Involvement of Estrogen in Rapid Pain Modulation in the Rat Spinal Cord
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The pivotal role of estrogens in the pain sensitivity has been investigated in many ways. Traditionally, it is ascribed to the slow genomic changes mediated by classical nuclear estrogen receptors (ER), ERα and ERβ, depending on peripheral estrogens. Recently, it has become clear that estrogens can also signal through membrane ERs (mERs), such as G-protein-coupled ER1 (GPER1), mediating the non-genomic effects. However, the spinal specific role played by ERs and the underlying cellular mechanisms remain elusive. The present study investigated the rapid estrogenic regulation of nociception at the spinal level. Spinal administration of 17β-estradiol (E2), the most potent natural estrogen, acutely produced a remarkable mechanical allodynia and thermal hyperalgesia without significant differences among male, female and ovariectomized (Ovx) rats. E2-induced the pro-nociceptive effects were partially abrogated by ICI 182,780 (ERs antagonist), and mimicked by E2-BSA (a mER agonist). Inhibition of local E2 synthesis by 1,4,6-Androstatrien-3,17-dione (ATD, a potent irreversible aromatase inhibitor), or blockade of ERs by ICI 182,780 produced an inhibitory effect on the late phase of formalin nociceptive responses. Notably, lumbar puncture injection of G15 (a selective GPER1 antagonist) resulted in similar but more efficient inhibition of formalin nociceptive responses as compared with ICI 182,780. At the cellular level, the amplitude and decay time of spontaneous inhibitory postsynaptic currents were attenuated by short E2 or E2-BSA treatment in spinal slices. These results indicate that estrogen acutely facilitates nociceptive transmission in the spinal cord via activation of membrane-bound estrogen receptors.
KeywordsEstrogen Estrogen receptors Nociceptive behavioral responses Inhibitory postsynaptic currents Spinal cord Rat
Estrogens regulate a wide range of cellular functions. It was originally identified as the sex steroid hormones from ovary and regulators of reproduction and sexual behavior, but it has long been suggested that estrogens have local effects on the somatosensory system at the spinal level related to pain processing . Natural estrogens include 17β-estradiol (E2), estriol and estrone. E2 is the strongest crude estrogen. Estrogens have been reported to play either pro-nociceptive [2, 3, 4, 5] or anti-nociceptive role [6, 7] in different species and treatments. Here, we confirmed that E2 facilitated the nociceptive responses at the spinal level in different gender rats.
In addition to ovarian E2, evidence suggests that the spinal cord is also a source of E2 via aromatization of testosterone [8, 9]. One likely function of neurosteroid E2 is to acutely modulate pain transmission . Recently, the important role of local synthesized E2 in spinal morphine analgesia has been demonstrated . In the present study, we further examined whether the inhibition of spinal E2 synthesis alters nociceptive responses.
It is known that three types of ERs: ERα, ERβ and GPER1 (also termed GPR30) are widely distributed in the spinal dorsal horn [11, 12, 13]. Estrogens not only modulate nociception via classical nuclear estrogen receptors (nERs), ERα and ERβ, which function as ligand-regulated transcription factors, they also initiate fast second-messenger cascades through GPER1 , a novel plasma membrane ER [15, 16, 17], which was proved promote mechanical hyperalgesia . Besides the nucleus localization, ERα and ERβ, were also reported localize at the plasma membrane to affect cellular physiology  or traffic to the plasma membrane, in which they associate with G-proteins [10, 20, 21, 22] and mediate activation of multiple membrane signaling cascades . In this study, all the ERα, ERβ and GPER1 were detected in cytoplasmic and/or membrane proteins of the spinal dorsal horn; and E2-induced facilitation on the spinal nociceptive transmission could be mediated by membrane binding ERs (mERs).
The superficial dorsal horn of the spinal cord, especially substantia gelatinosa (SG, lamina II) is a region critical for modulating nociceptive information. Moreover, E2 rapidly inhibits the function of glycine receptor (GlyR) in the spinal cord with a non-genomic characteristic  implying E2-induced disinhibition in superficial dorsal horn may underlie its modulation in nociception. Here, we further examined the acute effects of E2 on inhibitory transmission in SG neurons.
Materials and Methods
Experiments were performed on 165 adult (2–3 month old, male: 124, female: 41) or 8 young (3 week old, without estrous cycle) Sprague–Dawley rats. Rats were obtained from the Experimental Animal Center of the Chinese Academy of Science. Prior to experimental manipulation, rats were allowed to acclimate to the housing facilities for 1 week and maintained on a 12:12 h light–dark cycle and a constant room temperature of 21 °C with free access to food and water. Several female rats were bilaterally ovariectomized (Ovx) under isoflurane anesthesia using sterile surgical procedures. Three weeks later, behavioral tests were conducted on Ovx rats. All experimental protocols and animal handling procedures were approved by Animal Care and Use Committee of Fudan University, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering.
All reagents were purchased from Sigma (St Louis, MO) unless otherwise noted. The steroid 17β-estradiol (E2, a main form of estrogen) was dissolved in sesame oil (in vivo) or ethanol (in vitro). Fulvestrant (ICI 182,870) or G15 was dissolved in Dimethyl Sulphoxide (DMSO) and diluted in normal saline to a final concentration of 10 % DMSO. 1,4,6-Androstatrien-3,17-dione (ATD, Steraloids, Newport, RI 02849) were prepared in DMSO and diluted in sesame oil. E2-BSA was dissolved in normal saline. Bicuculline methiodide (BMI, 10 μM), strychnine (1 μM), AP5 (50 μM) and DNQX (10 μM) were prepared as stock solutions and diluted to the required concentration (1:1,000) with ACSF.
RNA Isolation and Real-Time PCR
The spinal cord dorsal horn from adult rats of both sexes were dissected and rapidly frozen in liquid nitrogen and stored at −80 °C until further processing. Frozen tissues were directly homogenized in TRIZOL reagent (Invitrogen). Total RNA was extracted following manufacturer’s protocol. The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide. Primer sequences for ERα, ERβ and GPER1 were acquired from the NCBI online database. All PCR reactions were performed using the PCR master mix reagents kit (Invitrogen Life Technologies) on a Promega MMLV Sequence Detection System (Promega Life Technologies). The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min, 50 cycles at 95 °C for 15 s and 65 °C for 1 min. Specific PCR amplification products were detected by the fluorescent double-stranded DNA-binding dye. Each sample was run at least in triplicate and Ct values were averaged and all of the samples with a coefficient of variation for Ct value higher than 1 % were retested. For controls, no-template reactions were run in which water replaced the cDNA template. β-actin was used as the reference gene to normalize expression levels. The relative gene expression level was computed from the target and β-actin using the following formula: mRNA relative expression = 2−(Ct of target–Ct of β-actin).
Under urethane (1.5 g/kg, i.p.) anesthesia, rats were rapidly sacrificed by decapitation. The L4-L6 lumbar spinal cord was rapidly removed, immediately frozen in liquid nitrogen and stored at −70 °C until use. Isolation of cytoplasmic and/or membrane and nuclear protein extracts were achieved using the Compartment Protein Extraction Kit (Chemicon) and protocol. Protein concentrations were measured using a BCA Protein Assay kit (Pierce). Equal amount of protein were loaded and separated in 10 % Tris-Tricine SDS-PAGE gel. The resolved proteins were transferred onto polyvinilidene difluoride (PVDF) membranes (Amersham Bioscience). The membranes were blocked with 5 % non-fat milk in Tris-buffered saline (pH 7.5) containing 0.1 % Tween 20 for 2 h at RT, and incubated overnight at 4 °C with rabbit anti-ERα (1:50, Upstate), rabbit anti-ERβ (1:200, Santa Cruz), rabbit anti-GPER1 (1:200, Abcam), rabbit anti-CREB (total CREB, 1:1,000, Sigma) and mouse anti-transferrin receptor (1:1,000, Invitrogen) primary antibody for 2 h. The blots were then incubated with the secondary antibody, goat anti- rabbit or goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (1:1,000, Pierce), for 2 h at 4 °C. Signals were finally visualized using enhanced chemiluminescence (ECL, Pierce) and the bands were visualized with the ChemiDox XRS system (Bio-Rad).
Under brief isoflurane anesthesia (2 % in oxygen), lumbar puncture (LP) injection was performed as previous described . Briefly, rats were anesthetized with isoflurane. The lumbar region was shaved, sterilized with iodine tincture, then 75 % alcohol, and the intervertebral spaces widened by placing the animal on a plexiglas tube. Animals were then injected at the L5-6 interspace using a 0.5-inch 30-gauge needle connected to a Hamilton syringe. Correct subarachnoid positioning of the tip of the needle was verified by a tail- or paw-flick test. Rats showed full recovery from anesthesia within 5 min after the acute injection. No abnormal motor behavior was observed after any injection. In the study, a volume of 15 μL E2 (0.15, 0.75, 1.5 nmol), E2-BSA (0.75 nmol), ICI 182,870 (100 pmol) or a mixture of ICI 182,870 (100 pmol) and E2 (0.75 nmol) were injected at a rate of 5 μL/min for 3 min 0.5 h before Hargreave’s test and von Frey test. Control animals received equivalent volume of vehicle. As to the formalin test experiments, ICI 182,870 (100 pmol), G15 (2.5 nmol), ATD (300 nmol) or respective vehicle were administered in the same manner. One hour after ICI injection, 30 min after G15 injection or 5 min after ATD injection, the rat was given a unilateral hindpaw intraplantar (i.pl.) injection of 5 % formalin (50 μL).
Hargreave’s Test for Thermal Hyperalgesia
After acclimation to the test chamber for about 30 min, thermal hyperalgesia was assessed by measuring the latency of paw withdrawal in response to a radiant heat source. Adult rats were placed individually into Plexiglas chamber on an elevated glass platform, under which a radiant heat source (model 336 combination units, IITC/life Science Instruments, Woodland Hill, CA, USA) was applied to the glabrous surface of the paw through the glass plate. The heat source was turned off when the rat lifted the foot, allowing the measurement of time from onset of radiant heat application to withdrawal of the rat’s hind paw. This time was defined as the hind paw withdrawal latency (PWL). The heat was maintained at a constant intensity, which produced a stable withdrawal latency of approximately 10–12 s in naïve rats. A 20 s cutoff was used to prevent tissue damage in the absence of a response. The hind paw was tested with 10 min interval between trials.
von Frey Test for Mechanical Allodynia
The hind paw withdrawal threshold (PWT) was determined using a calibrated series of von Frey hairs (Stoelting, IL, USA). Adult animals were placed individually into Plexiglass chamber with meshed wire platform. After acclimation to the test chamber for about 30 min, a series of 8 calibrated von Frey hairs were applied to the central region of the plantar surface of one hind paw in ascending order (1, 1.4, 2, 4, 6, 8, 10, and 15 g). A particular hair was applied until buckling of the hair occurred. This was maintained for approximately 2 s. The hair was applied only when the rat was stationary and standing on all four paws. A withdrawal response was considered valid only if the hind paw was completely removed from the platform. A trial consisted of application of a von Frey hair to the hind paw five times at 15-s intervals. If withdrawal did not occur during five applications of a particular hair, the next larger hair in the series was applied in a similar manner. When the hind paw was withdrawn from a particular hair in three out of the five consecutive applications, the value of that hair in grams was considered to be the withdrawal threshold. Once the threshold was determined for one hind paw, the same testing procedure was repeated on the right hind paw after 5 min. All behavioral tests were performed in a temperature controlled room (22 ± 2 °C).
Spinal Cord Slice Preparation and Whole-Cell Patch Clamp Recordings
The young rat spinal cord slices were prepared as described previously . Briefly, laminectomy was performed under diethyl ether deep anesthesia and the lumbosacral segment of the spinal cord was rapidly removed to ice-cold sucrose artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl, 80; KCl, 2.5; NaH2PO4, 1.25; CaCl2, 0.5; MgCl2, 3.5; NaHCO3, 25; sucrose, 75; ascorbate, 1.3; sodium pyruvate, 3.0; oxygenated with 95 % O2 and 5 % CO2; pH 7.4. Transverse slices (500 μm) with attached dorsal roots (8–12 mm) were prepared and then incubated at preoxygenated recording ACSF of the following composition (in mM): NaCl, 125; KCl, 2.5; CaCl2, 2; MgCl2, 1; NaH2PO4, 1.25; NaHCO3, 26; d-glucose, 25; ascorbate, 1.3; sodium pyruvate, 3.0; recovered at 32 ± 1 °C for 40 min and then at room temperature (RT) for an additional 1 h before experimental recordings. The slice was then transferred into a recording chamber and continuously perfused with recording solution at a rate of 5 ml/min prior to electrophysiological recordings at room temperature.
Whole-cell recordings were obtained from neurons in superficial dorsal horn of the spinal cord. Neurons were identified by infrared differential interference contrast (IR-DIC) video microscopy with an upright microscope (Leica DMLFSA, Germany) equipped with a 40×, 0.8 NA water-immersion objectives and a CCD camera (IR-1000E, USA). Patch pipettes (5–10 MΩ) from borosilicate glass were made on a horizontal micropipette puller (P-97, Sutter Instruments, Novato, CA, USA) and were filled with a solution having the following composition (in mM): CsCl 140, CaCl2 1, MgCl2 2, Na2ATP 2, NaGTP 0.5, HEPES 10, EGTA 1, pH 7.28 with KOH. Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded with normal ACSF containing CNQX (10 μM) and APV (50 μM) to block AMPA and NMDA receptor mediated excitatory synaptic currents. The membrane potential was held at −70 mV. After 5 min recording of basal sIPSCs, 10 μM E2 was bath applied for 5–10 min and sIPSCs were continuously recorded. Only one cell was recorded per slice to obviate the contamination of previous treatment. The sIPSCs were analyzed with MiniAnalysis 6.0.3 software (Synaptosoft, Decatur, GA, USA). sIPSCs were automatically identified as events at least 2 SD above the amplitude of baseline noise. Data were acquired using an Axopatch 200B amplifier and were low-pass filtered at 2 kHz, digitized at 5 kHz. The series resistance (Rs) was monitored during recording. Cells which Rs deviated >20 % or cells with Rs > 60 MΩ were excluded from analysis.
Data were expressed as mean ± SEM. Statistical comparisons were performed using Student’s t test, paired t test and two-way repeated measured ANOVA (treatment × time) followed by Holm-Sidak test. p < 0.05 was considered statistically significant.
The mRNA expression levels of different subtypes of ER in the spinal dorsal horn were assessed on 4 male and 9 female rats. The expression of ERα, ERβ and GPER1 in the isolation of cytoplasmic and/or membrane and nuclear protein extracts were detected on 2 male rats.
Forty-seven male rats were used to assess the effects of E2 in different doses on PWT. Rats were divided into 6 groups, including vehicle, E2 (0.15 nmol, 0.75 nmol and 1.5 nmol), ICI 100 pmol, and ICI (100 pmol) + E2 (0.75 nmol).
The effects of E2 (0.75 nmol) on PWL and PWT were compared in 16 males, 16 females and 16 Ovx rats.
The effects of mER agonist E2-BSA on PWL and PWT were assessed on 14 male rats.
The effects of endogenous E2 on formalin-induced biphasic nociceptive responses were examined in 41 male rats. Rats were divided into 5 groups: vehicle (n = 8)/ICI (100 pmol, n = 8)/G15 (2.5 nmol, n = 9) and vehicle (n = 8)/ATD (300 nmol, n = 8).
Eight young rats of both sexes were used to assess the acute effect of E2 (10 μM) and E2-BSA (10 μM) on sIPSCs.
Expression of Estrogen Receptors in the Spinal Cord
E2 Rapidly Induces Mechanical Allodynia and Thermal Hyperalgesia Via mERs
Endogenous E2 is Involved in the Formalin-Induced Nociceptive Responses
Mounting evidence indicates that estrogen can be synthesized locally in male and female brain and display functional characteristics of neuromodulators [28, 29]. In the spinal cord, estrogens could also be locally produced . We therefore examined the contribution of in situ synthesized estrogen in spinal nociceptive responses. LP injection of androstatrienedione (ATD, 300 nmol) that can inhibit estrogen biosynthesis, a potent irreversible aromatase inhibitor, also attenuated formalin-induced biphasic nociceptive responses (Fig 5b). These results suggest that endogenous synthesized estrogen in the spinal cord was involved in the acute pain modulation.
E2 Acutely Suppresses Spontaneous Inhibitory Postsynaptic Currents
The present study demonstrates that three types of ERs: ERα, ERβ and GPER1, were expressed in the spinal cord dorsal horn without sex specificity, and all of them were detected in cytoplasmic and/or membrane protein extracts. Exogenous E2 produced robust mechanical allodynia and thermal hyperalgesia at the level of the spinal cord, which might be mediated by mERs. Accordingly, no difference in pro-nociceptive effects of E2 between both sexes and Ovx rats were detected. Moreover, interruption of endogenous E2 also faded the formalin inflammatory pain. The disinhibition of GABA and/or Gly might be involved in E2-induced pro-nociception.
The classical estrogen action occurs through the entry of estrogen into the cell, interaction with the nuclear ER α/β, and transcriptional activation of estrogen-responsive genes. This cell signaling mechanism can take several hours or more to achieve its final downstream effects. Additional non-genomic estrogen-induced rapid cell signaling pathways via membrane bound receptors have been recognized as important contributors to the overall biological response. There is growing evidence that a subpopulation of the conventional nuclear steroid receptor localized at the cell membrane mediates many of the rapid signaling actions of steroid hormones [30, 31]. It is clear that GPER1 is the major membrane-associated ER and classical ERα or ERβ also traffic to the plasma membrane when exposed to estrogen to mediate the rapid non-genomic responses [32, 33, 34, 35]. Our western blot results revealed that all the ERα, ERβ and GPER1 were detected in isolation of membrane protein extracts of the spinal dorsal horn. Classical ER antagonist, ICI 182,780, blocks the pro-nociceptive effect induced by E2 indicating that nucleus-located and/or membrane-located ERα and ERβ were involved in pain modulation. By contrast with the previous studies showing long-term genomic effects of estrogens on nociception , the present finding indicates that estrogens can rapidly modulate behavioral responsiveness to painful stimuli at the spinal level. Rapid potentiation of nociceptive responses by E2 in the spinal cord suggests a non-genomic effect that requires the activation of a membrane estrogen-binding site . Furthermore, the membrane-impermeable E2-BSA conjugate gives an identical pro-nociceptive response to free E2, indicating that the hormone-binding site of this receptor is accessible from the extracellular surface of the plasma membrane. In support of this, GPER1 antagonist G15 efficiently relieved the both phases of formalin-induced nociceptive responses.
It is well established that women have a higher incidence of pain syndromes than men [35, 36, 37]. Most of nociceptive behavior tests are performed on humans and animals with hormonal fluctuation during the reproductive cycle, indicating sex differences of pain sensitivity [38, 39, 40]. Interestingly, when exogenous E2 was given by lumbar puncture in the present study, we fail to detect the differences of rapid pain modulation between both sexes and Ovx rats, suggesting that the effect of local E2 in the spinal cord is different from that in the periphery gonadal hormones system.
Although the ovary is the major source of estrogens released to the bloodstream, most evidence prove that E2 could be synthesis in the spinal cord via local aromatization of androgenic and cholesterol. Numerous tissues including the brain and spinal cord express aromatase . In according to our RT-PCR results that the basal expression of ERα, ERβ and GPER1 is similar in different sex, Evrard et al.  report the absence of sex differences in spinal aromatase expression and effects. The inhibitor of aromatase, ATD, relieves the late phase of formalin-induced responses, illustrating that local E2 plays an important role in acute inflammation pain. Consistently, inhibition of the endogenous E2 synthesis in quail spinal cord or blockade of ERs in neuropathic pain rats reduced the nociceptive behavioral responsiveness [8, 42].
Many studies have linked estrogen to GABAergic activity in various regions of the brain [43, 44, 45]. It is reported that E2 acts indirectly on pyramidal cells by causing a transient decrease of GABA synthesis in interneurons, which increases the excitatory activity on pyramidal cells in the hippocampus . Jiang et al.  demonstrated that E2 rapidly reduced the amplitude of glycine-activated currents (IGly) in cultured rat hippocampal and spinal dorsal horn neurons. They found that E2 similarly inhibited IGly in outside-out membrane patches from neurons devoid of nuclei, suggesting a non-genomic characteristic. According to our in vitro results, both E2 and E2-BSA reduce the amplitude and decay time of sIPSC mediated by GABAAR and/or GlyR, suggesting that E2 alters neuronal activity by suppressing inhibitory synaptic transmission via mERs.
This work was supported by National Basic Research Program of China (Grant 2007CB512502) and National Natural Science Fund of China (NSFC, 31121061, 31070973, 30900444 and 30830044).
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