adverse effects have been described, such as mild acne, seborrhea, facial hair growth and ankle swelling [37]. It has also been proposed that in animal models of chronic DHEA administration (10 mg/kg/ week for 5 weeks), it exerts a dual effect [38]. These effects can be antioxidant or pro-oxidant, depending on dose and on the tissue specificity [38]. After DHEA treatment, SOD activity (antioxidant enzyme) was significantly decreased on heart tissue [38]. On the other hand, lipid peroxidation in rat erythrocytes was significantly increased [38]. In another study, Jahn et al. [39] report that DHEA administration reduced the levels of proteins essential for cell survival (p-AKT) and also thioredoxin (the most important mechanism for the regulation of the redox balance) in muscle tissue, showing an environment conducive to redox
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imbalance. Moreover, in a study conducted in diabetic rats, chronic DHEA treatment was detrimental to renal tissue, since it reduced the glomerular filtration rate and renal medulla metabolism [40]. However, it is important to emphasize that these effects are observed in chronic DHEA administration. Furthermore, as our model uses a single dose of DHEA, adverse effects are unlikely to be observed. In conclusion, acute treatment with DHEA mimics the response to acute stress indexed by increased activity of the HPA axis. Treatment with DHEA before stress exposure may facilitate an adaptive stress response, prolonging the SIA, which may be a therapeutic strategy of interest to clinics. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This study was supported by the following Brazilian funding agencies: National Council for Scientific and Technological Development — CNPq (Dr. Torres, I.L.S. (grant 14-0103)); Committee for the Development of Higher Education Personnel — CAPES (Dr. Ribeiro, M.F. (grant PNPD3149/2010)). References [1] G.P. Chrousos, P.W. Gold, The concepts of stress system disorders: overview of behavioral and physical homeostasis, J. Am. Med. Assoc. 267 (1992) 1244–1252. [2] G.P. Chrousos, Stress and disorders of the stress system, Nat. Rev. Endocrinol. 5 (2009) 374–381. [3] K. Pacák, M. Palkovits, Stressor specificity of central neuroendocrine responses: implications for stress-related disorders, Endocr. Rev. 224 (2001) 502–548. [4] J.P. Herman, H. Figueiredo, N.K. Mueller, Y. Ulrich-Lai, M.M. Ostrander, D.C. Choi, W.E. Cullinan, Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary adrenocortical responsiveness, Front. Neuroendocrinol. 24 (2003) 151–180. [5] S.L. Lightman, W.S. Young, Changes in hypothalamic preproenkephalin A mRNA following stress and opiate withdrawal, Nature 6131 (1987) 643–645. [6] S.L. Lightman, W.S. Young, Corticotrophin-releasing factor, vasopressin and proopiomelanocortin mRNA responses to stress and opiates in the rat, J. Physiol. 403 (1988) 511–523. [7] R.K. Butler, D.P. Finn, Stress-induced analgesia, Prog. Neurobiol. 88 (2009) 184–202. [8] M. al'absi, M. Nakajima, J. Grabowski, Stress response dysregulation and stressinduced analgesia in nicotine-dependent men and women, Biol. Psychol. 93 (2013) 1–8. [9] L.B. Hough, J.W. Nalwalk, W. Yang, X. Ding, Significance of neuronal cytochrome P450 activity in opioid-mediated stress-induced analgesia, Brain Res. 1578 (2014) 30–37. [10] L.F. Vendruscolo, F.A. Pamplona, R.N. Takahashi, Strain and sex differences in the expression of nociceptive behavior and stress-induced analgesia in rats, Brain Res. 1030 (2004) 277–283. [11] L. Stárka, M. Dusková, M. Hill, Dehydroepiandrosterone: a neuroactive steroid, J. Steroid Biochem. Mol. Biol. 145 (2015) 254–260. [12] N. Maninger, O.M. Wolkowitz, V.I. Reus, E.S. Epel, S.H. Mellon, Neurobiological and neuropsychiatric effects of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), Front. Neuroendocrinol. 30 (2009) 65–91. [13] A.K. Lennartsson, M.M. Kushnir, J. Bergquist, I.H. Jonsdottir, DHEA and DHEA-S response to acute psychosocial stress in healthy men and women, Biol. Psychol. 90 (2012) 143–149. [14] M. Hill, M. Dusková, L. Stárka, Dehydroepiandrosterone, its metabolites and ion channels, J. Steroid Biochem. Mol. Biol. 145 (2015) 293–314. [15] P.D. Kroboth, F.S. Salek, A.L. Pittenger, T.J. Fabian, R.F. Frye, DHEA and DHEA-S: a review, J. Clin. Pharmacol. 39 (1999) 327–348. [16] G. Naert, T. Maurice, L. Tapia-Arancibia, L. Givalois, Neuroactive steroids modulate HPA axis activity and cerebral brain-derived neurotrophic factor (BDNF) protein levels in adult male rats, Psychoneuroendocrinol 32 (2007) 1062–1078. [17] T.A. Obut, M.V. Ovsyukova, T.Y. Dementeva, O.P. Cherkasova, S.K. Saryg, Effects of dehydroepiandrosterone sulfate on the conversion of corticosterone into 1dehydrocorticosterone in stress: a regulatory scheme, Neurosci. Behav. Physiol. 39 (2009) 695–699. [18] L.L. Chang, W.S. Wun, L.L. Ho, P.S. Wang, Effects of dehydroepiandrosterone on corticosterone release in rat zona fasciculata–reticularis cells, Naunyn Schmiedeberg's Arch. Pharmacol. 368 (2003) 487–495. [19] L. Givalois, S. Li, G. Pelletier, Age-related decrease in the hypothalamic CRH mRNA expression is reduced by dehydroepiandrosterone (DHEA) treatment in male and female rats, Brain Res. Mol. Brain Res. 48 (1997) 107–114.
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