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Published as doi: 10.1096/fj.07-8930com.
 (The FASEB Journal. 2008;22:93-104.)
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(The FASEB Journal. 2008;22:93-104.)
© 2008 FASEB

Biochemical and functional evidence for the control of pain mechanisms by dehydroepiandrosterone endogenously synthesized in the spinal cord

Cherkaouia Kibaly1, Laurence Meyer1, Christine Patte-Mensah and Ayikoe G. Mensah-Nyagan2

Equipe Stéroïdes et Système Nociceptif, Institut des Neurosciences Cellulaires et Intégratives, Unité Mixte de Recherche 7168/LC2-Centre National de la Recherche Scientifique, Université Louis Pasteur, Département Nociception et Douleur, Strasbourg Cedex, France

2Correspondence: Equipe Stéroïdes et Système Nociceptif, Institut des Neurosciences Cellulaires et Intégratives, Unité Mixte de Recherche 7168/LC2-Centre National de la Recherche Scientifique, Université Louis Pasteur, Département Nociception et Douleur, 21 rue René Descartes, 67084 Strasbourg Cedex, France. E-mail: gmensah{at}neurochem.u-strasbg.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the role and mechanism of action of dehydroepiandrosterone (DHEA) produced by the spinal cord (SC) in pain modulation in sciatic-neuropathic and control rats. Real-time polymerase chain reaction (PCR) after reverse transcription revealed cytochrome P450c17 (DHEA-synthesizing enzyme) gene repression in neuropathic rat SC. A combination of pulse-chase experiments, high performance liquid chromatography (HPLC), and flow-scintillation detection showed decreased DHEA biosynthesis from pregnenolone in neuropathic SC slices. Radioimmunoassays demonstrated endogenous DHEA level drop in neuropathic SC. Behavioral analysis showed a rapid pronociceptive and a delayed antinociceptive action of acute DHEA treatment. Inhibition of DHEA biosynthesis in the SC by intrathecally administered ketoconazole (P450c17 inhibitor) induced analgesia in neuropathic rats. BD1047 (sigma-1 receptor antagonist) blocked the transient pronociceptive effect evoked by acute DHEA administration. Chronic DHEA treatment increased and maintained elevated the basal nociceptive thresholds in neuropathic and control rats, suggesting that androgenic metabolites generated from daily administered DHEA exerted analgesic effects while DHEA itself (before being metabolized) induced a rapid pronociceptive action. Indeed, intrathecal administration of testosterone, an androgen deriving from DHEA, caused analgesia in neuropathic rats. Together, these molecular, biochemical, and functional results demonstrate that DHEA synthesized in the SC controls pain mechanisms. Possibilities are opened for pain modulation by drugs regulating P450c17 in nerve cells.—Kibaly, C., Meyer, L., Patte-Mensah, C., Mensah-Nyagan, A. G. Biochemical and functional evidence for the control of pain mechanisms by dehydroepiandrosterone endogenously synthesized in the spinal cord.


Key Words: biochemistry of steroidogenic enzymes • cytochrome P450c17 • HPLC • neurosteroids • real-time PCR


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FOR SEVERAL YEARS, dehydroepiandrosterone (DHEA) has been misleadingly advertised as capable of reducing various physiological deficits associated with aging. Paradoxically, scientific proof supporting these assertions is rare. It is true that DHEA and its sulfate derivative (DHEAS) are the most abundant steroids secreted by the human adrenal gland and that decreased DHEA plasma levels are observed during aging, but beneficial effects as well as mechanisms of action of DHEA remain a matter of controversy (1 , 2) . Because exogenous DHEA administration modulates various neurobiological mechanisms in animals (2) and a few clinical trials correlated DHEA plasma concentrations to the severity of symptoms in certain diseases (3 , 4) , DHEA has been considered by the media as a crucial endogenous modulator of numerous physiological functions. Therefore, despite the explicit reservations expressed by the clinical report of Baulieu et al. (5) about the potential actions of DHEA, millions of people in the United States and Europe continue to use DHEA (mostly without any medical supervision) as a molecule that may help them to cope with various physiological deficiencies. Unlike in humans, plasma levels of DHEA are extremely low or undetectable in adult rodents. Furthermore, cytochrome P450c17 (P450c17), the key DHEA-synthesizing enzyme, is expressed by the adrenals in humans but not in rodents (2 , 6) . Consequently, even though administration of synthetic DHEA affects several processes in the central nervous system (CNS) of adult rodents (1 , 2) , the role of DHEA as an endogenous neuromodulator is possible only if DHEA is produced by nerve cells. We have recently provided molecular and neurochemical evidence showing that the key DHEA-producing enzyme P450c17 is present in the adult rat spinal cord (SC) and that DHEA synthesis occurs in neural networks of the SC dorsal horn, which plays a pivotal role in nociception (7) .

The original aim of the present study is to verify whether there are direct links between important neurobiological functions such as nociception or pain sensitivity and cellular, molecular, and biochemical components determining DHEA synthesis in the CNS. In particular, we investigated whether or not DHEA endogenously synthesized in the SC (a pivotal structure controlling pain transmission) is involved in the regulation of nociception, which is well known as a crucial function in humans and several animal species. To reach our goal, we combined molecular, biochemical, behavioral, and pharmacological approaches to study the effects as well as the mechanism of action of DHEA produced by the SC on mechanical and thermal nociceptive thresholds in naive, sciatic neuropathic (8) and control (sham-operated) rats. Modifications of P450c17 gene expression were investigated in the SC of neuropathic rats using a highly sensitive tool, real-time polymerase chain reaction (rt-PCR) after reverse transcription. A well-validated method combining pulse-chase experiments, high-performance liquid chromatography analysis (HPLC), and flow scintillation detection (9 10 11 12) allowed in vitro investigation of P450c17 activity in the SC of neuropathic and control rats. Moreover, the effect of sciatic neuropathy on DHEA synthesis by the SC in vivo was assessed by radioimmunoassays after HPLC purification of tissue extracts. Behavioral tests were performed to determine the effects of subcutaneously or intrathecally administered DHEA or ketoconazole, a pharmacological P450c17 inhibitor (13 , 14) , on thermal and mechanical pain thresholds. Finally, the mechanism of action of DHEA in the control of nociception was determined by investigating the possible involvement of type 1 sigma receptors (r{sigma}1), which mediate DHEA-induced potentiation of glutamatergic neurotransmission (15 16 17 18) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male Sprague-Dawley rats weighing 250–350 g were used. Animal care and manipulations were performed according to the European Community Council Directives (86/609/EC) and under the supervision of authorized investigators. The animals were obtained from a commercial source (Harlan, Le Malcourlet, France) and housed under standard laboratory conditions in a 12 h light/dark cycle with food and water ad libitum. Surgical operations were made under ketamine (75 mg/kg)/xylasine (5 mg/kg) anesthesia. The neuropathic pain was produced according to the protocol described by Bennett and Xie (8) , and all experiments followed the International Association for the Study of Pain ethical guidelines (19) . Briefly, after dissection at the middle of the thigh, 4–5 mm of the common sciatic nerve was tied loosely with 3 ligatures spaced by 1 mm. A total of 69 neuropathic rats was used for the whole study. Various groups of control rats were used: some were not subjected to surgery (112 naive rats) and others (69 sham-operated rats) were subjected to sham operations (exposure of the right or left sciatic nerve without ligature). The animals were inspected every day to observe their recovery from the surgical operation and parameters indicating the occurrence of neuropathic pain including the gait, posture of the affected hind paw, and condition of claws (8) . Animals were euthanized 10 days after the sciatic nerve ligature for all experiments except for behavioral time-course studies or investigations of the effects of chronic drug administrations that were performed from days 3 to 18.

Chemicals and reagents
Synthetic steroids were purchased from Steraloids (Newport, RI, USA). Ketoconazole and (2-hydroxypropyl)-β-cyclodextrin 45% in water (CDEX) were supplied by Sigma. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen Corporation (Paisley, UK). Tritiated steroids were obtained from PerkinElmer (Boston, MA, USA). The selective r{sigma}1 antagonist BD1047 was kindly provided by Dr. Maurice (Montpellier, France).

Reverse transcription and real-time PCR
Extraction of RNA from the testis and SC and reverse transcription experiments were performed as described previously (7 , 20) . rt-PCR was performed using a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). The specific primer sequences for P450c17 were forward, 5'-GACCAAGGGAAAGGCGT-3' (nucleotides 351–368) and reverse, 5'-GCATCCACGATACCCTC-3' (nucleotides 636–653) (21) . The primers for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were forward, 5'-ACCACAGTCCATGCCATCAC-3' (nucleotides 3,069–3,088) and reverse, 5'-TCCACCACCCTGTTGCTGTA-3' (nucleotides 3,624–3,605; Clontech Inc., Palo Alto, CA, USA). PCR was performed according to the same procedure described by Kibaly et al. (7) . P450c17 mRNA concentration was calculated after normalization of rt-PCR P450c17 product to GAPDH. For each sample, the rt-PCR experiment was repeated four times.

Pulse-chase-HPLC
For each experiment, 220 mg of SC (lumbar segment) slices were preincubated for 15 min in 2 ml 0.9% NaCl at 37°C. The SC slices were incubated at 37°C for 3 h in 1.5 ml of DMEM (pH 7.4) containing 100 nM tritiated pregnenolone ([3H]PREG) supplemented with 1% propylene glycol. Details on the incubation procedure, extraction of newly synthesized neurosteroids released by SC slices, and prepurification of these neurosteroids have been published previously (7 , 11 , 12 , 20) .

HPLC-Flo/One characterization of steroids
The newly synthesized steroids extracted from the incubation medium or tissue slices were purified on Sep-Pak cartridges and then characterized using a previously validated method that combines HPLC analysis and flow scintillation detection (7 , 9 10 11 12) .

Quantification of steroid biosynthesis
The amount of radioactive steroids formed by the conversion of [3H]PREG was expressed as a percentage of the total radioactivity contained in all peaks resolved by the HPLC-Flo/One system, including [3H]PREG itself.

Radioimmunoassays
Endogenous DHEA was extracted from the lumbar SC with dichloromethane as described previously (7 , 11 , 12 , 20) . After HPLC purification of SC tissue extracts, DHEA concentrations were determined by radioimmunoassays using antiserum against DHEA courteously provided by Dr. Y. Akwa (Le Kremlin-Bicêtre, France). The final dilution of the anti-DHEA was 1:3500.

Direct transcutaneous intrathecal injection
Intrathecal injections were performed as described by Mestre et al. (22) . The animals were first anesthetized for no more than 2 min with a mixture of 4% halothane in O2/N2O (30:70 v/v). Then, a 26G x 1/2'' needle connected to a 50 µl Hamilton syringe was inserted through the vertebral column into the subarachnoid space between vertebrae L4 and L5. Successful placement was indicated by a stereotypical tail flick reflex: when the needle entered the subarachnoid space, a sudden lateral movement of the tail was observed (22) . DHEA, ketoconazole, BD1047 or testosterone was injected in a volume of 20 µl/250 g in CDEX (45% in water) vehicle.

Nociceptive behavioral tests
Thermal hyperalgesia was assessed by using a plantar test apparatus (Ugo Basile, Comerio, Italy) which measures the paw withdrawal latency in response to radiant heat (23) . The rats were first allowed to habituate to the experimental room for at least 2 h and then to the apparatus for 10 min before testing. After subcutaneous or intrathecal injection, each rat was placed individually in clear Plexiglas boxes (23x18x14 cm) positioned on a clear plastic surface. The heat source was then positioned under the plantar surface of the hind paw and activated with an infrared light beam. The heat source is connected to a timer that automatically switched off the heat when the paw was withdrawn. A cut-off time of 20 s was used to prevent tissue damage in absence of response. The mean paw withdrawal latencies (in seconds) for the ipsilateral and contralateral hind paws were determined from an average of three separate measures on each hind paw (a total of 6 measures per animal) at a given time point. The testing box was thoroughly cleaned between each test session.

The mechanical nociceptive sensitivity threshold was evaluated in individual rats placed in Plexiglas boxes (30x30x25 cm) on an elevated metal grid allowing access to the plantar surface of the hind paws. The presence of mechanical allodynia was assessed using a series of calibrated von Frey hairs (4, 6, 8, 10, 15, 26, 60, 100, 180, and 300 g; Stoelting, Wood Dale, IL, USA), which were applied to the plantar surface of the hind paw with increasing force until the individual filament used just started to bend. The filament was applied for a period of 1–2 s and the procedure was repeated five times at 4–5 s intervals. The threshold for paw withdrawal was calculated by taking the average of five repeated stimuli (in g), which induced a reflex paw withdrawal. Thus, at a given time point, five measures were obtained for each hind paw (a total of 10 measures per animal). In all behavioral experiments, both thermal and mechanical thresholds were measured in individual rats using the plantar test followed by the von Frey filament tests.

Acute administration of drugs
A single dose of each tested substance or vehicle was administered subcutaneously or intrathecally, and the time-response curve for the drug effects was determined from behavioral analysis performed at different time points within the 4 h interval after the injection.

Chronic administration of substances
The tested steroid (DHEA) or vehicle was administered subcutaneously once a day for 15 days. The basal nociceptive thresholds were assessed everyday in each animal group. In addition, at days 1, 8, and 15, time-response curves for the effects of chronic DHEA treatment were determined from behavioral analysis performed at different time points within the 4 h interval after the injection.

Statistical analyses
Student’s t tests or ANOVAs followed by Duncan post hoc comparisons were used. Behavioral data were analyzed with Statistica Software 5.1 (Statsoft, Maisons-Alfort, France).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of sciatic neuropathy on P450c17 gene expression in the SC
The levels of transcripts encoding P450c17 in the lumbar SC dorsal horn were examined by rt-PCR in sham-operated rats (controls) or in rats submitted to neuropathic pain generated by sciatic nerve ligature. Figure 1 shows representative amplification curves for P450c17 gene, GAPDH used as a housekeeping gene, and a negative control. The continuous fluorescence monitoring of DNA copy numbers within a large dynamic range resulted in highly sensitive measurement of specific template signals for P450c17 (Fig. 1A ) and GAPDH (Fig. 1B ) in the SC and testis, a classical steroidogenic tissue. Amplification reactions were followed by melting-curve analysis to ensure the specificity of PCR products (Fig. 1C, D ). The P450c17 mRNA concentrations detected in the nervous tissue were 100-fold lower than those measured in testis (Fig. 1A ). The sciatic neuropathy significantly decreased the level of P450c17 mRNA in the lumbar and cervical SC (Fig. 1E, F ). After normalization of rt-PCR P450c17 product to GAPDH, it appeared that P450c17 mRNA concentrations in the lumbar and cervical SC of neuropathic rats were 5- and 1.64-fold lower, respectively, than in controls (Fig. 1E, F ).


Figure 1
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  		Figure 1. Effect of neuropathic pain on P450c17 gene expression. rt-PCR amplification of P450c17 (A) and GAPDH (B) genes. rt-PCR obtained in testis and SC of neuropathic and control rats. Melting-curve analysis of P450c17 (C) and GAPDH (D) PCR products showing very high degree of specificity of amplified dsDNA. P450c17 mRNA level is expressed as a ratio of GAPDH mRNA amount in lumbar (E) and cervical (F) SC of control and neuropathic-pain rats. Each value is the mean ± SE of 4 independent experiments. *P < 0.05; ***P < 0.001.

Effect of sciatic neuropathy on P450c17 enzymatic activity in the SC in vitro
Reversed-phase HPLC analysis was coupled with flow scintillation detection to compare the conversion of [3H]PREG into [3H]DHEA by lumbar SC slices of control and neuropathic rats (Fig. 2 A, B). After 3 h of incubation, the amount of newly synthesized [3H]DHEA from [3H]PREG was 40% lower in SC slices of neuropathic-pain rats compared to controls (Fig. 2C ).


Figure 2
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  		Figure 2. Impact of neuropathic pain on P450c17 enzymatic activity in vitro and in vivo. A, B) Characterization of neurosteroids released in incubation medium by SC slices of control (A) and neuropathic-pain (B) rats after a 3 h incubation with [3H]PREG. C) Effects of neuropathic pain on P450c17 activity were determined in vitro by investigating [3H]PREG conversion into [3H]DHEA by SC slices of control and neuropathic rats. Before expressing data as percentage of control, each value was calculated as relative amount of [3H]DHEA compared with the total [3H]-compounds resolved by HPLC-Flo/One characterization (x100). (D) Endogenous concentrations of DHEA produced in vivo were determined by radioimmunoassays after HPLC analysis of SC extracts of control and neuropathic rats. Each value presented in C and D is the mean ± SE of 4 independent experiments. **P < 0.01; ***P < 0.001.

Effect of sciatic neuropathy on endogenous DHEA production in the SC in vivo
HPLC purification of the lumbar SC combined with radioimmunological detection of DHEA revealed an 89% decrease in the concentration of endogenous DHEA in the SC of neuropathic rats (i.e., 0.245±0.041 ng/g in neuropathic rats vs. 2.327±0.323 ng/g in controls; Fig. 2D ).

Effect of acute subcutaneous injection of DHEA on thermal and mechanical nociceptive thresholds
A dose-dependent thermal hyperalgesia was observed in naive rats 15 min (P<0.01 for 37.5 mg/kg; P<0.001 for 75 and 150 mg/kg) and 60 min (P<0.05 for 37.5 mg/kg; P<0.001 for 75 and 150 mg/kg) after subcutaneous injection of DHEA (Fig. 3 A). DHEA at 150 mg/kg produced a biphasic effect on the thermal sensitivity since the threshold decrease observed at 15 and 60 min after subcutaneous injection was followed by a significant increase at 150 min (P<0.05) and 195 min (P<0.001). A biphasic effect of DHEA was also observed on the mechanical sensitivity threshold, which decreased significantly (allodynia) in a dose-dependent manner at 30 min after DHEA injection (P<0.05 for 75 mg/kg; P<0.001 for 150 mg/kg) and increased from 120 to 210 min (P<0.05 for 75 mg/kg; P<0.01 for 37.5 mg/kg; P<0.001 for 150 mg/kg; Fig. 3B ).


Figure 3
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  		Figure 3. Effects of subcutaneous and intrathecal administrations of DHEA on nociceptive thresholds of naive rats. A, B) Dose- and time-related effects of subcutaneous injections of DHEA on thermal (A) and mechanical (B) nociceptive thresholds of naive rats. A) Each point is the mean ± SE of 6 observations in each of 8 rats. B) Each point is the mean ± SE of 10 observations in each of 8 rats. Naive animals were tested with 3 doses of DHEA (37.5, 75, and 150 mg/kg) compared to VEH. §,$,£P < 0.05; §§,$$P < 0.01; §§§,$$$,£££P < 0.001. § = VEH vs. DHEA (37.5 mg/kg); $ = VEH vs. DHEA (75 mg/kg); £ = VEH vs. DHEA (150 mg/kg). C, D) Time-response curves for effects of intrathecal injections of DHEA (10 mg/kg) or ketoconazole (KETO, 4 mg/kg) on thermal (C) and mechanical (D) nociceptive thresholds of naive rats. C) Each point is the mean ± SE of 6 measures in each of 9 rats. D) Each point is the mean ± SE of 10 measures in each of 9 rats. DHEA (10 mg/kg) or KETO (4 mg/kg) were compared to VEH. *P < 0.05; ##P < 0.01; ***,###P < 0.001. * = VEH vs. DHEA; # = VEH vs. KETO.

Effects of acute intrathecal injection of DHEA and ketoconazole on thermal and mechanical nociceptive thresholds
To determine whether the modulatory action of DHEA on nociceptive thresholds may involve sensory neural networks of the SC, naive rats were intrathecally injected at the lumbar level with DHEA at 10 mg/kg, a dose that is 7.5- and 15-fold lower than those (75 and 150 mg/kg) effective via the subcutaneous route. A significant thermal hyperalgesia was observed at 10 min after intrathecal injection of 10 mg/kg DHEA (P<0.001). In contrast, intrathecal injection of ketoconazole (4 mg/kg), a P450c17 inhibitor capable of blocking the local synthesis of DHEA in the SC, increased the thermal nociceptive threshold (P<0.001; Fig. 3C ).

Similarly, intrathecal injection of DHEA (10 mg/kg) induced a mechanical allodynia at 10 and 30 min (P<0.001) after administration, whereas intrathecal ketoconazole (4 mg/kg) significantly increased the mechanical sensitivity threshold (P<0.01; Fig. 3D ). The biphasic effect exerted by intrathecal injection of DHEA on mechanical sensitivity was similar to that produced by subcutaneous injection of DHEA. Indeed, the mechanical allodynia observed at 10 and 30 min after intrathecal injection of DHEA (10 mg/kg) was followed by a significant increase of the threshold value at 180 min (P<0.05; Fig. 3D ).

Effect of acute intrathecal injection of DHEA and ketoconazole on pain thresholds of sciatic neuropathic rats
As previously well demonstrated by Bennett and Xie (8) and also by numerous groups who used the same animal model, we observed a marked thermal hyperalgesia (P<0.001) and a mechanical allodynia (P<0.001) in sciatic neuropathic rats. Indeed, the withdrawal latency characterizing the thermal pain threshold was generally around 11.0 ± 0.2 s on each paw of naive and sham-operated rats and on the contralateral paw of neuropathic animals (Figs. 3 and 4 ). On the ipsilateral paw of sciatic neuropathic rats, the withdrawal latency to thermal stimulations was 7.0 ± 0.2 s (Fig. 4A ). Similarly, we observed that the withdrawal threshold for mechanical stimulations with the Von Frey filaments was 100 ± 7 g on naive and sham-operated paws while the values detected on the paws subjected to sciatic nerve ligature were equivalent to 37 ± 5 g (Figs. 3 and 4) . Treatment of these animals with an intrathecal injection of DHEA (10 mg/kg) potentiated neuropathy-evoked thermal hyperalgesia on the ipsilateral paw (P<0.01) and decreased the threshold on the contralateral paw (P<0.01) at 15 min after administration (Fig. 4A, B ). A pronociceptive effect of DHEA (10 mg/kg) was also observed in sham-operated rats, with DHEA decreasing thermal thresholds at 15 min (P<0.01) and 60 min (P<0.01) after injection (Fig. 4C ). Unlike DHEA, intrathecal ketoconazole (4 mg/kg) exerted a significant analgesic effect in neuropathic rats by increasing the thermal nociceptive threshold on the ipsilateral paw from 15 (P<0.001) to 105 min (P<0.001) after injection (Fig. 4A ). An antinociceptive action of ketoconazole was observed on the contralateral paw (P<0.05) of neuropathic rats (Fig. 4B ) and sham-operated animals (P<0.001 at 15, 60, and 150 min and P<0.05 at 105 min; Fig. 4C ).


Figure 4
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  		Figure 4. Time-response curves for effects of intrathecal injections of DHEA (10 mg/kg) or KETO (4 mg/kg) on the thermal (A–C) and mechanical (D–F) thresholds of ipsilateral (A, D) and contralateral paws (B, E) of neuropathic (A, B, D, E) and of sham-operated (C, F) rats. For thermal nociceptive thresholds (A–C), each point is the mean ± SE of 3 observations on each paw of 8 neuropathic or 8 sham-operated rats. For mechanical nociceptive thresholds (D–F), each point is the mean ± SE of 5 observations on each paw of 8 neuropathic or 8 sham-operated rats. DHEA (10 mg/kg) or KETO (4 mg/kg) compared to VEH. *,#P < 0.05; **,##P < 0.01; ***,###P < 0.001. * = VEH vs. DHEA; # = VEH vs. KETO.

In a similar manner to observations made on the thermal sensitivity, the mechanical threshold was differentially affected by DHEA and ketoconazole, as shown by two-way ANOVAs that revealed drugs x times interactions on the ipsilateral and contralateral paws of neuropathic rats (F10,535=3.17, P<0.001 and F10,535=4.95, P<0.001) and sham-operated rats (F10,585=15.36, P<0.001). Indeed, intrathecal injection of DHEA (10 mg/kg) enhanced the ipsilateral mechanical allodynia evoked by sciatic nerve ligature (Fig. 4D ) and decreased threshold values of the contralateral (Fig. 4E ) or pseudo-ligated (Fig. 4F ) paw in neuropathic or sham-operated rats, respectively. In contrast, by strongly increasing the mechanical threshold values, intrathecal injection of ketoconazole (4 mg/kg) completely abolished the neuropathy-evoked allodynia in neuropathic rats (Fig. 4D, E ) and produced an antinociceptive action in sham-operated animals (Fig. 4F ).

Mechanism of action of acute DHEA treatment on nociception
It is well-known that glutamate is the main excitatory neurotransmitter mediating pain transmission through activation of NMDA receptors (24) . Because DHEA potentiates glutamatergic transmission via r{sigma}1-induced phosphorylation of NMDA receptors (15 16 17 18) , we assessed the effects of acute DHEA administration on nociception in the presence or absence of BD1047, a selective r{sigma}1 antagonist. The rapid pronociceptive effect produced by acute intrathecal injection of DHEA on the thermal and mechanical sensitivity thresholds was completely abolished when DHEA was coadministered with BD1047 (0.6 mg/kg; Fig. 5 ). Administration of BD1047 alone did not modify the thermal (Fig. 5A ) and mechanical (Fig. 5B ) thresholds.


Figure 5
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  		Figure 5. Time-response curves for the effects of intrathecal injections of DHEA (10 mg/kg), BD1047 (0.6 mg/kg) or DHEA (10 mg/kg) + BD1047 (0.6 mg/kg) on thermal (A) and mechanical (B) nociceptive thresholds of naive rats. A) Each point is the mean ± SE of 6 measures in each of 6 rats. B) Each point is the mean ± SE of 10 measures in each of 6 rats. DHEA (10 mg/kg), BD 1047 (0.6 mg/kg) or DHEA (10 mg/kg) + BD1047 (0.6 mg/kg) compared to VEH. ***P < 0.001; VEH vs. DHEA.

Effect of chronic DHEA treatment on pain thresholds of sciatic neuropathic and control rats
Chronic subcutaneous administration of DHEA (75 mg/kg) significantly increased and maintained elevated the basal nociceptive thresholds of each paw in sham-operated (P<0.001) and neuropathic (ipsilateral P<0.05 and contralateral P<0.001) rats (Fig. 6 A). The starting day of the chronic treatment (day 1) the time-response curve for the effects of DHEA administration showed a biphasic action (rapid pronociceptive and delayed antinociceptive effects) in neuropathic and control rats (Fig. 6B1, C1, D1 ) as previously observed for acute DHEA treatment (Fig. 3) . After 1 wk of daily administration of DHEA (75 mg/kg), particularly from days 8 to 15, the rapid pronociceptive effect evoked by DHEA became undetectable when time course behavioral analyses were performed within the 4 h interval after the injection while the delayed antinociceptive action persisted (Fig. 6B2, B3, C2, C3, D2, D3 ).


Figure 6
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  		Figure 6. Effect of chronic DHEA subcutaneous administration on basal mechanical nociceptive thresholds (A) of ipsilateral and contralateral paws of sciatic-neuropathic and sham-operated rats at days 1, 8, and 15. Time-response curves for effects of DHEA on mechanical thresholds of the ipsilateral (B1–3) and contralateral (C1–3) paws of neuropathic (B1–3, C1–3) and sham-operated (D1–3) rats at day 1 (B1, C1, D1), day 8 (B2, C2, D2), and day 15 (B3, C3, D3) during chronic treatment. Each point is the mean + SE of 5 observations on each paw of 6 neuropathic or 6 sham-operated rats. DHEA (75 mg/kg) compared to VEH. *,#,$,£P < 0.05; **,##,$$,££P < 0.01; ***,###,$$$,£££P < 0.001. * = VEH vs. DHEA; # = time point vs. DHEA preinjection day 1; $ = time point vs. DHEA preinjection day 8; £ = time point vs. DHEA preinjection day 15.

Effect of intrathecal injection of testosterone, an androgenic metabolite of DHEA, on pain thresholds of sciatic-neuropathic rats
Testosterone (10 mg/kg), intrathecally administered in the SC, induced a significant analgesic effect in neuropathic rats by increasing the nociceptive thresholds on the ipsilateral (P<0.01 at 75 min and P<0.001 from 120 to 210 min) and contralateral (P<0.05 at 120 min and P < 0.001 from 165 to 210 min) paws (Fig. 7 A, B). An antinociceptive action of testosterone was also observed in sham-operated (P<0.001 from 120 to 210 min) animals (Fig. 7C ).


Figure 7
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  		Figure 7. Time-response curves for the effects of intrathecal injections of testosterone (10 mg/kg) on the mechanical nociceptive thresholds of the ipsilateral (A) and contralateral paws (B) of neuropathic (A, B) and sham-operated rats (C). Each point is the mean + SE of 5 observations on each paw of 6 neuropathic or 6 sham-operated rats. Testosterone (10 mg/kg) compared to VEH. *P < 0.05; **P < 0.01; ***P < 0.001.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Significant progress has been made recently in the medical treatment of pain but a variety of pathological pains, particularly neuropathic pain, can still not be alleviated because they are refractory to the currently available analgesics (25 26 27 28 29) . Therefore, biomedical researchers need to continue identifying key cellular and molecular factors affected in sensory pathways during chronic pain to characterize potential targets for new drugs. Thanks to the combination of several approaches, this report is the first to show that the gene expression and biological activity of P450c17, a key enzyme in DHEA synthesis, are significantly down-regulated in spinal sensory networks during neuropathic pain. Prior to the present study, we had used anatomical, molecular, and neurochemical tools to demonstrate that the rat SC dorsal horn, which plays a pivotal role in nociception, is an active steroid-producing center that contains various key steroid-synthesizing enzymes, including cytochrome P450 side-chain-cleavage, 3β-hydroxysteroid dehydrogenase, 5{alpha}-reductase, 3{alpha}-hydroxysteroid oxido-reductase, and P450c17 (7 , 11 , 12 , 20 , 30 31 32) .

Because of the particular interest evoked by DHEA in the media and the abusive use of this steroid all around the world, we sought to decipher the potential role of endogenous DHEA produced by the SC in pain modulation. The laboratory rat constitutes a suitable model in which to investigate the action of DHEA locally synthesized in the CNS, because in rodents, plasma concentrations of DHEA are undetectable and cannot interfere with the effect of endogenous DHEA produced by nerve cells (6 , 7) . In addition, the model of neuropathic pain used in this research (sciatic nerve tied loosely by ligatures) was validated by Bennett and Xie (8) has been used in several published studies on pain, and is generally considered to reproduce in rats the disorders of pain sensation seen in humans. By using quantitative real time RT-PCR, we observed that the transcripts encoding P450c17 were down-regulated in the SC dorsal horn of rats subjected to peripheral neuropathic pain. The down-regulation of P450c17 gene expression was accompanied by a marked decrease in P450c17 enzymatic activity in the SC, as revealed by in vitro and in vivo biochemical experiments. Therefore, it appeared that the local synthesis of DHEA by sensory neural networks of the SC (7) was dramatically reduced under a chronic pain situation. To understand the reason why DHEA synthesis decreased in the SC of neuropathic pain rats, we performed various series of behavioral and pharmacological studies using exogenous DHEA and ketoconazole, a pharmacological P450c17 inhibitor that blocks DHEA formation, therefore allowing the identification of the role played by DHEA endogenously produced by spinal nerve cells (13 , 14) .

In an initial series of behavioral experiments, we investigated the effects of acute subcutaneous administration of DHEA on nociceptive thresholds of naive rats. Injected concentrations in rats were determined on the basis of DHEA doses generally used in humans (33 34 35 36 37) . All behavioral measurements within the 1.5 h interval after acute subcutaneous administration revealed that each tested dose of DHEA was capable of decreasing both thermal and mechanical nociceptive thresholds. These observations indicate that the rapid action of DHEA on nociception is a pronociceptive effect. In support of this suggestion, our series of behavioral studies after acute intrathecal injections also showed that DHEA produced a rapid pronociceptive effect in naive rats and transiently potentiated the thermal hyperalgesia and mechanical allodynia characterizing neuropathic animals. The pronociceptive effect of acute DHEA treatment was very strong when DHEA was directly applied to the lumbar SC. Indeed, acute intrathecal injection of 10 mg/kg of DHEA (a dose 7.5- or 15-fold lower than those used for subcutaneous administration) was capable of producing a nociceptive threshold decrease similar to that obtained with acute subcutaneous injections of DHEA at 75 or 150 mg/kg. This result indicates that the SC sensory networks are strongly involved in the mediation of DHEA action on nociception. In addition, inhibition of the local synthesis of DHEA in spinal neural pathways by intrathecal administration of ketoconazole, a pharmacological P450c17 blocker (13 , 14) , produced analgesia in neuropathic rats and a potent antinociceptive effect in controls, demonstrating that DHEA produced by the SC (7) is an endogenous pronociceptive steroid. Therefore, about the question to know why DHEA synthesis decreased in the SC of neuropathic pain rats, it is possible to speculate that the down-regulation of P450c17 gene expression and DHEA formation in the SC may be an endogenous mechanism triggered by these animals to cope with the chronic pain condition. In support of this suggestion, suppression of DHEA synthesis in the SC by intrathecal injection of ketoconazole resulted in a significant analgesic effect that completely abolished in neuropathic rats the thermal hyperalgesia and mechanical allodynia evoked by sciatic nerve ligature. Interestingly, we previously observed that the neuropathic painful state also produced in the SC an up-regulation of the biosynthetic pathway of the neurosteroid allopregnanolone, a potent allosteric activator of GABAA receptors (20) . Thus, the process of neurosteroid biosynthesis appears to be a mechanism selectively regulated in the SC sensory networks during neuropathic pain to increase, on the one hand, the production of antinociceptive neurosteroids such as allopregnanolone and tetrahydrodeoxycorticosterone (40 , 41) and to reduce, on the other hand, the formation of pronociceptive neurosteroids such as DHEA. Investigations of the interactions between major neurotransmitters involved in pain transmission and P450c17 activity in the SC may certainly help to elucidate in the future the mechanisms underlying the inhibitory impact of neuropathic pain on DHEA biosynthesis in the SC.

Our results also revealed that, contrary to the rapid pronociceptive effect exerted by DHEA itself (before being metabolized), androgenic metabolites deriving from DHEA may induce a delayed analgesic or antinociceptive action in neuropathic pain or control rats, respectively. Indeed, we observed that the pronociceptive effect of acute DHEA treatment was followed by a delayed increase in the thermal and mechanical thresholds. In addition, chronic administration of DHEA, which is well-known to generate a permanently high level of androgens in the blood, significantly increased and maintained at elevated levels the basal nociceptive thresholds in neuropathic and control rats. In particular, after 1 wk of chronic DHEA treatment (especially from days 8 to 15), the rapid pronociceptive effect evoked by DHEA became undetectable when time-course behavioral analyses were performed within the 4 h interval after the injection while the delayed antinociceptive action persisted. Moreover, intrathecal administration of testosterone, one of the major androgens deriving from DHEA (38) , induced a significant analgesic effect in neuropathic rats by increasing the nociceptive thresholds on the ipsilateral and contralateral paws. In agreement with our results, previous investigations have reported androgen-induced analgesic effects and discussed the possible mechanisms of action of testosterone and its 5{alpha}-reduced metabolites in pain modulation (39 , 42) . However, the rapid pronociceptive action exerted by DHEA itself before being metabolized, as well as the occurrence of a biphasic effect of acute DHEA treatment, has never been described. Therefore, to clarify as much as possible our findings, we performed a series of pharmacological analyses to provide valuable clues on the mechanism of action underlying the rapid pronociceptive effect of acute DHEA treatment described herein. In fact, until now, a specific receptor for DHEA has not been characterized. DHEA acts as an allosteric modulator of NMDA and P2X receptors that play a pivotal role in the control of nociceptive transmission (24 , 37 , 43 , 44) . Thus, the pronociceptive effect of DHEA described herein may be explained by DHEA action on NMDA or P2X receptors localized in the SC dorsal horn (45 46 47) . In particular, it has been clearly demonstrated that DHEA activates the glutamatergic transmission by potentiating NMDA responses via r{sigma}1 (16) . Indeed, several studies that identified functional interactions between r{sigma}1 and NMDA receptors revealed that DHEA triggers through r{sigma}1 intracellular cascades, leading to phosphorylation of NMDA receptors (15 16 17 18) . Therefore, based on the crucial role played by the glutamatergic system in nociception (24) , we determined whether the mechanism of action of DHEA on pain modulation involves the process of r{sigma}1-evoked NMDA receptor activation. Our results clearly demonstrate that BD1047, a selective antagonist of r{sigma}1 (48) , completely blocks the rapid pronociceptive effect of DHEA on thermal and mechanical pain thresholds. Since the presence of r{sigma} has been well-established in the SC sensory networks, where DHEA is locally synthesized by P450c17-positive cells (7 , 49) , our data strongly indicate that endogenous DHEA may control spinal nociceptive mechanisms through paracrine or autocrine modulation of the process of r{sigma}1-induced NMDA receptor activation.

In conclusion, this report provides six novel findings that may constitute major advances in various areas of biomedical research: 1) endogenous DHEA produced via the process of neurosteroidogenesis in the spinal cord is a key factor involved in the regulation of pain mechanisms; 2) during chronic neuropathic pain (a largely unmet therapeutic need), a down-regulation occurred in the gene expression and biological activity of cytochrome P450c17 (the key DHEA-synthesizing enzyme) in spinal neural networks controlling nociception; 3) acute DHEA treatment exerts a biphasic effect on nociception: a rapid pronociceptive action and a delayed antinociceptive effect; 4) in vivo blockade of DHEA biosynthesis in the SC by intrathecal injection of ketoconazole, a P450c17 inhibitor, suppressed the pronociceptive action of endogenous DHEA and induced analgesia in neuropathic rats; 5) the mechanism of action of DHEA on pain regulation involves sigma receptors because the pronociceptive effect evoked by acute DHEA treatment was abolished by BD1047, a selective antagonist of sigma-1 receptors; and 6) chronic DHEA treatment increased and maintained elevated the basal nociceptive thresholds in neuropathic and control rats, suggesting that androgenic metabolites generated from daily administered DHEA exerted analgesic effects, while DHEA itself caused a rapid pronociceptive action. Indeed, intrathecal administration of testosterone, an androgen deriving from DHEA, induced analgesia in neuropathic rats. This study opens interesting possibilities for the development of strategies against neuropathic pain by using different pharmacological agents to target P450c17 activity in nerve cells.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS, France) and Université Louis Pasteur (Strasbourg, France). C.K. was a recipient of a fellowship from Ministère de l’Education Nationale et de la Recherche. L.M. is a postdoctoral fellow supported by Association Titoine (Normandie, France). Thanks to A. Lacaud for technical assistance in surgical operations. Thanks also to M. Barrot for the loan of the plantar test apparatus


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication May 10, 2007. Accepted for publication July 19, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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