Uterine cervical distension underlies labor pain, yet its neurophysiology and pharmacology of inhibition remain unexplored. The authors examined uterine cervical distension-evoked cFos immunoreactivity in rat spinal cords, and the inhibitory effect of spinal cyclo-oxygenase inhibition on cFos expression.
Female rats were anesthetized with halothane, and pairs of metal rods were inserted in each cervical os through a mid-line laparotomy. A submaximal distension force (75 g) was applied for either 30 or 60 min, or, in control animals, no force was applied. Other animals received cervical lidocaine infiltration prior to uterine cervical distension. At the end of the experiments, the spinal cord at T12 to L2 levels was harvested and immunostained for cFos protein. Other animals received intrathecal ketorolac (0, 5, 25, and 50 microg; n = 5-6 for each group) prior to uterine cervical distension.
Uterine cervical distension significantly increased cFos immunoreactivity in the spinal cord from T12 to L2, with most cFos expression in the deep dorsal and central canal regions. Surgical preparation alone without uterine cervical distension resulted in minimal cFos expression, primarily in the superficial dorsal horn. Uterine cervical distension-evoked cFos expression was prevented by prior infiltration of lidocaine into the cervix. Intrathecal ketorolac produced a dose-dependent inhibition of uterine cervical distension-induced cFos expression.
The present study demonstrates that uterine cervical distension results in a similar pattern of spinal cord neuronal activation as seen with other noxious visceral stimuli. The inhibition of cFos expression by intrathecal ketorolac suggests that spinal cyclo-oxygenase plays a role in uterine cervical distension-induced nociception.
ALTHOUGH labor pain is effectively treated clinically by administration of local anesthetics alone or with opioids in the epidural and/or spinal spaces, 1–5this is very labor-intensive and carries potential adverse effects to the fetus and mother. For these reasons, new, simpler approaches to the treatment of labor pain are needed. Advances in understanding and treatment for labor pain are, in general, hampered by our limited understanding of visceral pain because most laboratory work has focused on somatic pain.
Ness and Gebhart pioneered the recent investigation of visceral pain, developing a model of colorectal distension in the rat in which distention of this hollow organ produces stimulation-dependent cardiovascular and evoked electromyographic responses. 6This investigation was followed by development of other models, including bladder and esophageal distension, and intraperitoneal inflammation by injection of acetic acid. 7–12Although Berkley has performed a series of neurophysiologic and behavioral studies in female rats to assess nociception in the reproductive tract, these investigations focused on uterine distension and chronic inflammation rather than acute uterine cervical distension, the source of labor pain. 12–16Thus, this common, severe pain syndrome has been essentially neglected in fundamental investigation.
cFos, an immediate-early gene, transcribes the protein cFos in spinal cord neurons following peripheral noxious stimulation, and cFos immunoreactivity in the spinal cord has been widely used as an indicator of acute and chronic nociceptive spinal transmission. Many studies have demonstrated that cFos expression in spinal cord neurons increases shortly after the onset of a noxious peripheral stimulation. cFos expression after somatic stimulation is anatomically tightly localized in the superficial laminae of the spinal cord, and this cFos expression can be inhibited, although not totally abolished, by treating the animal with a variety of known analgesic drugs. Therefore, immunostaining for cFos protein is established as a biologic marker for the study of nociception and analgesia. 17–20
We recently developed a novel animal model of uterine cervical distension (UCD) to lay the foundation for the study of neurophysiology and pharmacology of labor pain. 21In this model, noxious stimulation is produced by manually distending the rat uterine cervix, thereby evoking reflex electromyographic activity in a force-dependent manner. This reflex muscle contraction is inhibited in a dose-dependent manner by μ- and κ-opioid agonists. 22,23
One of the purposes of the current study was to further validate this UCD model by determining the distribution of spinal neurons activated by this stimulation. In addition, because spinal cyclo-oxygenase (COX) is known to be activated by prolonged noxious stimulation and to drive central sensitization, 24,25we examined the role of spinal COX in this UCD model of visceral pain.
Materials and Methods
The study protocol was approved by our institution's Animal Care and Use Committee, and all experiments were conducted in accordance with guidelines of animal care from the National Institutes of Health and the International Association for the Study of Pain.
Sprague-Dawley Harlan, Indianapolis, IN) adult virgin female rats (220–250 g) were studied. Animals were housed two per cage on a 12h–12-h light–dark cycle. Ambient temperature was kept at 22°C, and animals had free access to standard food and tap water. Animals were allowed to habituate to the housing facilities for at least 1 week before the study.
Uterine Cervical Distension.
Animals were anesthetized with inhalation of halothane and spontaneous ventilation. A lower abdominal laparotomy was performed to expose the uterus, and two fine metal rods were placed through the cervical osses, with one side attached to a metal stand and the other side connected to a force transducer (FT03, Grass Instruments, Quincy, MA). Then, the halothane concentration was adjusted to 0.5–0.7%, at which the animal showed no gross movement during UCD stimulation. A constant UCD stimulation of 75 g was applied for either 30 or 60 min, as determined by the study protocol. Body temperature was maintained in the range of 37–38°C throughout the experiment with a circulating water heating pad. At the end of UCD, the abdominal wall was closed with 4–0 silk, and the animals were allowed to recover for 1 h before euthanasia. This time delay was included to allow an adequate period for cFos expression to develop.
Intrathecal Catheter Placement.
Intrathecal catheters were inserted according to the method of Yaksh and Rudy 26with a slight modification. In brief, rats were anesthetized with inhalational halothane with spontaneous ventilation. A small incision was made above the cervical vertebrae, followed by blunt dissection of paraspinal fasciae and muscle to expose the dura at the atlanto-occipital junction. A small incision was made in the dura that allowed the insertion of a polyethylene catheter caudally (6.5 cm), with the tip located at the thoracolumbar region. All incision layers were closed with 4–0 suture and the polyethylene tubing was externalized at the back of neck. Animals were allowed 5–7 days recovery and were euthanized immediately if they showed any signs of motor deficit.
UCD was applied tonically with a distension force of 75 g. The effect of time of UCD was studied by UCD distension of 30 min (UCD 30, n = 5) or 60 min (UCD 60, n = 6). Controls included rats with a lower abdominal laparotomy incision without UCD (n = 6), those with UCD preceded by lidocaine infiltration in the cervix (n = 4), and those with an intrathecal catheter, but no surgery or UCD (n = 2). To study the role of spinal COX inhibition, some animals received intrathecal saline or ketorolac (ketorolac tromethamine, preservative-free; (Allergan, Palo Alto, CA), 5, 25, or 50 μg, prior to UCD stimulation (n = 5–7 per group).
One h after UCD, animals were deeply anesthetized with sodium pentobarbital (50–70 mg/kg) intraperitoneally and were perfused with 250 ml of 0.01 M sodium nitrite intracardially, followed by 500 ml of 4% paraformaldehyde in 0.01 M phosphate buffer solution (PBS). The thoracolumbar spinal cord was removed and postfixed up to 4 h in paraformaldehyde PBS and then cryoprotected in 30% sucrose solution. Tissue was sectioned at 40 μm at −20°C using a cryostat; then, slices were stored at −70°C until the time of immunohistochemistry processing.
A free-floating technique was used for cFos-like protein staining, as previously described. 18In brief, sections were washed with 0.3% hydrogen peroxide for 45 min, followed by two washes with 0.2% Triton-X in 0.01 M PBS (10 min each); then, sections were incubated overnight with rabbit anti-Fos polyclonal antibody (1:5,000; Santa Cruz Biotetchnology, CA). After three washes with 0.01 M PBS, sections were incubated in biotinylated rabbit antigoat antibody (1:200; Vector, Burlingame, CA) in 10% goat serum for 60 min. Sections were washed with PBS twice, then incubated for 60 min with avidin–biotin horseradish peroxidase complex (1:100; Vectastain ABC-Elite, Vector Burlingame, CA). After the final wash with PBS, the sections were reacted with hydrogen peroxide, 0.01%, and diaminobenzidine, 0.05%, as chromogen. Sections were mounted on gelatin-subbed slides and air-dried. Slides were dehydrated in an ascending alcohol series, defatted in xylene, and then cover-slipped.
Sections from T12 to L2 were examined under a light microscope, and labeled nuclei were counted using a manual drawing technique. Each section was divided into (1) superficial dorsal horn (laminae I–II), (2) deep dorsal horn (laminae III–V), (3) ventral horn (laminae VI–IX), and (4) central canal region (lamina X). The average of randomly selected three sections from each level was used for data analysis.
Data are expressed as mean ± SEM and were analyzed by two-way ANOVA with the use of a factorial design. Corrections were made for multiple comparisons by means of Fisher's protected least significant difference approach with Bonferroni corrections when appropriate. P < 0.05 was considered significant.
UCD significantly increased cFos immunoreactivity in the dorsal spinal cord from T12 to L2, consistent with uterine cervical innervation by the hypogastric nerve, 13which is composed of afferent entering the cord at these levels. Sham surgery, in which there was laparotomy but no UCD, resulted in expression of cFos immunostaining in the superficial laminae, but minimal staining in the deep dorsal horn. In contrast, UCD resulted in time-dependent cFos immunoreactivity in the deep dorsal horn (laminae III–V) and in the central canal (lamina X). Indeed, cFos expression was only increased significantly by UCD compared with sham in these regions, rather than in the superficial or ventral laminae (fig. 1). Local infiltration with lidocaine into the uterine cervix significantly blocked cFos immunoreactivity from UCD stimulation, and, in the presence of cervical lidocaine infiltration, the cFos immunostaining was similar to the sham surgery group (fig. 2).
Animals with a chronic intrathecal catheter only, but without surgery or UCD, exhibited similar cFos immunostaining to those without an intrathecal catheter (data not shown). Compared with intrathecal saline, intrathecal administration of ketorolac significantly inhibited spinal cord cFos expression induced by UCD in a dose-dependent manner (figs. 3). The effect of ketorolac, 50 and 25 μg, differed from that of 5 μg; however, the difference between ketorolac, 50 and 25 μg, was not significant. CFos immunostaining in animals receiving intrathecal ketorolac, 50 μg, was similar to that of control animals without UCD, with only a few cFos immunoreactive cells in the superficial dorsal horn and minimal staining in the ventral and central canal regions (fig. 4).
Quantification of cFos expression by summing all cFos expressing cells in the spinal cord from T12 to L2 segments among different groups revealed that animals that received UCD 60 min and UCD with intrathecal saline induced a four- to fivefold greater number of spinal cFos neurons than animals receiving cervical lidocaine and intrathecal ketorolac, 25 or 50 μg, the latter being similar to sham surgery (fig. 5).
The present study demonstrates that visceral noxious stimulation by UCD increases the expression of cFos in rat spinal cords, with the greatest cFos expression in the deep dorsal and central regions, which contrasts with cFos expression observed after noxious somatic stimulation that concentrated in the superficial laminae. In addition, we provide novel evidence for the role of spinal COX in the response to UCD.
Despite the effective management of labor pain clinically with spinal and epidural administration of local anesthetics, few studies address the underlying mechanisms and physiology of labor pain to indicate how it might be more simply treated. 1The current study adds to our previous investigations to support the use of acute UCD in the rat as a method to study the physiology of this noxious visceral input. UCD produces a force-dependent increase in neural activity in the hypogastric nerve 21as well as evoked abdominal muscle reflex electromyographic activity 22,23reminiscent of the sacral neural activity and reflex electromyographic activity evoked by noxious colorectal distension. 6UCD-induced reflex electromyography and afferent fiber firing are inhibited by systemic opioids in doses thought to be antinociceptive in rats, further suggesting that UCD is a truly noxious stimulus. 23
The current study used cFos to indicate strong and/or sustained neuronal activation, consistent with nociceptive input in the spinal cord, to further support and validate this model. CFos expression at T12 to L2 is consistent with spinal entry sites of the hypogastric nerve, and with the region of referred pain of women in the first stage of labor. Berkley recorded afferents following stimulation of the uterine horns, cervix, and genital canal in the rat and concluded that the lower uterine segment and uterine aspect of the cervix are mostly innervated by the hypogastric nerve, which was further confirmed by retrograde tracing. 12,13The spinal levels of T12 to L2 were chosen mainly to examine the hypogastric nerve pathway in activation of cFos expression in the spinal cord.
UCD increases spinal cFos expression mostly in the deep dorsal horn and central canal regions, similar to previous observations with other visceral noxious stimuli, 17and quite different from the highly localized, superficial dorsal horn expression observed with somatic stimuli. 19,20Our study suggests an increase in spinal cFos expression with increasing duration of UCD, although we cannot exclude the influence of greater time from onset of UCD to perfusion in the UCD 60-min group compared with the UCD 30-min group to increase cFos expression. Local infiltration of the cervix with a local anesthetic effectively blocked cFos activation in the spinal cord, further confirming that UCD, as performed in this model, reflects noxious input from the uterine cervix rather than traction on other visceral or somatic structures by deformation of the tissue during distension.
COX is constitutively expressed in spinal horn neurons in glia, with both isoforms being present in the rat and human dorsal spinal cord. Activation of spinal COX occurs with peripheral inflammation, and spinally synthesized prostaglandins are thought to drive central sensitization and enhance the perception of pain. 24,25The role of spinal COX in response to acute or chronic visceral noxious stimulation has not been previously described. This study clearly showed that intrathecal ketorolac significantly inhibited spinal cFos expression induced by tonic UCD for 60 min, and did so in a dose-dependent manner. Arguably the marked efficacy of COX inhibitors, including aspirin and ibuprofen, in treating menstrual cramping pain reflects in part a spinal inhibitory mechanism against this visceral input. Possibly intrathecal administration of COX inhibitors could provide a useful treatment, alone or with traditional anesthetics or analgesics, for labor pain.
In summary, this study provides novel evidence that UCD stimulates spinal cord cFos expression, which is significantly inhibited by intrathecal ketorolac. These data suggest that UCD could serve as a useful animal model for the study of acute visceral pain such that during labor, and that intrathecal injection of COX inhibitors might be useful in the treatment of such pain.