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Education  |   July 2003
Uterine Cervical Distension Induces cFos Expression in Deep Dorsal Horn Neurons of the Rat Spinal Cord
Author Affiliations & Notes
  • Chuanyao Tong, M.D.
    *
  • Weiya Ma, M.D., Ph.D
    *
  • Sang-Wook Shin, M.D.
  • Robert L. James, M.S.
  • James C. Eisenach, M.D.
    §
  • * Assistant Professor of Anesthesiology, † Research Postdoctoral Fellow, ‡ Biostatistician, Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, North Carolina. § F.M. James III Professor of Anesthesiology, Department of Anesthesiology and Center for the Study of Pharmacologic Plasticity in the Presence of Pain, Wake Forest University School of Medicine.
  • Received from the Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, North Carolina.
Article Information
Education
Education   |   July 2003
Uterine Cervical Distension Induces cFos Expression in Deep Dorsal Horn Neurons of the Rat Spinal Cord
Anesthesiology 7 2003, Vol.99, 205-211. doi:
Anesthesiology 7 2003, Vol.99, 205-211. doi:
ALTHOUGH labor pain is effectively treated clinically by administration of local anesthetics alone or with opioids in the epidural and/or spinal spaces, 1–5 this 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. 6 This investigation was followed by development of other models, including bladder and esophageal distension, and intraperitoneal inflammation by injection of acetic acid. 7–12 Although 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. 12–16 Thus, 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. 21 In 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, 24,25 we 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.
Animals
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.
Surgery
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 26 with 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.
Experimental Groups
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).
Immunohistochemistry
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. 18 In 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.
Statistics
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.
Results
UCD significantly increased cFos immunoreactivity in the dorsal spinal cord from T12 to L2, consistent with uterine cervical innervation by the hypogastric nerve, 13 which 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).
Fig. 1. Comparison of cFos expression in spinal cord in animals receiving uterine cervical distension for either 60 min (UCD 60, n = 6) or 30 min (UCD 30, n = 5), laparotomy without uterine cervical distension (sham, n = 6), or uterine cervical lidocaine infiltration (n = 4) prior to 60 min UCD. SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (laminae X). *P  < 0.05 compared with sham surgery and cervical infiltration of lidocaine. ΦP  < 0.05 compared with UCD 30 min.
Fig. 1. Comparison of cFos expression in spinal cord in animals receiving uterine cervical distension for either 60 min (UCD 60, n = 6) or 30 min (UCD 30, n = 5), laparotomy without uterine cervical distension (sham, n = 6), or uterine cervical lidocaine infiltration (n = 4) prior to 60 min UCD. SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (laminae X). *P 
	< 0.05 compared with sham surgery and cervical infiltration of lidocaine. ΦP 
	< 0.05 compared with UCD 30 min.
Fig. 1. Comparison of cFos expression in spinal cord in animals receiving uterine cervical distension for either 60 min (UCD 60, n = 6) or 30 min (UCD 30, n = 5), laparotomy without uterine cervical distension (sham, n = 6), or uterine cervical lidocaine infiltration (n = 4) prior to 60 min UCD. SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (laminae X). *P  < 0.05 compared with sham surgery and cervical infiltration of lidocaine. ΦP  < 0.05 compared with UCD 30 min.
×
Fig. 2. Photomicrograph showing cFos expression induced by uterine cervical distension. There is a significant increase in cFos expression in the deep dorsal horn regions of animals receiving 60 min of distension (A  ) compared with animals having surgery but no distension (B  ) and lidocaine infiltration of the cervix prior to distension (C  ). Photomicrographs (D  ), (E  ), and (F  ) show the cFos immunostaining in the central canal region (lamina X) in animals with 60 min of distension, surgery without distension, or distension after local lidocaine infiltration, respectively.
Fig. 2. Photomicrograph showing cFos expression induced by uterine cervical distension. There is a significant increase in cFos expression in the deep dorsal horn regions of animals receiving 60 min of distension (A 
	) compared with animals having surgery but no distension (B 
	) and lidocaine infiltration of the cervix prior to distension (C 
	). Photomicrographs (D 
	), (E 
	), and (F 
	) show the cFos immunostaining in the central canal region (lamina X) in animals with 60 min of distension, surgery without distension, or distension after local lidocaine infiltration, respectively.
Fig. 2. Photomicrograph showing cFos expression induced by uterine cervical distension. There is a significant increase in cFos expression in the deep dorsal horn regions of animals receiving 60 min of distension (A  ) compared with animals having surgery but no distension (B  ) and lidocaine infiltration of the cervix prior to distension (C  ). Photomicrographs (D  ), (E  ), and (F  ) show the cFos immunostaining in the central canal region (lamina X) in animals with 60 min of distension, surgery without distension, or distension after local lidocaine infiltration, respectively.
×
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).
Fig. 3. The effects of intrathecal ketorolac on uterine cervical distension–induced spinal cFos expression. Animals received intrathecal ketorolac, 50, 25, or 5 μg (Keto 50, Keto 25, Keto 5  ; n = 5 per group), or intrathecal saline with UCD 60 (IT Sal  , n = 7). SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (lamina X). *P  < 0.05 compared with intrathecal ketorolac, 50 μg and 25 μg; ΦP  < 0.05 compared with intrathecal ketorolac, 25 μg.
Fig. 3. The effects of intrathecal ketorolac on uterine cervical distension–induced spinal cFos expression. Animals received intrathecal ketorolac, 50, 25, or 5 μg (Keto 50, Keto 25, Keto 5 
	; n = 5 per group), or intrathecal saline with UCD 60 (IT Sal 
	, n = 7). SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (lamina X). *P 
	< 0.05 compared with intrathecal ketorolac, 50 μg and 25 μg; ΦP 
	< 0.05 compared with intrathecal ketorolac, 25 μg.
Fig. 3. The effects of intrathecal ketorolac on uterine cervical distension–induced spinal cFos expression. Animals received intrathecal ketorolac, 50, 25, or 5 μg (Keto 50, Keto 25, Keto 5  ; n = 5 per group), or intrathecal saline with UCD 60 (IT Sal  , n = 7). SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (lamina X). *P  < 0.05 compared with intrathecal ketorolac, 50 μg and 25 μg; ΦP  < 0.05 compared with intrathecal ketorolac, 25 μg.
×
Fig. 4. Representative photomicrograph showing the effect of intrathecal ketorolac on spinal cFos expression. There is a significant decrease in cFos immunostaining with 50 μg ketorolac treatment (A  ) compared with saline (B  ) in the deep dorsal horn region; similar differences were present in the central canal region (lamina X), (C  ) = 50 μg ketorolac, (D  ) = saline.
Fig. 4. Representative photomicrograph showing the effect of intrathecal ketorolac on spinal cFos expression. There is a significant decrease in cFos immunostaining with 50 μg ketorolac treatment (A 
	) compared with saline (B 
	) in the deep dorsal horn region; similar differences were present in the central canal region (lamina X), (C 
	) = 50 μg ketorolac, (D 
	) = saline.
Fig. 4. Representative photomicrograph showing the effect of intrathecal ketorolac on spinal cFos expression. There is a significant decrease in cFos immunostaining with 50 μg ketorolac treatment (A  ) compared with saline (B  ) in the deep dorsal horn region; similar differences were present in the central canal region (lamina X), (C  ) = 50 μg ketorolac, (D  ) = saline.
×
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).
Fig. 5. Quantification of cFos expression in spinal cord across T12 to L2 levels. An average of cFos expressing cells in randomly selected sections from each level here and the expression from T12 to L2 were summed. Animals receiving intrathecal ketorolac, 25 (n = 5) or 50 μg (n = 6), had similar cFos expression compared with uterine cervical lidocaine infiltration and sham surgery, whereas those receiving uterine cervical distension with intrathecal saline had a four- to fivefold increase in cFos compared with control groups. *P  < 0.05 compared with sham surgery, cervical lidocaine injection, and intrathecal ketorolac, 50 μg.
Fig. 5. Quantification of cFos expression in spinal cord across T12 to L2 levels. An average of cFos expressing cells in randomly selected sections from each level here and the expression from T12 to L2 were summed. Animals receiving intrathecal ketorolac, 25 (n = 5) or 50 μg (n = 6), had similar cFos expression compared with uterine cervical lidocaine infiltration and sham surgery, whereas those receiving uterine cervical distension with intrathecal saline had a four- to fivefold increase in cFos compared with control groups. *P 
	< 0.05 compared with sham surgery, cervical lidocaine injection, and intrathecal ketorolac, 50 μg.
Fig. 5. Quantification of cFos expression in spinal cord across T12 to L2 levels. An average of cFos expressing cells in randomly selected sections from each level here and the expression from T12 to L2 were summed. Animals receiving intrathecal ketorolac, 25 (n = 5) or 50 μg (n = 6), had similar cFos expression compared with uterine cervical lidocaine infiltration and sham surgery, whereas those receiving uterine cervical distension with intrathecal saline had a four- to fivefold increase in cFos compared with control groups. *P  < 0.05 compared with sham surgery, cervical lidocaine injection, and intrathecal ketorolac, 50 μg.
×
Discussion
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. 1 The 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 21 as well as evoked abdominal muscle reflex electromyographic activity 22,23 reminiscent of the sacral neural activity and reflex electromyographic activity evoked by noxious colorectal distension. 6 UCD-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. 12,13 The 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, 17 and quite different from the highly localized, superficial dorsal horn expression observed with somatic stimuli. 19,20 Our 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. 24,25 The 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.
References
Eisenach JC: Pain physiology and pharmacology: Clinical relevance (refresher course lecture). American Society of Anesthesiologists 2001; 511: 1–7Eisenach, JC
Zakowski M: Complications associated with regional anesthesia in the obstetric patient. Semin Perinatol 2002; 26: 154–68Zakowski, M
Nelson KE, Rauch T, Terebuh V, D'Angelo R: A comparison of intrathecal fentanyl and sufentanil for labor analgesia. A nesthesiology 2002; 96: 1070–73Nelson, KE Rauch, T Terebuh, V D'Angelo, R
Bucklin BA, Chestnut DH, Hawkins JL: Intrathecal opioids versus epidural local anesthetics for labor analgesia: A meta-analysis. Reg Anesth Pain Med 2002; 27: 23–30Bucklin, BA Chestnut, DH Hawkins, JL
Thallon A, Shennan A: Epidural and spinal analgesia and labour. Curr Opin Obstet Gynecol 2001; 13: 583–87Thallon, A Shennan, A
Ness TJ, Gebhart GF: Colorectal distention as a noxious visceral stimulus: Physiologic and pharmacologic characterization of pseudoaffective reflexes in the rat. Brain Res 1988; 450: 153–69Ness, TJ Gebhart, GF
Aziz Q, Thompson DG, Ng VW, Hamdy S, Sarkar S, Brammer MJ, Bullmore ET, Hobson A, Tracey I, Gregory L, Simmons A, Williams SC: Cortical processing of human somatic and visceral sensation. J Neurosci 2000; 20: 2657–63Aziz, Q Thompson, DG Ng, VW Hamdy, S Sarkar, S Brammer, MJ Bullmore, ET Hobson, A Tracey, I Gregory, L Simmons, A Williams, SC
Castroman PJ, Ness TJ: Ketamine, an N-methyl-D-aspartate receptor antagonist, inhibits the reflex responses to distension of the rat urinary bladder. A nesthesiology 2002; 96: 1401–9Castroman, PJ Ness, TJ
Rong W, Spyer KM, Burnstock G: Activation and sensitisation of low and high threshold afferent fibres mediated by P2X receptors in the mouse urinary bladder. J Physiol 2002; 541: 591–600Rong, W Spyer, KM Burnstock, G
Behrens E, Schramm J, Zenter J, Konig R: Surgical and neurological complications in series of 708 epilepsy surgery procedures. Neurosurgery 1997; 41: 1–10Behrens, E Schramm, J Zenter, J Konig, R
Ikeda Y, Ueno A, Naraba H, Oh-ishi S: Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci 2001 69; 2911–19Ikeda, Y Ueno, A Naraba, H Oh-ishi, S
Berkley KJ, Robbins A, Sato Y: Afferent fibers supplying the uterus in the rat. J Neurophysiol 1988; 59: 142–63Berkley, KJ Robbins, A Sato, Y
Berkley KJ, Wood E, Scofield SL, Little M: Behavioral responses to uterine or vaginal distention in the rat. Pain 1995; 61: 121–31Berkley, KJ Wood, E Scofield, SL Little, M
Berkley KJ, Robbins A, Sato Y: Functional differences between afferent fibers in the hypogastric and pelvic nerves innervating female reproductive organs in the rat. J Neurophysiology 1993; 69: 533–44Berkley, KJ Robbins, A Sato, Y
Bradshaw HB, Temple JL, Wood E, Berkley KJ: Estrous variations in behavioral responses to vaginal and uterine distention in the rat. Pain 1999; 82: 187–97Bradshaw, HB Temple, JL Wood, E Berkley, KJ
Temple JL, Bradshaw HB, Wood E, Berkley KJ: Effects of hypogastric neurectomy on escape response to uterine distention in the rat. Pain 1999Aug; Suppl 6: S13–S20Temple, JL Bradshaw, HB Wood, E Berkley, KJ
Clement CI, Keay KA, Podzebenko K, Gordon BD, Bandler R: Spinal sources of noxious visceral and noxious deep somatic afferent drive onto the ventrolateral periaqueductal gray of the rat. J Comp Neurol 2000; 425: 323–44Clement, CI Keay, KA Podzebenko, K Gordon, BD Bandler, R
Yi DK, Barr GA: The induction of Fos-like immunoreactivity by noxious thermal, mechanical and chemical stimuli in the lumbar spinal cord of infant rats. Pain 1995; 60: 257–65Yi, DK Barr, GA
Buritova J, Besson JM: Effects of flurbiprofen and its enantiomers on the spinal c-Fos protein expression induced by noxious heat stimuli in the anaesthetized rat. Eur J Pharmacol 2000; 406: 59–67Buritova, J Besson, JM
Catheline G, Le Guen S, Besson JM: Effects of U-69,593, a kappa-opioid receptor agonist, on carrageenin-induced peripheral oedema and Fos expression in the rat spinal cord. Eur J Pharmacol 1999; 370: 287–96Catheline, G Le Guen, S Besson, JM
Sandner-Kiesling A, Pan HL, Chen SR, James R, DeHaven-Hudkin DL, Dewan D, Eisenach JC: Effect of kappa opioid agonists on visceral nociception induced by uterine cervical distension in rats. Pain 2002; 96: 13–22Sandner-Kiesling, A Pan, HL Chen, SR James, R DeHaven-Hudkin, DL Dewan, D Eisenach, JC
Sandner-Kiesling A, Eisenach JC: Estrogen reduces efficacy of mu- but not kappa-opioid agonist inhibition in response to uterine cervical distension. A nesthesiology 2002; 96: 375–80Sandner-Kiesling, A Eisenach, JC
Sandner-Kiesling A, Eisenach JC: Pharmacology of opioid inhibition of noxious uterine cervical distension. A nesthesiology 2002; 97: 966–71Sandner-Kiesling, A Eisenach, JC
Svensson CI, Yaksh TL: The spinal phospholipase-cyclooxygenase-prostanoid cascade in nociceptive processing. Annu Rev Pharmacol Toxicol 2002; 42: 553–83Svensson, CI Yaksh, TL
Yamamoto T, Sakashita Y: The role of the spinal opioid receptor-like–1 receptor, the NK-1 receptor, and cyclooxygenase-2 in maintaining postoperative pain in the rat. Anesth Analg 1999; 89: 1203–8Yamamoto, T Sakashita, Y
Yaksh TL, Rudy TA: Chronic catheterization of the spinal subarachnoid space. Physiol Behavior 1976; 17: 1031–36Yaksh, TL Rudy, TA
Fig. 1. Comparison of cFos expression in spinal cord in animals receiving uterine cervical distension for either 60 min (UCD 60, n = 6) or 30 min (UCD 30, n = 5), laparotomy without uterine cervical distension (sham, n = 6), or uterine cervical lidocaine infiltration (n = 4) prior to 60 min UCD. SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (laminae X). *P  < 0.05 compared with sham surgery and cervical infiltration of lidocaine. ΦP  < 0.05 compared with UCD 30 min.
Fig. 1. Comparison of cFos expression in spinal cord in animals receiving uterine cervical distension for either 60 min (UCD 60, n = 6) or 30 min (UCD 30, n = 5), laparotomy without uterine cervical distension (sham, n = 6), or uterine cervical lidocaine infiltration (n = 4) prior to 60 min UCD. SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (laminae X). *P 
	< 0.05 compared with sham surgery and cervical infiltration of lidocaine. ΦP 
	< 0.05 compared with UCD 30 min.
Fig. 1. Comparison of cFos expression in spinal cord in animals receiving uterine cervical distension for either 60 min (UCD 60, n = 6) or 30 min (UCD 30, n = 5), laparotomy without uterine cervical distension (sham, n = 6), or uterine cervical lidocaine infiltration (n = 4) prior to 60 min UCD. SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (laminae X). *P  < 0.05 compared with sham surgery and cervical infiltration of lidocaine. ΦP  < 0.05 compared with UCD 30 min.
×
Fig. 2. Photomicrograph showing cFos expression induced by uterine cervical distension. There is a significant increase in cFos expression in the deep dorsal horn regions of animals receiving 60 min of distension (A  ) compared with animals having surgery but no distension (B  ) and lidocaine infiltration of the cervix prior to distension (C  ). Photomicrographs (D  ), (E  ), and (F  ) show the cFos immunostaining in the central canal region (lamina X) in animals with 60 min of distension, surgery without distension, or distension after local lidocaine infiltration, respectively.
Fig. 2. Photomicrograph showing cFos expression induced by uterine cervical distension. There is a significant increase in cFos expression in the deep dorsal horn regions of animals receiving 60 min of distension (A 
	) compared with animals having surgery but no distension (B 
	) and lidocaine infiltration of the cervix prior to distension (C 
	). Photomicrographs (D 
	), (E 
	), and (F 
	) show the cFos immunostaining in the central canal region (lamina X) in animals with 60 min of distension, surgery without distension, or distension after local lidocaine infiltration, respectively.
Fig. 2. Photomicrograph showing cFos expression induced by uterine cervical distension. There is a significant increase in cFos expression in the deep dorsal horn regions of animals receiving 60 min of distension (A  ) compared with animals having surgery but no distension (B  ) and lidocaine infiltration of the cervix prior to distension (C  ). Photomicrographs (D  ), (E  ), and (F  ) show the cFos immunostaining in the central canal region (lamina X) in animals with 60 min of distension, surgery without distension, or distension after local lidocaine infiltration, respectively.
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Fig. 3. The effects of intrathecal ketorolac on uterine cervical distension–induced spinal cFos expression. Animals received intrathecal ketorolac, 50, 25, or 5 μg (Keto 50, Keto 25, Keto 5  ; n = 5 per group), or intrathecal saline with UCD 60 (IT Sal  , n = 7). SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (lamina X). *P  < 0.05 compared with intrathecal ketorolac, 50 μg and 25 μg; ΦP  < 0.05 compared with intrathecal ketorolac, 25 μg.
Fig. 3. The effects of intrathecal ketorolac on uterine cervical distension–induced spinal cFos expression. Animals received intrathecal ketorolac, 50, 25, or 5 μg (Keto 50, Keto 25, Keto 5 
	; n = 5 per group), or intrathecal saline with UCD 60 (IT Sal 
	, n = 7). SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (lamina X). *P 
	< 0.05 compared with intrathecal ketorolac, 50 μg and 25 μg; ΦP 
	< 0.05 compared with intrathecal ketorolac, 25 μg.
Fig. 3. The effects of intrathecal ketorolac on uterine cervical distension–induced spinal cFos expression. Animals received intrathecal ketorolac, 50, 25, or 5 μg (Keto 50, Keto 25, Keto 5  ; n = 5 per group), or intrathecal saline with UCD 60 (IT Sal  , n = 7). SDH = superficial dorsal horn (laminae I–II); DDH = deep dorsal horn (laminae III–V); VH = ventral horn (laminae VI–IX); CR = central region (lamina X). *P  < 0.05 compared with intrathecal ketorolac, 50 μg and 25 μg; ΦP  < 0.05 compared with intrathecal ketorolac, 25 μg.
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Fig. 4. Representative photomicrograph showing the effect of intrathecal ketorolac on spinal cFos expression. There is a significant decrease in cFos immunostaining with 50 μg ketorolac treatment (A  ) compared with saline (B  ) in the deep dorsal horn region; similar differences were present in the central canal region (lamina X), (C  ) = 50 μg ketorolac, (D  ) = saline.
Fig. 4. Representative photomicrograph showing the effect of intrathecal ketorolac on spinal cFos expression. There is a significant decrease in cFos immunostaining with 50 μg ketorolac treatment (A 
	) compared with saline (B 
	) in the deep dorsal horn region; similar differences were present in the central canal region (lamina X), (C 
	) = 50 μg ketorolac, (D 
	) = saline.
Fig. 4. Representative photomicrograph showing the effect of intrathecal ketorolac on spinal cFos expression. There is a significant decrease in cFos immunostaining with 50 μg ketorolac treatment (A  ) compared with saline (B  ) in the deep dorsal horn region; similar differences were present in the central canal region (lamina X), (C  ) = 50 μg ketorolac, (D  ) = saline.
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Fig. 5. Quantification of cFos expression in spinal cord across T12 to L2 levels. An average of cFos expressing cells in randomly selected sections from each level here and the expression from T12 to L2 were summed. Animals receiving intrathecal ketorolac, 25 (n = 5) or 50 μg (n = 6), had similar cFos expression compared with uterine cervical lidocaine infiltration and sham surgery, whereas those receiving uterine cervical distension with intrathecal saline had a four- to fivefold increase in cFos compared with control groups. *P  < 0.05 compared with sham surgery, cervical lidocaine injection, and intrathecal ketorolac, 50 μg.
Fig. 5. Quantification of cFos expression in spinal cord across T12 to L2 levels. An average of cFos expressing cells in randomly selected sections from each level here and the expression from T12 to L2 were summed. Animals receiving intrathecal ketorolac, 25 (n = 5) or 50 μg (n = 6), had similar cFos expression compared with uterine cervical lidocaine infiltration and sham surgery, whereas those receiving uterine cervical distension with intrathecal saline had a four- to fivefold increase in cFos compared with control groups. *P 
	< 0.05 compared with sham surgery, cervical lidocaine injection, and intrathecal ketorolac, 50 μg.
Fig. 5. Quantification of cFos expression in spinal cord across T12 to L2 levels. An average of cFos expressing cells in randomly selected sections from each level here and the expression from T12 to L2 were summed. Animals receiving intrathecal ketorolac, 25 (n = 5) or 50 μg (n = 6), had similar cFos expression compared with uterine cervical lidocaine infiltration and sham surgery, whereas those receiving uterine cervical distension with intrathecal saline had a four- to fivefold increase in cFos compared with control groups. *P  < 0.05 compared with sham surgery, cervical lidocaine injection, and intrathecal ketorolac, 50 μg.
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