The Journal of Pain
Volume 12, Issue 1 , Pages 71-83, January 2011

Progesterone Prevents Allodynia After Experimental Spinal Cord Injury

  • María F. Coronel

      Affiliations

    • Laboratorio de Bioquímica Neuroendócrina, Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina
    • Facultad de Ciencias Biomédicas, Universidad Austral, Buenos Aires, Argentina
    • ,
  • Florencia Labombarda

      Affiliations

    • Laboratorio de Bioquímica Neuroendócrina, Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina
    • Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
    • ,
  • Marcelo J. Villar

      Affiliations

    • Facultad de Ciencias Biomédicas, Universidad Austral, Buenos Aires, Argentina
    • ,
  • Alejandro F. De Nicola

      Affiliations

    • Laboratorio de Bioquímica Neuroendócrina, Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina
    • Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
    • ,
  • Susana L. González

      Affiliations

    • Laboratorio de Bioquímica Neuroendócrina, Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina
    • Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
    • Corresponding Author InformationAddress reprint requests to Susana Laura González, PhD, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina.

Received 9 January 2010; received in revised form 18 March 2010; accepted 29 April 2010. published online 05 August 2010.

Article Outline

Abstract 

Chronic pain after spinal cord injury represents a therapeutic challenge. Progesterone, a neuroprotective steroid, has been shown to modulate nociceptive thresholds, whereas its effect on neuropathic pain needs to be further explored. In this study, we evaluated whether progesterone could ameliorate pain-associated behaviors in animals subjected to a spinal cord hemisection. The development of mechanical and cold allodynia was assessed in injured male rats treated with daily injections of progesterone or vehicle. The expression of N-methyl-D-aspartate receptor (NMDAR) subunits, protein kinase C gamma (PKCγ), preprodynorphin (ppD), and kappa opioid receptor (KOR), key players in chronic pain mechanisms, was determined in the dorsal spinal cord. Twenty-eight days after injury, all vehicle-treated animals presented allodynic behaviors and a marked increase in NMDAR subunits, PKCγ, and ppD mRNA levels, with no changes in KOR mRNA levels. Progesterone prevented the development of mechanical allodynia and reduced the painful responses to cold stimulation. In correlation with the attenuation of pain behaviors, the steroid prevented NMDAR subunits and PKCγ mRNAs upregulation, did not modify the elevated ppD mRNA levels, but increased KOR expression. In conclusion, progesterone modulates neuropathic pain after spinal cord injury, creating a favorable molecular environment that may decrease spinal nociceptive signaling.

Perspective

The present study suggests that progesterone administration could represent an interesting strategy to modulate neuropathic pain circuits after spinal cord injury. Further studies are needed to investigate the potential progesterone receptors involved in these actions.

Key words: Chronic pain, protein kinase C, N-methyl-D-aspartate receptor, preprodynorphin, kappa opioid receptors

 

Chronic pain is a major concern for patients with spinal cord injury, with an estimated incidence that ranges from 40 to 60%.3, 9 These patients, already burdened with the disability of paralysis, emotional trauma and spasticity, must contend with severe unrelenting pain.9, 24 Below-level mechanical and thermal allodynia, 2 neuropathic pain-associated behaviors, are commonly observed after spinal cord injury in humans.9, 24 Unfortunately, most of the currently available drugs for the treatment of neuropathic pain are relatively ineffective.3

Although the precise mechanisms underlying chronic pain after spinal cord injury remain elusive, several maladaptive molecular events are known to contribute to the observed pain-related behaviors following injury.17, 49, 97 In this regard, both the increased expression and/or the activity of the N-methyl-D-aspartate receptor (NMDAR) play a critical role in the development and maintenance of chronic pain.6, 37

The functional NMDAR contains an obligatory NR1 subunit in combination with at least 1 of the 4 NR2 subunit family members, of which NR2A and NR2B are the most abundant in the adult rat dorsal horn.60, 66 Previous studies have found that several conditions such as diabetes,88 excitotoxic10 or traumatic30 spinal cord injuries, or morphine tolerance52 alter the expression of NMDAR subunits in the spinal cord contributing to abnormal pain processing. Moreover, increased phosphorylation of the NR1 subunit correlates with the presence of neuropathic pain behaviors after excitotoxic spinal lesions10 and peripheral nerve injuries.26, 89

Furthermore, dynorphin and the gamma isoform of the protein kinase C (PKCγ), both key players in neuropathic pain signaling, have been shown to amplify the NMDAR-mediated circuit by either direct or indirect activation of this receptor.48, 50, 58

A recent report from our laboratory indicates that spinal cord hemisection induces a time-dependent upregulation of NMDAR subunits, PKCγ and preprodynorphin (ppD), the dynorphin precursor peptide, in the dorsal horn of animals exhibiting allodynic behaviors.43 Therefore, drugs that could either prevent or attenuate the maladaptive molecular changes that arise after injury could represent an effective treatment for central neuropathic pain.

Several reports support the crucial role of neuroactive steroids in the modulation of pain sensation.62 Progesterone, in particular, mediates gestational antinociception,29 contributes to sex-related differences in pain,29, 45, 54 and reduces pain sensitivity in intact rats.25 In addition, progesterone attenuates neuropathic pain-associated behaviors in animals displaying a peripheral nerve injury82 or diabetic neuropathy.51

Interestingly, several neuroactive effects of progesterone involve the recruitment of the opioid system,20, 29, 74 the modulation of dynorphin levels,84 and the activity of NMDAR78 and PKC,5 suggesting that this steroid could have the ability to influence pain sensitivity through the modulation of these molecules.

The present study was designed to examine the role of progesterone on the onset of chronic pain in male rats subjected to a spinal cord hemisection, a well-recognized model of central neuropathic pain.14 Furthermore, we investigated the spinal expression of NMDAR subunits, PKCγ, ppD, and KOR, all key players in the process of central sensitization, trying to provide insight into the potential molecular mechanisms involved in progesterone analgesic effects. Our current investigations suggest further applications for progesterone-based therapies and may open new avenues for the treatment of chronic pain after central injuries.

Back to Article Outline

Methods 

Spinal Cord Injury and Progesterone Administration 

All experimental procedures were reviewed by the local Animal Care and Use Committee (Assurance Certificate N A5072-01, Instituto de Biología y Medicina Experimental) and followed the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Care was taken to minimize animal discomfort and to limit the number of animals used. Male Sprague-Dawley rats (200–220 g), bred at the colony of the Instituto de Biología y Medicina Experimental (Buenos Aires, Argentina), were deeply anesthetized with chloral hydrate (400 mg/kg, ip). In a group of rats, the spinal cord was exposed and unilaterally hemisected at thoracic T13 level (n = 32),43 as originally described by Christensen et al.14 In hemisected animals, the ipsilateral hindlimb is acutely paralyzed while the contralateral limb is unimpaired.14 For this reason, all animals presenting acute contralateral hindlimb involvement after the surgery—a demonstration of overhemisection or ischemic complication—were excluded from the study. Postoperative care included control of body temperature and antibiotic administration.42 Injured animals received daily subcutaneous injections of natural progesterone (16 mg/kg/day, n = 12, HX+PG; Proluton, Schering Laboratories, Buenos Aires, Argentina), vehicle (Ricine oil; Ewe, Sanitas, AR; n = 10), or none (n = 10, HX). This protocol of progesterone administration has been shown to prevent oedema and neuronal loss and improve cognitive responses following brain-contusion injury,18 and to induce oligodendrogenesis and remyelination after spinal cord injury.44 Since progesterone may exert sex-based divergent analgesic properties,46, 54 further studies are in progress in order to analyze the effects of long-term progesterone administration and the influence of the oestrous cycle in female rats with spinal cord injury. Control groups included sham-operated rats (n = 32) receiving either progesterone (n = 12), vehicle (n = 10), or none (n = 10), and intact animals (n = 26) receiving either progesterone (n = 10), vehicle (n = 8), or none (n = 8).

Behavioral Assessment 

Behavioral testing was performed in all animals before surgery (day 0) and at different time points after spinal cord hemisection or sham operation, as previously described.16, 43 Briefly, the animals were placed in their acrylic testing chambers for 15 minutes for adaptation, and mechanical sensitivity was assessed with von Frey hairs (Stoelting, Wood Dale, IL). The hairs were applied in ascending order (1, 2, 4, 6, 8, 10, 15, 26 g) onto the plantar surface of both ipsilateral and contralateral hindpaws.11 Each hair was delivered 3 times with 5-second intervals. The lowest force at which application elicited a brisk paw withdrawal was taken as the mechanical response threshold. A paw withdrawal reflex obtained with 6 g or less was considered as an allodynic response. Cold sensitivity of the hindpaw to acetone (Choi test)13 was quantified by foot withdrawal frequency. Thus, 100 μl of acetone was applied to the plantar surface of the paw using a plastic tubule connected to a 1 mL syringe. Acetone was applied 5 times to each paw at an interval of at least 5 minutes. The number of brisk foot withdrawals was recorded. Only rats showing normal responses to mechanical and thermal stimulation before surgery were included in the experiments. After spinal cord hemisection, the ipsilateral hindlimb was acutely paralyzed but presented a considerable return to motor function by 5 days postsurgery, when the animals entered an early phase of recovery with return of paw placement and frequent-to-consistent weight supporting steps. On the following days, the return of motor function included predominant paw position, trunk stability, and tail position, allowing a complete and reliable behavioral testing of mechanical and thermal allodynia.14 As previously reported, results were analyzed using the Friedman Repeated Measures of Analysis of Variance followed by Multiple Comparison Test.43

Immunofluorescence 

Tissue Preparation 

Twenty-eight days after spinal hemisection, the animals were deeply anesthetized with an overdose of chloral hydrate (800 mg/kg ip) and perfused through the heart with 60 mL of .9% NaCl, followed by 60 mL of fixative (4% paraformaldehide in .16 M phosphate buffer, pH 7) at 4°C. Spinal lumbar segments caudal to the injury site (L4–L5) were removed and postfixed in the same fixative for 90 minutes at 4°C. Tissues were then rinsed in 20% sucrose in phosphate buffer (pH 7.2) and stored in the same solution at 4°C.

Immunofluorescence Procedure 

Tissues were embedded in OCT compound (Tissue Tek, Miles Laboratories, Elkhart, IN) and cut transversally at 14-μm thickness in a cryostat (HM505N, Microm, Heidelberg, Germany). Sections were mounted onto positively charged microscope slides, allowed to dry for at least 1 hour, and rinsed twice in phosphate-buffered saline (PBS). After preincubation in 10% goat serum for 10 minutes at 37°C, sections were incubated overnight at 4°C with antibodies raised against PKCγ (1:500, rabbit; Santa Cruz Biotechnology, Santa Cruz, CA), NR1 (1:500, mouse; Upstate-Millipore, Billerica, MA) or pNR1 (1:100, rabbit; Ser 896, Upstate-Millipore), all diluted in PBS containing 2% goat serum and .2% Triton X-100. Sections were then rinsed twice in PBS containing .1% Triton X-100 and incubated for 1 hour at room temperature with goat anti-rabbit or goat anti-mouse secondary antibodies conjugated with FITC (1:200; Vector Laboratories, Burlingame, CA). Sections were given 3 rinses in PBS and coverslipped using Fluoromount G (Southern Biotech, Birmingham, AL) as mounting media. Negative controls were prepared omitting the primary or secondary antibodies. Sections were examined under a Zeiss Axioplan fluorescence microscope (Zeiss, Jena, Germany) and images were taken using a Nikon Eclipse E-800 confocal scanning laser microscope (Nikon, Tokyo, Japan). Resolution, brightness, and contrast of the digital images were optimized using the Adobe Photoshop software (Adobe Systems Inc, San Jose, CA).

Quantification and Statistical Analysis 

The number of neuronal profiles exhibiting PKCγ, NR1, or pNR1 immunoreactive (IR) signal was determined in the lumbar L4–L5 segments of the dorsal horn by counting immunostained profiles in randomly, systematically sampled sections throughout the spinal cord (every 10th section, 10 sections per spinal cord). In order to determine the individual spinal cord laminae, the gray matter landmarks were first identified.64 In the case of PKCγ, almost all immunoreactive signal was located in lamina II, and the average number of PKCγ-immunopositive neuronal profiles per section was determined for each animal. Data presented correspond to the mean ± SEM of the number of PKCγ immunolabeled profiles detected in each experimental group. On the contrary, NR1- and pNR1-IR neuronal profiles were observed throughout the dorsal horn and were therefore quantified in the following 3 regions: superficial dorsal horn (laminae I and II), nucleus propius (laminae III and IV), and neck region (laminae V and VI), as previously described.83 These regions were identified by cytoarchitectonic criteria,64 evaluating digital images visualized and analyzed with a computer-assisted image analysis system attached to the microscope (Bioscan Optimas II software; Bioscan, Edwards, WA), as previously reported.44 The microscope illumination and data acquisition parameters were fixed throughout the entire analysis. The mean number of NR1- and pNR1-IR neuronal profiles per section was determined for each animal. These values were averaged within each experimental group and presented as group data. The mean area of each dorsal horn region was determined (superficial dorsal horn, .093 ± .004; nucleus propius, .161 ± .008; neck region, .185 ± .006 mm2) using the computer-assisted image analysis system and the results were therefore expressed as the mean number of NR1- or pNR1-IR neuronal profiles per unit area (1 mm2), as previously described.83 The mean area of each of the dorsal horn regions evaluated did not change across the different experimental groups. Statistical analysis was carried out by applying One-way Analysis of Variance (ANOVA) and Newman–Keuls Multiple Comparison Post-Test.

Real Time–Polymerase Chain Reaction 

Tissue Preparation 

After behavioral assessment, animals were deeply anesthetized with chloral hydrate (800 mg/kg, ip) and decapitated. Spinal lumbar segments caudal to the injury site (L4–5) were immediately removed and the dorsal spinal cord was dissected by cutting through the central canal.43 Tissues were frozen and stored at –70°C until gene-expression studies were performed. Samples from different experimental groups were run at the same time.

Reverse Transcription 

RNA was extracted using the TRIzol (Invitrogen, Carlsbad, CA) method, as previously described.43 The concentration and purity of total RNA was determined by measuring the optical density at 260 and 280 nm. All samples were precipitated with ethanol and then dissolved in distilled water at a concentration of 1 μg/μL, and their quality was verified by gel electrophoresis. Total RNA was subjected to DNAse 1 (Invitrogen) treatment to remove residual contaminating genomic DNA. Reverse transcription was performed from 2 μg of total RNA using a SuperScript II Rnase H reverse transcriptase kit (Invitrogen) for 1 hour at 42°C in the presence of random hexamer primers (Promega, Madison, WI).

Real Time–Polymerase Chain Reaction 

As previously described,43 nucleotide sequences of forward (F) and reverse (R) primers used for amplification were: NR1: F: AGA TGG CCC TGT CAG TGT GT, R: GTG AAG TGG TCG TTG GGA GT;4 NR2A: F: GTG ATC GTG CTG AAC AAG GA, R: GCT CGC AGT CAG AAA AGG AC;4 NR2B: F: TCC GAA GCT GGT GAT AAT CC, R: TGG TCA TCC TCT TGC TCC TC;4 PKCγ: F: AGC CTC CTC CAG AAG TTT GGG, R: CCT TTC CCT AGA ACC ATG AGG;81 preprodynorphin (ppD): F: GGG TTC GCT GGA TTC AAA TA, R: TGT GTG GAG AGG GAC ACT CA;34 KOR: F: GCC ATC CCT GTT ATC ATC AC, R: GGT CTT CAT CTT TGT GTA TCG C;90 Cyclophilin (Cyc) F: GTG GCA AGA TCG AAG TGG AGA AAC, R: TAA AAA TCA GGC CTG TGG AAT GTG; accession number: NM_022536) was chosen as housekeeping gene43 and designed using the Oligo Primer Analysis software version 6.54 (Molecular Biology Insights Inc, Cascade, CO). Relative gene expression was determined using Syber green master mix and the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA). The change in the target mRNA was calculated using the 2–(ΔΔCt) method56 and expressed as fold-increase relative to CTL. Linearity and efficiency of PCR amplification were validated before quantification as previously described.42 The correlation coefficients (r) (r: Cyc: .997; NR1: .991; NR2A: .999; NR2B: .995; PKCγ: .996; ppD: .993; KOR: .990), and the PCR efficiency values (Ex) (Ex: Cyc: 100.08; NR1: 94.17; NR2A: 91.63; NR2B: 100.92; PKCγ: 99.25; ppD: 110.93; KOR: 105.06, calculated as Ex = (10-1/slope) –1 ∗ 100),71 allowed an accurate quantification of targeted gene mRNAs. The specificity of PCR amplification and the absence of dimer formation were confirmed by melting-curve analysis and controlled with high resolution gel electrophoresis. All PCR amplifications lead to a single and specific product of the predicted size. PCR was performed using 2–4 ng cDNA/μl of reaction under optimized conditions: 95°C at 10 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 min. Primers were used in a 5-μM final concentration. Seven animals were included in each experimental group and samples were run in triplicate. Data shown correspond to the mean ± SEM of relative mRNA levels. Statistical comparisons were made with the use of One-way Analysis of Variance (ANOVA) and Newman–Keuls Test for post hoc comparisons.

Back to Article Outline

Results 

Behavioral Evaluation of Neuropathic Pain: Mechanical and Cold Allodynia 

All the control groups included in this study (see Methods) showed similar behavioral patterns when evaluated using the von Frey and Choi Tests. No statistically significant differences were observed between any of these control groups at any of the time points evaluated (P > .05 in all cases). In particular, progesterone administration did not induce any changes in the nociceptive behavior when administered to sham-operated animals (P > .05 at all time points evaluated). Thus, in order to facilitate the visualization of the results, the behavioral assessment corresponding only to sham-operated animals without any treatment (from now on referred as CTL) were included in the graphs (Figs 1A, B) and used for statistical analysis.

In accordance with previous reports,14, 43 spinal hemisection induced a gradual decrease in mechanical withdrawal threshold that was observed in both the ipsilateral and contralateral hindpaws (P < .01 versus CTL at day 14, Fig 1A), reaching allodynic values 21 days after injury (P < .001 versus CTL, Fig 1A). At this time point, all the hemisected animals receiving oil (vehicle) or none (HX) showed guarding behaviors and changes in the posture, including plantar flexion and toe clenching. When stimulated, paw withdrawals were accompanied by active attention to the stimulus, abrupt head turning and attack, vocalization, and/or body reposturing in order to avoid the stimuli. These aversive behaviors were well established by 14 days after injury. The inclusion of these complex nocifensive behaviors excludes simple hyperreflexia, which is a segmental response, and represents a strong and convincing indicator that noxious stimuli have been detected supraspinally. In fact, after spinal hemisection at T13, several pathways remain intact and provide the anatomical substrate for the transmission of evoked stimuli caudal to the lesion site both ipsilaterally and contralaterally to supraspinal centers.14, 15

  • View full-size image.
  • Figure 1 

    Effect of progesterone administration on nociceptive behavior after spinal cord hemisection. Spinal cord injury induced the development of mechanical (A) and thermal (B) allodynia in both the ipsilateral and contralateral hindpaws. It is noticeable that the administration of progesterone prevented mechanical allodynia (A) and significantly reduced the number of nociceptive responses to cold stimulation (B). Values show mean ± SEM and were analyzed using the Friedman Repeated Measures of Analysis of Variance followed by Multiple Comparison Test. Only statistically significant differences between treated and nontreated hemisected animals are stated in the graphs, using the following symbols to represent P values: ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. CTL, control animals (sham operated, receiving no treatment); HX, hemisected animals; HX+PG, hemisected animals treated with progesterone; ipsi, ipsilateral hindpaw; contra, contralateral hindpaw.

The allodynic response was still observed on day 28 (P < .001 versus CTL, Fig 1A). A similar behavioral pattern was detected when cold sensitivity was assessed: There was a gradual and clear increase in the number of positive nociceptive responses in both hindpaws starting 14 days after the surgery (P < .01 versus CTL, Fig 1B), with the highest number of allodynic responses detected 21 and 28 days after the lesion (P < .01 versus CTL at both time points, Fig 1B). Interestingly, hemisected animals receiving progesterone (HX+PG) presented behavioral responses to both mechanical and thermal stimuli that were similar to those of control animals (P > .05 versus CTL at all time points evaluated, Figs 1A, B). The HX+PG group did not develop mechanical allodynia (P < .05 at day 14, P < .001 at days 21 and 28 versus Hx in both hindpaws (Fig 1A), and showed a significantly lower number of positive nociceptive responses to cold stimuli when compared to the HX group (P < .05 at days 14 and 21, P < .01 at day 28 in both hindpaws, Fig 1B).

Relative mRNA Levels 

As we have previously reported,43 a significant increase in NMDAR subunits (NR1, NR2A and NR2B), PKCγ and ppD mRNA levels could be detected in the lumbar dorsal spinal cord 28 days after hemisection as compared with control levels (P < .05 versus CTL in all cases, Fig 2). No changes were detected in KOR expression after the spinal cord injury (P > .05 versus CTL, Fig 2). Interestingly, in the HX+PG group, the mRNA levels corresponding to NR1, NR2A, NR2B, and PKCγ resulted similar to those observed in control animals (P > .05 versus CTL and P < .05 versus HX in all cases, Fig 2). On the contrary, progesterone administration did not change ppD mRNA expression; that remained up-regulated (P < .05 versus CTL and P > .05 versus Hx, Fig 2) but produced a marked increase in KOR mRNA levels (P < .05 versus CTL and Hx, Fig 2). All the control groups included in the study showed similar levels of expression of the different markers (P > .05 in all cases). In particular, we did not find significant changes in the expression of NMDAR subunits or PKC, not even an upward trend in KOR mRNA levels in the dorsal horn of sham-operated animals receiving progesterone as compared to sham animals receiving oil or none. Thus, mRNA levels corresponding only to sham-operated animals receiving no treatment were included in the graphs (CTL, Fig 2).

  • View full-size image.
  • Figure 2 

    Relative mRNA levels corresponding to NMDAR subunits NR1, NR2A, and NR2B, PKCγ, ppD, and KOR detected in the dorsal spinal cord 28 days after either spinal hemisection or sham operation. Note the significant increase in NMDAR subunits (NR1, NR2A and NR2B), PKCγ, and ppD mRNA levels observed in hemisected animals. Interestingly, progesterone administration prevented injury-induced increase in NMDAR subunits and PKCγ expression, did not modify ppD mRNA levels, and induced KOR upregulation. Values show mean ± SEM and represent mRNA levels relative to control values. Statistical comparisons were performed by One-way ANOVA, followed by Newman–Keuls post hoc test. The following symbols were used to represent P values: ns P > .05; ∗P < .05 and ∗∗P < .01. CTL, control animals (sham operated, receiving no treatment); HX, hemisected animals; HX+PG, hemisected animals treated with progesterone.

PKCγ Immunoreactivity 

PKCγ immunoreactivity was observed in a dense band, with the IR signal highly restricted to a subset of neuronal profiles mainly confined to the inner region of lamina II, with the exception of a few cells present in lamina III (Fig 3A). Twenty-eight days after spinal hemisection, we detected a marked bilateral increase in the number of PKCγ-IR profiles located in the lumbar segments caudal to the injury site (P < .001 versus CTL, Figs 3B, D). Interestingly, progesterone administration counteracted this increase (P < .05 versus HX, Figs 3C, D), and the number of PKCγ-IR neuronal profiles in treated animals did not differ from that detected in control ones (P > .05 versus CTL, Figs 3C, D). Neither sham operation nor progesterone or vehicle administration induced changes in the number of PKCγ positive cells in control animals (P > .05 in all cases when comparing the 6 control groups). Only sham-operated animals receiving no treatment were included in the corresponding figure (CTL, Figs 3A, D).

  • View full-size image.
  • Figure 3 

    Photomicrographs (A-C) and number of neuronal profiles (D) exhibiting PKCγ immunoreactivity in the dorsal horn of the lumbar (L4-L5) spinal cord from animals that were either sham operated (A, D), hemisected (B, D), or hemisected and treated with progesterone (C, D). In order to facilitate the visualization of the immunoreative neuronal profiles, images taken using high magnification and showing just a part of the entire lamina II are shown (A-C). Boxed area in (B) is shown at a higher magnification in the corresponding inset. As it can be observed, in the 3 experimental groups, PKCγ immunoreactive signal was highly restricted to a subset of neuronal profiles mainly confined to the inner region of lamina II (A-C). Note that the great increase in the number of PKCγ-IR neuronal profiles observed after the lesion (B, D) is prevented by progesterone administration (C, D). In (D), values show mean ± SEM and represent the average number of immunoreactive profiles per section detected in each experimental group. Statistical comparisons were performed by One-way ANOVA, followed by Newman–Keuls post hoc test. The following symbols were used to represent P values: ns P > .05 and ∗∗P < .01. CTL, control animals (sham operated, receiving no treatment); HX, hemisected animals; HX+PG, hemisected animals treated with progesterone. Calibration bar: 50 μm in (A, B, C).

NR1 and pNR1 Immunoreactivity 

NR1- and pNR1-immunopositive neuronal profiles were distributed throughout the 3 dorsal horn regions evaluated. As in the previous sections, only the data corresponding to sham-operated animals without any treatment were included in the graphs as a control group (Figs 4A, B) and used for statistical analysis. Spinal cord injury produced a dramatic increase in the number of NR1- and pNR1-IR profiles in all the spinal cord regions evaluated (P < .001 versus CTL for both markers in the 3 regions, Figure 4, Figure 5). However, injured animals receiving progesterone presented a significantly lower number of neuronal profiles exhibiting NR1 immunoreactivity, resulting similarly to the values detected in control animals (P < .01 versus HX, P > .05 versus CTL in the 3 dorsal horn areas evaluated, Figure 4, Figure 5). Progesterone administration after spinal hemisection also resulted in a lower number of pNR1-immunopositive profiles (P < .001 versus HX, P < .01 versus CTL in laminae I-II and V-VI, P < .05 versus HX, P < .05 versus CTL in laminae III-IV, Figure 4, Figure 5).

  • View full-size image.
  • Figure 4 

    Number of NR1- (A) and pNR1-IR (B) neuronal profiles per unit area detected in the 3 dorsal horn regions evaluated: lamina I-II, III-IV and V-VI. Note the significant increase in the number of NR1- (A) and pNR1-IR (B) neuronal profiles after spinal hemisection, and the significantly lower number of cells exhibiting either NR1 (A) or pNR1 (B) immunoreactivity after progesterone administration. Values show mean ± SEM and represent the mean number of NR1 or pNR1-IR neuronal profiles per unit area (1 mm2). Statistical analysis was carried out by applying One-way Analysis of Variance (ANOVA) and Newman–Keuls Multiple Comparison Post Test. The following symbols were used to represent P values: ns P > .05, ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. CTL, control animals (sham operated, receiving no treatment); HX, hemisected animals; HX+PG, hemisected animals treated with progesterone.

  • View full-size image.
  • Figure 5 

    Representative photomicrographs of L4-L5 spinal cord sections illustrating NR1 (A-F) and p-NR1 (G-L) immunoreactivity in the dorsal horn of animals subjected to sham operation (A, D, G, J) or spinal cord hemisection followed (C, F, I, L) or not (B, E, H, K) by progesterone administration. In order to facilitate the observation of the immunoreactive cells, representative boxed areas in (A, B, C, G, H, I) are shown at a higher magnification in (D, E, F, J, K, L), respectively. Note the increase in the number of NR1- and pNR1-IR neuronal profiles detected after the spinal cord lesion (B and H, respectively), as well as the significantly lower number of immunoreactive cells observed in lesioned animals treated with progesterone (C for NR1; I for pNR1). CTL, control animals; HX, hemisected animals; HX+PG, hemisected animals treated with progesterone. Calibration bars: 200 μm in (A, B, C, G, H, I) and 50 μm in (D, E, F, J, K, L).

Back to Article Outline

Discussion 

The present study is the first to show that progesterone administration: 1) inhibited the onset of mechanical allodynia and reduced the number of aversive responses to cold stimuli after experimental spinal cord injury; and 2) prevented the maladaptive expression of several pain-related molecules with crucial roles in spinal nociceptive processing.

Thus, the behavioral and molecular changes taking place during progesterone administration suggest that this steroid could be favoring a scenario that inhibits and/or attenuates the onset of neuropathic pain after experimental spinal cord injury.

Our observations extend previous reports that support the idea that neuroactive steroids significantly influence pain perception.62 In particular, progesterone has been shown to reduce pain sensitivity in intact rats25, 54 and during pregnancy,29 and may act as a major determinant of sex-related differences in pain.29, 45, 54 Our results are in line with recent reports showing an attenuation of neuropathic pain behaviors after progesterone administration to animals with peripheral nerve injury82 or diabetic neuropathy,51 although there is no general consensus.41

The present data shows that progesterone administration was able to prevent the injury-induced upregulation of NMDAR subunits and PKCγ mRNAs, and the increased number of PKCγ- and NR1-IR neuronal profiles. In addition, animals receiving progesterone presented a lower number of profiles displaying the phosphorylated NR1 subunit, a posttranslational modification that is essential to enhance NMDAR activity and facilitates neuropathic pain.26, 89 In accordance with the proposed role of these molecules in the pain circuit, animals exhibiting reduced PKCγ and NMDAR expression after progesterone treatment did not display aversive responses to mechanical and cold stimuli. These observations are of upmost importance, since inhibition of both PKCγ and NMDAR expression and/or activity might provide a valuable tool for alleviating allodynia in the clinical setting.

Several other conditions, like morphine exposure,52 diabetes,88 and excitotoxic injury10 produce an altered expression of NMDAR subunits within the dorsal horn, contributing to reinforce their role in pain and tolerance mechanisms. Furthermore, the increased expression and phosphorylation of the NR1 subunit in the spinal cord83 is associated with increased neuronal responsiveness, which underlies the process of central sensitization.6, 37

PKCγ is restricted to a population of excitatory local interneurons in lamina II and has been implicated in injury-induced allodynia, a condition wherein pain is produced by non-noxious stimuli.67 The activation of these PKCγ neurons may open the gate signaling in the superficial dorsal horn63 and activate NMDAR-dependant spinal circuits underlying injury-induced persistent pain.58 Along this line, the spinal inhibitory dysfunction that occurs after SCI6, 37 might be producing allodynia through the involvement of PKCγ neurons in an NMDAR-dependent circuit.

Furthermore, after spinal cord injury, increased dynorphin levels may facilitate NMDAR function and cause neurotoxicity and pain.47, 50, 91 Dynorphin, an endogenous opioid peptide, was originally identified as a ligand for the KOR with analgesic properties, but it was later found to be required for the maintenance of neuropathic pain.28, 47 This switch from antinociceptive to pronociceptive properties has been related to the ability of ppD-derived peptides to bind to the NR1 subunit94 through a nonopioid mediated mechanism that in turn activates the NMDAR.47, 50 Accordingly, the increased levels of ppD mRNA observed in the chronic phase after spinal hemisection, coincident with the enhanced transcription of NMDAR subunits, could result in the stimulation of this receptor's activity and the induction of pain-related behaviors.

Injured animals receiving progesterone sustained an enhanced expression of ppD transcripts. However, this coincided with a basal transcription level of the NMDAR subunits, a decreased number of pNR1-IR neuronal profiles and a concomitant enhanced level of KOR mRNAs expression, situation probably favoring dynorphin binding to the opioid receptor and at least partially underlying the observed inhibition of pain behaviors.

The relationship between the opioid system and steroid hormones in pain modulation has been extensively reported.1, 20, 55 In this regard, progesterone-induced elevation of nociceptive response thresholds in intact animals results from the activation of multiple spinal antinociceptive systems,12, 25 including the dynorphin-KOR system.20 Our observations suggest that the recruitment of the KOR system after progesterone administration to male rats with spinal cord injury could be mediating the steroid-mediated analgesia.

Taken together, our results demonstrate that progesterone, by preventing the potential deleterious consequences of injury-induced maladaptive changes in the CNS, may afford pain relief after spinal cord injury.

It is important to remark that the pathophysiology of neuropathic pain after spinal cord injury involves multiple factors such as the imbalance of excitatory and inhibitory neurotransmission, an exacerbated inflammatory response and cell death.6, 37, 97 Recent reports suggest that activated glial cells both at spinal33, 35 and supraspinal levels80, 92 may modulate neuronal activity and contribute to persistent pain. One of the consequences of this neuronal-glial cross-talk is the induction of PKC32 and the activation of NMDAR-dependent and independent mechanisms.18, 93 Strong evidence supports the idea that the manifestations of chronic pain depend on descending facilitatory and/or inhibitory pathways arising from supraspinal sites.8, 36, 76, 79 In particular, descending pathways from the rostral ventromedial medulla may be responsible for regulating dynorphin content in the dorsal horn and eliciting central sensitization.27 Our studies on mRNA expression allow neither ascribing the extent of these influences to the ipsilateral and/or contralateral side, nor determining how descending control systems interface with the dorsal horn nociceptive process to contribute to the pathological pain state. However, it is likely that the convergence of descending modulation, spinal and supraspinal plasticity, and afferent drive23, 27, 79, 92 may underlie the complex changes observed in the dorsal horn after spinal cord injury.

Therefore, drugs used for the treatment of chronic pain should be able to block the multiple cellular and molecular events leading to damage, at the different control levels. In this context, progesterone is an excellent candidate since, after spinal or brain injury, it exerts concerted beneficial influences on multiple processes, resulting in better functional and histological outcomes,87 promoting neuroprotection and remyelination,22, 44, 86 and reducing inflammatory mediators and glial activation.18, 22 Thus, progesterone may be contributing to limit the spread of pathological changes in the injured spinal cord, avoiding the onset of spontaneous pain behaviors.97 In addition, progesterone-linked changes in the excitability of descending systems arising from supraspinal sites may contribute to the modulation of the descending control over spinal nociceptive processing.57

Progesterone has been shown to exert those pleiotropic effects within the nervous system by regulating gene transcription, intracellular signaling pathways, and neurotransmission.7 The spinal cord expresses an array of progesterone receptors, including the classic nuclear receptor (PR), that may act as a ligand-activated transcription factor to regulate the expression of target genes.22, 86 It still remains unclear whether the response elements in the promoters of the genes encoding the studied molecules allow a direct control of their expression through the PR. However, multiple signals have been involved in the regulation of PKC and the NMDAR subunits genes even in the absence of steroid-response elements.38, 52, 53, 77 Thus, the possibility exists that their transcription may be regulated through the cross-talk of PR with members of the AP-1, NFκB, and Sp-family of transcription factors19, 73 or through its interactions with the Src/Ras/MAPK and the c-AMP signaling pathways.85 It should be noted that we did not find significant changes in the expression of the studied molecules in sham animals receiving progesterone, suggesting that the neurosteroid may be modulating their expression by targeting signaling events that are triggered after injury.

The newly cloned progesterone membrane receptors and the membrane-associated protein PGRMC131 localized in dorsal horn neurons also offer multiple sites for progesterone action in pain mechanisms. Furthermore, other steroid mediated actions derive from the rapid conversion of progesterone into 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone in the dorsal spinal cord.22, 42, 61, 69, 75, 86 These reduced metabolites, acting as positive allosteric modulators of GABAA receptor complex,25, 68 or by enhancing specific GABAA receptor subunits,72 may play an important role in progesterone induced-analgesia, a notion that is in line with the key results reported by Patte-Mensah et al70 regarding the crucial role of these derivatives in spinal pain processing.

Other mechanisms could not be precluded when considering progesterone analgesic effects. The sigma-1 receptor (Sig-1R), another target of progesterone,59, 65 is strongly expressed in the dorsal spinal cord2 and is associated with central sensitization and pain.21 A recent picture suggests that the activation of the spinal Sig-1R enhances NMDAR-induced pain via PKC-dependent phosphorylation of the NR1 subunit.40 Our results suggest that progesterone, a competitive inhibitor of the Sig-1 R binding site,59, 65 may have a potential role in this mechanism. This idea is in perfect agreement with a previous report demonstrating a crucial role of Sig-1R in the modulation of pain after dehydroepiandrosterone administration.39

Further studies are needed in order to uncover the diverse signaling mechanisms involved in progesterone observed effects. However, the results shown here are promising and provide compelling evidence that supports the early use of progesterone to prevent the development of neuropathic pain. Our results also add new data that further stimulate the study of steroid-based treatments for traumatic, neurodegenerative, and autoimmune diseases.22, 62 In fact, the neuroprotective effects of natural progesterone have already been tested in 2 clinical trials, showing favorable neurological outcomes in patients receiving the steroid following moderate traumatic brain injury.95, 96 Natural progesterone has several features that make it an attractive potential drug for the treatment of injuries or diseases of the nervous system: Its pharmacokinetic properties are well known due to the wide experience derived from its administration in hormone replacement therapy; it is inexpensive and widely available; it is able to rapidly cross the blood-brain barrier; and it has limited adverse effects.96 In conclusion, our present results suggest that progesterone-based strategies might offer a novel perspective for the prevention of chronic pain after central injuries.

Back to Article Outline

Acknowledgments 

We deeply thank Ms. Paulina Roig for excellent technical assistance.

Back to Article Outline

References 

  1. Aloisi AM, Bonifazi M. Sex hormones, central nervous system and pain. Horm Behav. 2006;50:1–7
  2. Alonso G, Phan V, Guillemain I, Saunier M, Legrand A, Anoal M, et al. Immunocytochemical localization of the sigma(1) receptor in the adult rat central nervous system. Neuroscience. 2000;97:155–170
  3. Baastrup C, Finnerup NB. Pharmacological management of neuropathic pain following spinal cord injury. CNS Drugs. 2008;22:455–475
  4. Bajo M, Crawford EF, Roberto M, Madamba SG, Siggins GR. Chronic morphine treatment alters expression of N-methyl-D-aspartate receptor subunits in the extended amygdala. J Neurosci Res. 2006;83:532–537
  5. Balasubramanian B, Portillo W, Reyna A, Chen JZ, Moore AN, Dash PK, et al. Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats. Endocrinology. 2008;149:5509–5517
  6. Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell. 2009;139:267–284
  7. Baulieu EE. Neurosteroids, their role in brain physiology: Neurotrophycity, memory, aging. J Bull Acad Natl Med. 2001;185:349–372
  8. Bee LA, Dickenson AH. Rostral ventromedial medulla control of spinal sensory processing in normal and pathophysiological states. Neuroscience. 2007;147:786–793
  9. Burchiel KJ, Hsu FP. Pain and spasticity after spinal cord injury: Mechanisms and treatment. Spine. 2001;26:146–160
  10. Caudle RM, Perez FM, King C, Yu CG, Yezierski RP. N-methyl-D-aspartate receptor subunit expression and phosphorylation following excitotoxic spinal cord injury in rats. Neurosci Lett. 2003;349:37–40
  11. Chaplan S, Bach F, Pogrel J, Chung J, Yaksh T. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci. 1994;16:7711–7724
  12. Charlet A, Lasbennes F, Darbon P, Poisbeau P. Fast non-genomic effects of progesterone-derived neurosteroids on nociceptive thresholds and pain symptoms. Pain. 2008;139:603–609
  13. Choi Y, Yoon YW, Na HS, Kim SH, Chung JM. Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain. Pain. 1994;59:369–376
  14. Christensen MD, Everhart AW, Pickelman JT, Hulsebosch CE. Mechanical and thermal allodynia in chronic central pain following spinal cord injury. Pain. 1996;68:97–107
  15. Christensen MD, Hulsebosch CE. Chronic central pain after spinal cord injury. J Neurotrauma. 1997;14:517–537
  16. Coronel MF, Hernando-Insúa A, Rodriguez JM, Elías F, Chasseing NA, Montaner AD, et al. Oligonucleotide IMT504 reduces neuropathic pain after peripheral nerve injury. Neurosci Lett. 2008;444:69–73
  17. Costigan M, Scholz J, Woolf CJ. Neuropathic pain: A maladaptive response of the nervous system to damage. Annu Rev Neurosci. 2009;32:1–32
  18. Cutler SM, Cekic M, Miller DM, Wali B, VanLandingham JW, Stein DG. Progesterone improves acute recovery after traumatic brain injury in the aged rat. J Neurotrauma. 2007;24:1475–1486
  19. Dai D, Litman ES, Schonteich E, Leslie KK. Progesterone regulation of activating protein-1 transcriptional activity: A possible mechanism of progesterone inhibition of endometrial cancer cell growth. J Steroid Biochem Mol Biol. 2003;87:123–131
  20. Dawson-Basoa ME, Gintzler AR. Gestational and ovarian sex steroid antinociception: Synergy between spinal kappa and delta opioid systems. Brain Res. 1998;794:61–67
  21. De la Puente B, Nadal X, Portillo-Salido E, Sánchez-Arroyos R, Ovalle S, Palacios G, et al. Sigma-1 receptors regulate activity-induced spinal sensitization and neuropathic pain after peripheral nerve injury. Pain. 2009;145:294–303
  22. De Nicola AF, Labombarda F, Gonzalez Deniselle MC, Gonzalez SL, Garay L, Meyer M, et al. Progesterone neuroprotection in traumatic CNS injury and motoneuron degeneration. Front Neuroendocrinol. 2009;30:173–187
  23. Dogrul A, Ossipov MH, Porreca F. Differential mediation of descending pain facilitation and inhibition by spinal 5HT-3 and 5HT-7 receptors. Brain Res. 2009;1280:52–59
  24. Finnerup NB, Johannesen IL, Sindrup SH, Bach FW, Jensen TS. Pain and dysesthesia in patients with spinal cord injury: A postal survey. Spinal Cord. 2001;39:256–262
  25. Frye CA, Duncan JE. Progesterone metabolites, effective at the GABAA receptor complex, attenuate pain sensitivity in rats. Brain Res. 1994;643:194–203
  26. Gao X, Kim HK, Chung JM, Chung K. Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats. Pain. 2005;116:62–72
  27. Gardell LR, Vanderah TW, Gardell SE, Wang R, Ossipov MH, Lai J, et al. Enhanced evoked excitatory transmitter release in experimental neuropathy requires descending facilitation. J Neurosci. 2003;23:8370–8379
  28. Gardell LR, Ibrahim M, Wang R, Wang Z, Ossipov MH, Malan TP, et al. Mouse strains that lack spinal dynorphin upregulation after peripheral nerve injury do not develop neuropathic pain. Neuroscience. 2004;123:43–52
  29. Gintzler AR, Liu NJ. The maternal spinal cord: Biochemical and physiological correlates of steroid-activated antinociceptive processes. Prog Brain Res. 2001;133:83–97
  30. Grossman SD, Wolfe BB, Yasuda RP, Wrathall JR. Changes in NMDA receptor subunit expression in response to contusive spinal cord injury. J Neurochem. 2000;75:174–184
  31. Guennoun R, Meffre D, Labombarda F, Gonzalez SL, Gonzalez Deniselle MC, Stein DG, et al. The membrane-associated progesterone-binding protein 25-Dx: Expression, cellular localization and up-regulation after brain and spinal cord injuries. Brain Res Rev. 2008;57:493–505
  32. Guo W, Wang H, Watanabe M, Shimizu K, Zou S, LaGraize SC, et al. Glial-cytokine-neuronal interactions underlying the mechanisms of persistent pain. J Neurosci. 2007;27:6006–6018
  33. Gwak YS, Hulsebosch CE. Remote astrocytic and microglial activation modulates neuronal hyperexcitability and below-level neuropathic pain after spinal injury in rat. Neuroscience. 2009;161:895–903
  34. Haberny SL, Carr KD. Comparison of basal and D-1 dopamine receptor agonist-stimulated neuropeptide gene expression in caudate-putamen and nucleus accumbens of ad libitum fed and food-restricted rats. Brain Res Mol Brain Res. 2005;141:121–127
  35. Hains BC, Waxman SG. Activated microglia contribute to the maintenance of chronic pain after spinal cord injury. J Neurosci. 2006;26:4308–4317
  36. Heinricher MM, Tavares I, Leith JL, Lumb BM. Descending control of nociception: Specificity, recruitment and plasticity. Brain Res Rev. 2009;60:214–225
  37. Hulsebosch CE, Hains BC, Crown ED, Carlton SM. Mechanisms of chronic central neuropathic pain after spinal cord injury. Brain Res Rev. 2009;60:202–213
  38. Jing H, Iwasaki Y, Nishiyama M, Taguchi T, Tsugita M, Taniguchi Y, et al. Multisignal regulation of the rat NMDA1 receptor subunit gene–a pivotal role of glucocorticoid-dependent transcription. Life Sci. 2008;82:1137–1141
  39. Kibaly C, Meyer L, Patte-Mensah C, Mensah-Nyagan AG. Biochemical and functional evidence for the control of pain mechanisms by dehydroepiandrosterone endogenously synthesized in the spinal cord. FASEB J. 2008;22:93–104
  40. Kim HW, Roh DH, Yoon SY, Seo HS, Kwon YB, Han HJ, et al. Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice. Br J Pharmacol. 2008;154:1125–1134
  41. Kondo D, Yabe R, Kurihara T, Saegusa H, Zong S, Tanabe T. Progesterone receptor antagonist is effective in relieving neuropathic pain. Eur J Pharmacol. 2006;541:44–48
  42. Labombarda F, Pianos A, Liere P, Eychenne B, González S, Cambourg A, et al. Injury elicited increase in spinal cord neurosteroid content analyzed by gas chromatography mass spectrometry. Endocrinology. 2006;147:1847–1859
  43. Labombarda F, Coronel MF, Villar MJ, De Nicola AF, Gonzalez SL. Neuropathic pain and temporal expression of preprodynorphin, protein kinase C and N-methyl-D-aspartate receptor subunits after spinal cord injury. Neurosci Lett. 2008;447:115–119
  44. Labombarda F, Gonzalez SL, Lima A, Roig P, Guennoun R, Schumacher M, et al. Effects of progesterone on oligodendrocyte progenitors, oligodendrocyte transcription factors and myelin proteins following spinal cord injury. Glia. 2009;57:884–897
  45. Lacroix-Fralish ML, Tawfik VL, Nutile-McMenemy N, DeLeo JA. Progesterone mediates gonadal hormone differences in tactile and thermal hypersensitivity following L5 nerve root ligation in female rats. Neuroscience. 2006;138:601–608
  46. Lacroix-Fralish ML, Tawfik VL, Nutile-McMenemy N, Deleo JA. Neuregulin 1 is a pronociceptive cytokine that is regulated by progesterone in the spinal cord: Implications for sex specific pain modulation. Eur J Pain. 2008;12:94–103
  47. Lai J, Ossipov MH, Vanderah TW, Malan TP, Porreca F. Neuropathic pain: The paradox of dynorphin. Mol Interv. 2001;1:160–167
  48. Lan JY, Skeberdis VA, Jover T, Grooms SY, Lin Y, Araneda RC, et al. Protein kinase C modulates NMDA receptor traffiking and gating. Nat Neurosci. 2001;4:382–390
  49. Latremoliere A, Woolf CJ. Central sensitization: A generator of pain hypersensitivity by central neural plasticity. J Pain. 2009;10:895–926
  50. Laughlin TM, Vanderah TW, Lashbrook J, Nichols ML, Ossipov M, Porreca F, et al. Spinally administered dynorphin A produces long-lasting allodynia: Involvement of NMDA but not opioid receptors. Pain. 1997;72:253–260
  51. Leonelli E, Bianchi R, Cavaletti G, Caruso D, Crippa D, García-Segura LM, et al. Progesterone and its derivatives are neuroprotective agents in experimental diabetic neuropathy: A multimodal analysis. Neurscience. 2007;144:1293–1304
  52. Lim G, Wang S, Zeng Q, Sung B, Yang L, Mao J. Expression of spinal NMDA receptor and PKCgamma after chronic morphine is regulated by spinal glucocorticoid receptor. J Neurosci. 2005;25:11145–11154
  53. Liu A, Hoffman PW, Lu W, Bai G. NF-kappaB site interacts with Sp factors and up-regulates the NR1 promoter during neuronal differentiation. J Biol Chem. 2004;279:17449–17458
  54. Liu NJ, Gintzler AR. Prolonged ovarian sex steroid treatment of male rats produces antinociception: Identification of sex-based divergent analgesic mechanisms. Pain. 2000;85:273–281
  55. Liu NJ, von Gizycki H, Gintzler AR. Sexually dimorphic recruitment of spinal opioid analgesic pathways by the spinal application of morphine. J Pharmacol Exp Ther. 2007;322:654–660
  56. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔCT) method. Methods. 2001;25:402–408
  57. Lovick TA, Devall AJ. Progesterone withdrawal-evoked plasticity of neural function in the female periaqueductal grey matter. Neural Plast. 2008 Dec 2;[Epub ahead of print]
  58. Martin WJ, Malmberg AB, Basbaum AI. PKCgamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain. J Neurosci. 2001;21:5321–5327
  59. Maurice T, Grégoire C, Espallergues J. Neuro(active)steroids actions at the neuromodulatory sigma1 (sigma1) receptor: Biochemical and physiological evidences, consequences in neuroprotection. Pharmacol Biochem Behav. 2006;84:581–597
  60. McBain CJ, Mayer ML. N-methyl-D-aspartic acid receptor structure and function. Physiol Rev. 1994;74:723–760
  61. Mensah-Nyagan AG, Kibaly C, Schaeffer V, Venard C, Meyer L, Patte-Mensah C. Endogenous steroid production in the spinal cord and potential involvement in neuropathic pain modulation. J Steroid Biochem Mol Biol. 2008;109:286–293
  62. Mensah-Nyagan AG, Meyer L, Schaeffer V, Kibaly C, Patte-Mensah C. Evidence for a key role of steroids in the modulation of pain. Psychoneuroendocrinology. 2009;34(Suppl 1):S169–S177
  63. Miraucourt LS, Dallel R, Voisin DL. Glycine inhibitory dysfunction turns touch into pain through PKCgamma interneurons. PLoS One. 2007;2:1116
  64. Molander C, Grant G. Spinal Cord Cytoarchitecture. In: The Rat Nervous System. 2nd ed.. San Diego, CA: Academic Press; 1995;p. 39–45
  65. Monnet FP, Maurice T. The sigma1 protein as a target for the non-genomic effects of neuro(active)steroids: Molecular, physiological, and behavioral aspects. J Pharmacol Sci. 2006;100:93–118
  66. Nagy GG, Watanabe M, Fukaya M, Todd AJ. Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique. Eur J Neurosci. 2004;20:3301–3312
  67. Neumann S, Braz JM, Skinner K, Llewellyn-Smith IJ, Basbaum AI. Innocuous, not noxious, input activates PKCgamma interneurons of the spinal dorsal horn via myelinated afferent fibers. J Neurosci. 2008;28:7936–7944
  68. Pathirathna S, Brimelow BC, Jagodic MM, Krishnan K, Jiang X, Zorumski CF, et al. New evidence that both T-type calcium channels and GABAA channels are responsible for the potent peripheral analgesic effects of 5alpha-reduced neuroactive steroids. Pain. 2005;114:429–443
  69. Patte-Mensah C, Li S, Mensah-Nyagan AG. Impact of neuropathic pain on the gene expression and activity of cytochrome P450side-chain-cleavage in sensory neural networks. Cell Mol Life Sci. 2004;61:2274–2284
  70. Patte-Mensah C, Kibaly C, Mensah-Nyagan AG. Substance P inhibits progesterone conversion to neuroactive metabolites in spinal sensory circuit: A potential component of nociception. PNAS. 2005;102:9044–9049
  71. Peinnequin A, Mouret C, Birot O, Alonso A, Mathieu J, Clarençon D, et al. Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green. BMC Immunol. 2004;5:3
  72. Peng HY, Chen GD, Lee SD, Lai CY, Chiu CH, Cheng CL, et al. Neuroactive steroids inhibit spinal reflex potentiation by selectively enhancing specific spinal GABA(A) receptor subtypes. Pain. 2009;143:12–20
  73. Pettus EH, Wright DW, Stein DG, Hoffman SW. Progesterone treatment inhibits the inflammatory agents that accompany traumatic brain injury. Brain Res. 2005;1049:112–119
  74. Pluchino N, Luisi M, Lenzi E, Centofanti M, Begliuomini S, Freschi L, et al. Progesterone and progestins: Effects on brain, allopregnanolone and beta-endorphin. J Steroid Biochem Mol Biol. 2006;102:205–213
  75. Poisbeau P, Patte-Mensah C, Keller AF, Barrot M, Breton JD, Luis-Delgado OE, et al. Inflammatory pain upregulates spinal inhibition via endogenous neurosterois production. J Neurosci. 2005;25:11768–11776
  76. Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci. 2002;25:319–325
  77. Qiang M, Ticku MK. Role of AP-1 in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene up-regulation in mouse cortical neurons. J Neurochem. 2005;95:1332–1341
  78. Ren K, Dubne R, Murphy A, Hoffman GE. Progesterone attenuates persistent inflammatory hyperalgesia in females rats: Involvement of spinal NMDA receptor mechanism. Brain Res. 2000;865:272–277
  79. Ren K, Dubner R. Descending modulation in persistent pain: An update. Pain. 2002;100:1–6
  80. Roberts J, Ossipov MH, Porreca F. Glial activation in the rostroventromedial medulla promotes descending facilitation to mediate inflammatory hypersensitivity. Eur J Neurosci. 2009;30:229–241
  81. Rodriguez Parkitna JM, Bilecki W, Mierzejewski P, Stefanski R, Ligeza A, Bargiela A, et al. Effects of morphine on gene expression in the rat amygdala. J Neurochem. 2004;91:38–48
  82. Roglio I, Bianchi R, Gotti S, Scurati S, Giatti S, Pesaresi M, et al. Neuroprotective effects of dihydroprogesterone and progesterone in an experimental model of nerve crush injury. Neuroscience. 2008;155:673–685
  83. Roh DH, Kim HW, Yoon SY, Seo HS, Kwon YB, Han HJ, et al. Depletion of capsaicin-sensitive afferents prevents lamina-dependent increases in spinal N-methyl-D-aspartate receptor subunit 1 expression and phosphorylation associated with thermal hyperalgesia in neuropathic rats. Eur J Pain. 2008;12:552–563
  84. Rometo AM, Rance NE. Changes in prodynorphin gene expression and neuronal morphology in the hypothalamus of postmenopausal women. J Neuroendocrinol. 2008;20:1376–1381
  85. Schumacher M, Guennoun R, Ghoumari A, Massaad C, Robert F, El-Etr M, et al. Novel perspectives for progesterone in hormone replacement therapy, with special reference to the nervous system. Endocr Rev. 2007;28:387–439
  86. Schumacher M, Sitruk-Ware R, De Nicola AF. Progesterone and progestins: Neuroprotection and myelin repair. Curr Opin Pharmacol. 2008;8:740–746
  87. Thomas AJ, Nockels RP, Pan HQ, Shaffrey CI, Chopp M. Progesterone is neuroprotective after experimental acute spinal cord trauma in rats. Spine. 1999;24:2134–2138
  88. Tomiyama M, Furusawa K, Kamijo M, Kimura T, Matsunaga M, Baba M. Upregulation of mRNAs coding for AMPA and NMDA receptor subunits and metabotropic glutamate receptors in the dorsal horn of the spinal cord in a rat model of diabetes mellitus. Brain Res Mol Brain Res. 2005;136:275–281
  89. Ultenius C, Linderoth B, Meyerson BA, Wallin J. Spinal NMDA receptor phosphorylation correlates with the presence of neuropathic signs following peripheral nerve injury in the rat. Neurosci Lett. 2006;399:85–90
  90. Vats ID, Dolt KS, Kumar K, Karar J, Nath M, Mohan A, et al. YFa, a chimeric opioid peptide, induces kappa-specific antinociception with no tolerance development during 6 days of chronic treatment. J Neurosci Res. 2008;86:1599–1607
  91. Wang Z, Gardell LR, Ossipov MH, Vanderah TW, Brennan MB, Hochgeschwender U, et al. Pronociceptive actions of dynorphin maintain chronic neuropathic pain. J Neurosci. 2001;21:1779–1786
  92. Wei F, Guo W, Zou S, Ren K, Dubner R. Supraspinal glial-neuronal interactions contribute to descending pain facilitation. J Neurosci. 2008;28:10482–10495
  93. Weyerbacher AR, Xu Q, Tamasdan C, Shin SJ, Inturrisi CE. N-Methyl-d-aspartate receptor (NMDAR) independent maintenance of inflammatory pain. Pain. 2010;148(2):237–246
  94. Woods AS, Kaminski R, Oz M, Wang Y, Hauser K, Goody R, et al. Decoy peptides that bind dynorphin noncovalently prevent NMDA receptor-mediated neurotoxicity. J Proteome Res. 2006;5:1017–1023
  95. Wright DW, Kellermann AL, Hertzberg VS, Clark PL, Frankel M, Goldstein FC, et al. ProTECT: A randomized clinical trial of progesterone for acute traumatic brain injury. Ann Emerg Med. 2007;49:391–402
  96. Xiao G, Wei J, Yan W, Wang W, Lu Z. Improved outcomes from the administration of progesterone for patients with acute severe traumatic brain injury: A randomized controlled trial. Crit Care. 2008;12(2):R61
  97. Yezierski RP. Spinal cord injury pain: Spinal and supraspinal mechanisms. J Rehabil Res Dev. 2009;46:95–107

 Supported by the University of Buenos Aires (M808) and the National Research Council of Argentina (CONICET, PIP 5542).

PII: S1526-5900(10)00533-X

doi:10.1016/j.jpain.2010.04.013

The Journal of Pain
Volume 12, Issue 1 , Pages 71-83, January 2011

Article Tools

* Image Usage & Resolution