| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
| ||||||||||||||
| ||||||||||||||
|
|
|
|
||||||||||||
|
||||||||||||||
From the Department of Anesthesiology and the Center for the Study of Pharmacologic Plasticity in the Presence of Pain, Wake Forest University School of Medicine, Winston-Salem, North Carolina.
Address correspondence and reprint requests to Dr. James C. Eisenach, Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157-1009. Address e-mail to jim{at}eisenach.us.
Abstract
INTRODUCTION: Estrogen sensitizes responses to painful stimuli, but its contribution to acute and chronic pain from the uterine cervix is unknown. Previous studies link the excitatory transient receptor potiential-1 channel (TRPV-1) to sensitization in viscera, and show that estrogen increases TRPV-1 expression in afferents from the uterine cervix. Here, we tested whether estrogen enhanced responses to uterine cervical distension in rats, and whether this involved TRPV-1 channels.
METHODS: Ovariectomized rats, with or without estrogen replacement, were anesthetized and hypogastric nerve and abdominal muscle contraction reflex responses to graded uterine cervical distension were recorded. Single unit hypogastric nerve fiber firing was measured before and after acute treatment with the TRPV-1 antagonist, capsaizepine, or vehicle.
RESULTS: Abdominal muscle contraction reflex responses to uterine cervical distension were enhanced in estrogen-treated rats. Hypogastric afferent responses to cervical distension were reduced by capsaizepine in estrogen-treated animals, but were unaffected in ovariectomized animals without estrogen replacement.
CONCLUSIONS: These data suggest that the TRPV-1 channel is unimportant for normal mechanosensation in the cervix in the absence of estrogen, since capsaizepine failed to reduce responses to uterine cervical distension in rats without estrogen replacement. In contrast, TRPV-1 function is important for estrogen-induced sensitization. These data raise the possibility that acute and chronic pain coming from the cervix, such as labor or cancer, may be enhanced by estrogen and might be reduced by antagonists of TRPV-1.
The uterine cervix is a major source of physiologic pain in the first stage of labor, pathologic pain of cervical cancer, and iatrogenic pain of acute cervical dilation. Little is known, however, regarding the neurophysiologic basis of pain from female pelvic organs, especially from the uterine cervix. We recently (1,2) developed a model of uterine cervical distension in rats in order to gain such understanding.
Estrogen receptor signaling dramatically affects uterine cervical structure, and may also enhance pain responses. Estrogen is thought to underlie observations that women have increased sensitivity to pain stimuli compared to men, and that they exhibit a higher incidence of some chronic pain conditions, including irritable bowel syndrome, a visceral pain condition (3). We recently (4) showed that chronic estrogen treatment in rats increases spontaneous activity of afferents which innervate the uterine cervix, and increases afferent firing in response to cervical distension. That study, however, examined only the firing frequency of peripheral nerve fibers, not central nervous system activation by cervical distension. One goal of the current study was to determine whether more complex integrated reflexes to cervical distension are also enhanced by chronic estrogen treatment.
Estrogen might sensitize afferents by altering expression or function of mechanosensitive ion channels on nerve endings in the cervix. Of such channels, the transient receptor potential-1 (TRPV-1) appears relevant to visceral pain. Initially described as responding to the active ingredient in chili peppers, capsaicin, and as a noxious heat and acid sensor (5), TRPV-1 is now thought necessary for normal response to distension of the small and large bowel (6,7) and urinary bladder (8). Additionally, TRPV-1 expression increases in colorectal biopsy tissue in patients with irritable bowel syndrome (9). TRPV-1 knockout mice lack the normal sensitization to inflammation in the colon (7) and bladder (8). We recently (10) showed that the proportion of hypogastric afferents innervating the uterine cervix, which express TRPV-1 increases with chronic estrogen treatment. The second goal of the current study was to determine, using the selective TRPV-1 antagonist, capsaizepine, whether estrogen-induced sensitization of cervical afferents relies on function of this ion channel. We hypothesized that uterine cervical distension would result in a stimulus-dependent activation of a motor reflex, that this would be amplified by estrogen, and that the effect of estrogen would be reduced by acute treatment with capsaizepine. A power analysis was performed in order to observe a difference in visceromotor reflex by estrogen treatment of five-fold, resulting in a group size of 8.
METHODS
Animals
Female Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 225–250 g were used. The study protocol was approved by the Animal Care and Use Committee of Wake Forest University (Winston-Salem, NC). Animals were housed in our institutional central animal resource facilities, two per cage with a 12 hr light–dark cycle and free access to food and water.
Ovariectomy and Estrogen Replacement
Anesthesia was induced with 5% and maintained with 2% halothane in oxygen with spontaneous ventilation. A midline skin incision was made lateral to the lumbar paraspinous muscles bilaterally, and both ovaries and surrounding tissues were dissected and removed through this retroperitoneal approach. Animals were randomized to have a pellet containing either estrogen or vehicle implanted subcutaneously at the time of surgery. We previously (11) showed that these estrogen-containing pellets resulted in circulating concentrations of 17ß-estradiol of 50–75 pg/mL, similar to high proestrus concentrations in nonpregnant animals, and that this level is maintained for at least 3 wk. Incisions were then closed with 4-0 silk, animals recovered from anesthesia, and were studied 3 wk later.
Electromyographic (EMG) Response to Uterine Cervical Distension
The visceromotor reflex contraction (guarding reflex) of the rectus abdominus muscle from controlled uterine cervical distension was measured as previously described (2). In brief, under halothane anesthesia, the right carotid artery and jugular vein were cannulated for arterial blood pressure monitoring and for drug or fluid administration and a tracheotomy was performed for mechanical ventilation. The uterus was exposed via a midline abdominal incision and two fine metal rods were inserted under direct vision through a small incision in the lower uterine segment, passing through the cervical osses, and into the vaginal canal. One end of each rod was connected to a 1-0 silk suture. One suture was passed around a rod attached to a metal stand for applying manual distension and the other was connected to a force transducer (FT03, Grass Instruments, Quincy, MA) for simultaneous measurement of distension force. The metal stand and force transducer were positioned perpendicular to the longitude axis of the uterine cervix, thereby maintaining a constant angle of force without torsion of the tissue. Surgery and implantation of the rods were done by a separate investigator from the one who performed the distension experiment, and the incision was covered by saline-soaked gauze, so that the experimenter was blinded to estrogen or placebo treatment. The halothane concentration was then decreased to 0.5%–0.7%. Rectal temperature was monitored continuously and maintained at 37.5–39°C using a circulating water heating pad.
Two uninsulated needle electrodes were inserted bilaterally near the inguinal margins of the rectus abdominus muscles, and EMG activity was rectified and recorded online for later analysis (BIOPAC Systems, Goleta, CA). A 10 sec, 80 g distension was applied every 3 min for 7–9 cycles until a stable response was obtained. Animals which did not respond to the 80 g stimulus with an integrated EMG value of >50 µV sec were considered unresponsive and not further studied. Then a stimulus response was performed by applying distension forces of 20, 40, 60, and 80 g in escalating order with stimuli separated by 3 min. This cycle was repeated three times and the average used for data analysis. The EMG was amplified, rectified, and integrated over the 10 sec before and during each stimulus, and the distension-evoked response calculated as the integrated response during stimulation minus that of the preceding 10 sec.
Single Unit Afferent Recording
Hypogastric nerve afferents were isolated and recorded as previously reported (2). Animals were prepared as described for EMG recording, and the retroperitoneal space dorsal to the uterus was dissected and the right hypogastric nerve was identified, cut near the aortic bifurcation, and draped on a platform covered with warm mineral oil. The nerve sheath was removed and nerve filaments carefully dissected under a microscope and draped over the recording electrode until single unit activity was obtained. The halothane concentration was then reduced to 1% for the remainder of the experiment. At the end of EMG or afferent experiments, rats were euthanized with IV sodium pentobarbital.
Single unit activity was recorded with a unipolar platinum electrode. A single unit was identified initially by examining the waveform and the spike amplitude using a window discriminator (Sciworks 3.0, Datawave Technology, Longmont, CO) at a rapid sweep speed as well as by checking the recorded sound frequency related to each spike activity. Units were classified as low threshold if they responded to distension of 20 g or high-threshold if they responded to distension 40 g. Since estrogen selectively sensitizes high threshold units (4), only high threshold units were studied. The spike number during a 10 sec distension was determined, and distension-induced response calculated as this number minus the spontaneous frequency. Conduction velocity was measured using a pair of bipolar electrodes applied to the uterine cervix.
Animals with estrogen treatment were randomized to receive, after the baseline series of cervical distension, either IV capsaizepine, 200 µg, or vehicle. Since pilot studies demonstrated a lack of capsaizepine effect in placebo pellet-treated animals, a group randomized to vehicle rather then capsaizepine was not included in placebo pellet-treated animals. The experimenter was blinded to estrogen status and treatment.
Drugs
17ß-estradiol (1.5 mg, sustained release) or placebo pellets were obtained from Innovative Research of America (Sarasota, Fl). Capsaizepine was obtained from Sigma-Aldrich (St. Louis, MO), and dissolved in DMSO/ soy oil (10:90, v/v), at a concentration of 20 mg/L.
Statistics
EMG responses were not normally distributed, are presented as median ±25th and 75th percentile, and were analyzed by a nonparametric two way analysis of variance for repeated measures. Afferent spike frequencies were normally distributed, are presented as mean ± sem and were analyzed by one and two way analysis of variance for repeated measures. P < 0.05 was considered significant.
RESULTS
All animals recovered rapidly from ovariectomy surgery, groomed normally, and gained weight over the ensuing three weeks. At the time of surgical insertion of the cervical rods for distension, there was a clear difference in morphology of the uterus and cervix according to treatment, with small, atrophic structures in rats randomized to placebo pellets and larger, more obviously vascularized structures in rats randomized to estrogen pellets.
Reflex Responses to Uterine Cervical Distension
Overall, a similar proportion of rats with estrogen replacement (8 of 12) met criteria for EMG reflex responsiveness to cervical distension as rats with ovariectomy without estrogen (8 of 15). Uterine cervical distension produced a stimulus-dependent increase in EMG activity in estrogen and placebo pellet-treated animals (Fig. 1). Estrogen-treated animals, however, exhibited a greater response than placebo-treated animals, and post hoc testing showed groups to differ at the 80 g stimulus intensity (Fig. 1). As previously noted (12), the mean arterial blood pressure increase to cervical distension was small and variable, only achieved significance in estrogen-treated animals, but overall did not differ between estrogen and placebo-treated animals (Table 1).
|
|
Afferent Responses and Effects of Capsaizepine
All afferents conducted at C-fiber velocity, similar to previous reports (2,4). High threshold hypogastric nerve afferents in estrogen-treated animals (n = 7) exhibited spontaneous activity and a stimulus-dependent increase in activity from uterine cervical distension (Fig. 2). Capsaizepine treatment did not affect spontaneous activity of these afferents, but significantly reduced their response to cervical distension (Fig. 2A). In contrast, treatment with vehicle failed to alter spontaneous activity or distension-induced responses in high threshold afferents in estrogen-treated animals (Fig. 2B). Capsaizepine failed to reduce response to distension in animals without estrogen treatment (response to 80 g stimulus before capsaizepine = 1.6 ± 0.5 spikes/sec and after capsaizepine = 1.8 ± 0.7 spikes/sec, P = NS).
|
DISCUSSION
Clinicians often treat women who have acute or chronic pain originating from the lower uterine segment and cervix. The current study in animals enlarges our understanding of the properties of these pain nerves and points to ways they are sensitized, and how this pain might be treated. Key findings are estrogen sensitization to integrated reflex measures from uterine cervical distension and dependence of estrogen sensitization of uterine cervical nerve endings on increased TRPV-1 channel function.
Assessment of pain is difficult in animals, particularly to stimuli like uterine cervical distension, which cannot be applied in a controlled manner in the awake, behaving animal. Peripheral nerve activity increases with stimuli intensity within the presumed noxious range for cervical distension. Although this observation is consistent with pain, measuring peripheral nerve activity does not assess the central nervous system response to the stimulus. We previously (4) showed that estrogen sensitizes the response of hypogastric afferents to cervical distension. The current study shows a parallel sensitization by estrogen of high threshold afferents and motor reflexes. These data strongly support the notion that pain increases as a result of stimulation of the uterine cervix when estrogen is present, consistent with studies of estrogen- induced sensitization of visceromotor and hemodynamic reflexes with distension of the colon (13) and uterus (14).
A sizeable minority of animals did not respond to uterine cervical distension with a motor reflex in the current study, similar to our previous experience (2). One could argue that this model examines only animals with abnormally high sensitivity to this stimulus. Rather, we believe that this lack of response in some animals represents the fine line between providing adequate anesthesia to assure lack of gross motor response to the surgical preparation, but not so deep a level of anesthesia as to abolish the fine EMG response in this visceromotor reflex.
The TRPV-1 channel transduces mechanical stimuli, since genetic deletion of this channel, or treatment of wildtype mice with the TRPV-1 antagonist, capsaizepine, reduces afferent and visceromotor responses to distension of the bladder (8), small bowel (6), and large bowel (7). This is not universally observed, however, since sensitivity to mechanical stimulation of the skin is not affected in mice lacking the TRPV-1 channel (15). Although approximately half of uterine cervical afferents express TRPV-1 immunoreactivity (10), and a similar proportion respond to noxious heat (4), the current study does not support a role for this ion channel in mechanosensitivity in the absence of estrogen, since capsaizepine failed to alter responses to distension in ovariectomized animals without estrogen treatment.
Estrogen treatment increases pain sensitivity to somatic stimuli in women (3). We previously showed that estrogen increases afferent responses in rats to cervical distension (4). We speculate that one reason why uterine contractions in early term labor are more painful than Braxton Hicks contractions reflects increased estrogen receptor signaling at the onset of cervical ripening (16), which sensitizes nerves to the uterine cervix. Since estrogen signaling also increases spontaneous activity of cervical afferents (4), it is conceivable that their release of neuropeptides may participate in the cervical ripening process itself (17). Estrogen also reduces to effectiveness of morphine to block responses to uterine cervical distension (11), which could partially explain the mediocre analgesia obtained from systemic opioids administered in the first stage of labor (18).
TRPV-1 expression increases during inflammation of other viscera, and TRPV-1 knockout or antagonism reduces this inflammation associated sensitization (7,19). We previously showed that TRPV-1 expression increases in uterocervical afferents of rats treated with estrogen (10), accompanied by an increase in the proportion of heat-sensitive units which are also mechanosensitive (4). The current study directly demonstrates a role for TRPV-1 receptors in estrogen-induced sensitization.
In summary, uterine cervical distension in rats results in stimulus-dependent afferent activity and motor reflexes which are increased by chronic estrogen treatment. This sensitization is blocked by the TRPV-1 channel antagonist, capsaizepine. Together, these data suggest that states of high estrogen receptor signaling could enhance pain from the cervix, and that TRPV-1 antagonists might prevent or treat such enhanced pain.
Footnotes
Accepted for publication January 29, 2007.
Supported in part by National Institutes of Health Grants NS48065 and NS41386.
REFERENCES
-opioid agonist inhibition in response to uterine cervical distension. Anesthesiology 2002;96:375–9.[Web of Science][Medline]This article has been cited by other articles:
|
|
 |
|
  H.-Y. Peng, H.-M. Chang, S.-D. Lee, P.-C. Huang, G.-D. Chen, C.-H. Lai, C.-Y. Lai, C.-H. Chiu, K.-C. Tung, and T.-B. Lin TRPV1 mediates the uterine capsaicin-induced NMDA NR2B-dependent cross-organ reflex sensitization in anesthetized rats Am J Physiol Renal Physiol, November 1, 2008; 295(5): F1324 - F1335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-Y. Peng, P.-C. Huang, J.-M. Liao, K.-C. Tung, S.-D. Lee, C.-L. Cheng, J.-C. Shyu, C.-Y. Lai, G.-D. Chen, and T.-B. Lin Estrous cycle variation of TRPV1-mediated cross-organ sensitization between uterus and NMDA-dependent pelvic-urethra reflex activity Am J Physiol Endocrinol Metab, September 1, 2008; 295(3): E559 - E568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-Y. Peng, H.-M. Chang, S. Y. Chang, K.-C. Tung, S.-D. Lee, D. Chou, C.-Y. Lai, C.-H. Chiu, G.-D. Chen, and T.-B. Lin Orexin-A modulates glutamatergic NMDA-dependent spinal reflex potentiation via inhibition of NR2B subunit Am J Physiol Endocrinol Metab, July 1, 2008; 295(1): E117 - E129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-F. Pan, H.-Y. Peng, C.-C. Chen, M.-J. Chen, S.-D. Lee, C.-L. Cheng, J.-C. Shyu, J.-M. Liao, G.-D. Chen, and T.-B. Lin Nicotine-activated descending facilitation on spinal NMDA-dependent reflex potentiation from pontine tegmentum in rats Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1195 - F1204. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
P < 0.05 compared to placebo pellet treated animals by two way nonparametric ANOVA followed by Dunn's test.
