ACUTE UTERINE IRRITATION PROVOKES PAINFUL
COLONIC MOTILITY VIA TRANSIENT RECEPTOR
POTENTIAL A1-DEPENDENT SPINAL NR2B
PHOSPHORYLATION IN RATS
Short title: Painful uterus-colon crosstalk
aHsien-Yu Peng, Ph.D., bChou-Ming Yeh, M.D., M.Sc., cJen-Kun Cheng,
M.D., Ph.D., dYat-Pang Chau, Ph.D., eTing Ruan, Ph.D., fGin-Den Chen,
M.D., g,hMing-Chun Hsieh, M.Sc., I,jCheng-Yuang Lai, B.S., k,l,mTzer-Bin
Lin, Ph.D.* a
Assistant Professor Department of Medicine, Mackay Medical College, New Taipei, Taiwan
b
Director Division of Thoracic Surgery, Department of Health, Taichung Hospital, Executive Yuan, Taichung, Taiwan
c
Chair Department of Anesthesiology, Mackay Memorial Hospital, New Taipei, Taiwan
d
Professor Department of Medicine, Mackay Medical College, New Taipei, Taiwan
e
Assistant Professor School of Medicine, Fu-Jen Catholic University, New Taipei, Taiwan
f
Professor Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, Chung-Shan Medical University, Taichung, Taiwan
g
Ph.D. Student Department of Physiology, School of Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
h
Research Assistant Department of Medicine, Mackay Medical College, New Taipei, Taiwan
i
Ph.D. Student Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan
j
Research Assistant Department of Medicine, Mackay Medical College, New Taipei, Taiwan
k
Professor Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
l
Professor Department of Biotechnology, Asia University, Taichung, Taiwan
m
Professor Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
Disclosure: We declare no conflicts of interest in this study.
Grants support:
This research was founded by the Grant from National Science Council , Taipei, Taiwan (NSC 101-2320-B-715-001-MY3 and NSC 102-2628-B-715-001) and Mackay Memorial Hospital, New Taipei, Taiwan (MMH-MM-10206)
to Dr HY Peng , Department of Health, Executive Yuan, Taipei, Taiwan
(PG10107-0052) to Dr CM Yeh, Mackay Memorial Hospital, New Taipei, Taiwan (MMH-10153) and National Science Council , Taipei, Taiwan (NSC 101-2314-B-195-002-MY2) to Dr JK Cheng and National Science Council , Taipei, Taiwan (NSC 101-2320-B-039-013-MY3) to Dr TB Lin .
Drs. Peng and Lin contributed equally to this work.
Words count: There are 247 words in the abstract; 412 words in
the introduction, and 1050 words in the discussion.
Correspondence:
Hsien-Yu Peng
Department of Medicine, Mackay Medical College No.46, Sec. 3, Zhongzheng Rd, Sanzhi Dist New Taipei, Taiwan 25245
Tel: +886-2-2636-0303 ext 1239 E-mail:[email protected] Tzer-Bin Lin
Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University No. 250, Wu-Hsing Street, Taipei, Taiwan 11031 Tel: +886-2-2736-1661
Author contributions:
Hsien-Yu Peng: Study design, analysis and interpretation of data, technical
support and study supervision.
Chou-Ming Yeh: Critical revision of the manuscript. Jen-Kun Cheng: Critical revision of the manuscript. Yat-Pang Chau: Critical revision of the manuscript. Gin-Den Chen: Drafting of the manuscript.
Ting Ruan: Acquisition and analysis of data
Ming-Chun Hsieh: Acquisition and analysis of data Cheng-Yuang Lai: Acquisition and analysis of data Tzer-Bin Lin: Study design and supervision.
ABSTRACT
Background: Patients with inflammatory gynecological/obstetrical problems
often complain of irritable bowel syndrome. We examined whether acute uterus irritation reflexively provokes painful colonic motility in rat preparations.
Methods: A modified colon manometry and striated abdominal muscle
electromyogram activity in response to mustard oil instillation into the uterine horn were continuously recorded in anesthetized rats. The lumbosacral (L6-S1) dorsal horn was dissected to assess the level and the cellular location of phosphorylated NR2B subunit using Western blotting and immunofluorescence analysis, respectively. Finally, the uterine transient receptor potential A 1 or spinal NR2B subunit was pharmacologically blocked
to elucidate their roles.
Results: ustard oilM (0.1%, 0.2 ml) injected into the lower uterine horn dramatically provoked painful colonic hyper-motility characterized by rhythmic colonic contractions (about 3-4 contractions/10 min, n=7) in accompanied by synchronized electromyogram firing in the abdominal muscle (about 4-5 folds of control, n=7). In addition to provoking colonic hyper-motility, mustard oil
administration also up-regulated phosphorylated (about 2-3 folds of control, n=7), but not total, NR2B expression in the dorsal horn neurons. Both intra-thecal Ro 25-6981 (a selective NR2B subunit antagonist; 10 μM, 10 μl) and intra-uterine HC-030031 (a selective transient receptor potential A 1 receptor
antagonist; 30 mg/kg, 0.2 ml) injected prior to the mustard oil instillation attenuated the mustard oil-induced colonic hyper-motility and spinal NR2B phosphorylation.
CONCLUSIONS: The co-morbidity of gynecological/obstetrical and
gastrointestinal problems is not coincidental but rather causal in nature, and clinicians should investigate for gynecological/urological diseases on the setting of bowel problems with no known pathological etiology.
INTRODUCTION
Acute irritation of pelvic organs was shown to provoke phosphorylation of the NR2B subunit of glutamatergic N-methyl-D-aspart ate receptors (NMDARs)
in the lumbosacral dorsal horn.1,2-4 Focal knockdown of spinal NR2B
expression3 and spinal administration of reagents selectively antagonizing
NR2B phosphorylation1,2,3 both thwarted the irritation-induced visceral
hyperreflexia, suggesting that spinal NR2B phosphorylation plays a crucial role in visceral pain pathology.
Although the underlying mechanism remains unclear, epidemiological studies demonstrate patients with inflammatory pain-associated gynecological problems, such as endometriosis or dysmenorrhea, often complain of irritable bowel syndrome (IBS).5,6,7 Cross-organ sensitization, a phenomenon in which
inflammation/injury enhances the N MDAR-mediated neurotransmission in the dorsal horn of both the damaged viscera itself and in unaffected neighboring organs, has recently emerged as an important theme in researches investigating the concurrence of problems in pelvic viscera.8,9,10 Our laboratory
has demonstrated acute uterus irritation dynamically enhances pain-related urethra electromyogram activity via spinal NR2B phosphorylation.1,11
Nevertheless, whether damages in the uterus could also reflexively affect bowel functions, demonstrating the co-morbidity of gynecological problems and IBS, and the possible role of spinal NR2B subunit has not yet been clearly investigated.
While there is sparse direct immunohistochemical evidence demonstrated
t he transient receptor potential subfamily ankyrin 1 (TRPA1)12-expressing
afferents innervates the pelvic viscera,13 pharmacological activation of TRPA 1
modifies the function of the urinary bladder,14 urethra,15 colon,16,17 and uterus.18
This suggests a role of TRPA1 in the neural regulation of lower urogenital and
gastrointestinal tracts. Knockout of mouse TRPA1 prevents the enhanced
visceromotor response caused by 2,4,6-trinitrobenzenesulfonic acid-induced colitis and colon irritation induced by acute mustard oil (MO , a selective TRPA1 agonist) instillation.19 Moreover, TRPA1 activation caused by a brief
intra-colonic MO application increased spontaneous excitatory post-synaptic potentials in a spinal slice through the selective potentiation of glutamate-mediated neurotransmission in the substantia gelatinosa, a key site for noxious input integration.20 Interestingly, by applying MO into the descending
colon, studies have demonstrated the MO-induced spinal NR2B phosphorylation is an important determinant for the painful crosstalk among pelvic viscera.3,4,21,22 These several studies prompt us to investigate whether
and how the activation of nociceptive TRPA1-expressing afferents and the
subsequent spinal NR2B phosphorylation participate in uterus-colon crosstalk, a possible mechanism underlying the concurrence of gynecological pain and IBS.
W
e hypothesized acute uterus irritation provokes colonic hyper-motility via the spinal NR2B phosphorylation. Thereby, we recorded the colon motility and analyzed the spinal expression and location of phosphorylated NR2B in response to intra-uterine MO instillation. Moreover, uterine TRPA1 and spinal
NR2B were pharmacologically antagonized to elucidate their roles in the uterus-colon crosstalk.
MATERIALS and METHODS
Animal preparations
A
ll procedures for animal studies were reviewed and approved by the Institutional Review Board of National Chung-Hsing University, Taichung, Taiwan . Two hundred and sixty-three female Sprague-Dawley rats, weighing 250–300 g, were randomly assigned to groups and used throughout this study. Animals were individually housed in wood chip-lined plastic cages, had free access to water and food, and were maintained on a 12:12-h light-dark cycle with lights on at 07:00 am. The estrous stage was assessed daily at 9:00 am by vaginal lavage using the traditional stage nomenclature.23 Rats
that had two complete, regular 4-day estrous cycles before the day of the experiment were used for this study. Measurements were made approximately 5 to 8 h after the lights were turned on and when the rats were in the diestrus stage (when both estradiol and progesterone are low).23
Surgical preparations
In order to quickly achieve adequate anesthesia for surgical procedures, animals were first anesthetized with isoflurane (5% for induction and 2% for surgery). A PE-50 catheter (Portex, Hythe, Kent, U nited K ingdom) was placed in the left jugular vein for anesthetic administration. A midline abdominal incision was made to expose the pelvic viscera. Two wide-bore uterine cannulae, one for drug injections and the other for fluid drainage, were inserted into the lumen of the right lower uterine horn through small incisions made on the uterine horn and secured with cotton thread. After all surgical procedures were completed, urethane (1.2 g/kg Sigma-Aldrich, St. Louis, MO) was injected intravenously to maintain anesthesia, as this reagent produces a
long duration of anesthesia following a single bolus while causing minimal changes in the visceral and reflex response.24
Colonic motility recording
The motility of the descending colon was continuously recorded as changes in intra-colonic pressure (ICP) using an approach modified from Gourcerol et al.25 and Carini et al.26 In brief, a catheter with a 4 cm deflated
flexible latex balloon lubricated with medical-grade lubricant in the tip was inserted intra-anally into the descending colon such that the end of the balloon was 1 cm proximal to the anus. The balloon was then filled with normal saline (approximately 0.5 ml), and the ICP was continuously recorded via a catheter connected to a pressure transducer (P23 ID; Gould-Statham, Quincy, IL) on a computer system (MP30,Biopac, Santa Barbara, CA) through a preamplifier (7P1, Grass, Cleveland, OH). Because our pilot experiments showed the maximal ICP provoked by the colonic irritation was approximately 30 mmHg (28.15±3.21 mmHg), the motility of the descending colon was then quantified off-line by the number of contractions in which the amplitude was higher than 15 mmHg (about half the amplitude of the maximal contraction) using a program built in the recording software (Student Lab BSL PRO 3.7, Biopac,
Santa Barbara, CA . Figure 1A). Because balloon distension initially provoked rhythmic colonic contractions that gradually subsided, drug pretreatments, including intra-uterine HC-030031 (a selective TRPA1 antagonist) or
intra-thecal Ro 25-6981 (a NMDAR NR2B subunit selective antagonist) injection was carried out after a equilibrium period of 90 min following balloon distension . In all the cases (including animals received no pretreated reagents), t he ICP in response to intra-uterine either mustard oil or corn oil
instillation was continuously recorded for 60 min after a period of pretreatment (30 min). T herefore, experiment usually finished 1 8 0 min after the starting of balloon distension.
Electromyogram recording
In some experiments, electromyogram activity was picked up by Teflon-coated stainless steel wire electrodes stitched into the external oblique musculature immediately superior to the inguinal ligament. The
electromyogram signals were continuously recorded on a computer system (MP30, Biopac,Santa Barbara, CA) through a preamplifier (P511AC, Grass,
Cleveland, OH) using a band-pass filter with a frequency range of 30-3000 Hz. The electromyogram activity was quantified off-line by integrating the area under the rectified electromyogram signal using a program built in the recording software (Student Lab BSL PRO 3.7, Biopac, Santa Barbara, CA ,
Figure 1B).
Intrathecal catheter
Implantation of an intrathecal cannula was performed as described in our previous study.4 Briefly, the occipital crest of the skull was exposed, and the
atlanto-occipital membrane was incised at the midline with the tip of an 18-gauge needle. A PE-10 catheter was inserted through the slit and passed caudally to the L6-S1 level of the dorsal aspect of the arachnoid space. The volume of fluid within the cannula was kept constant at 10 μl in all experiments. A single, 10 μl volume of drug solution was administered followed by a 10 μl flush of vehicle solution. At the end of the experiment, a laminectomy was performed to verify the location of the cannula tip. In 5 out of 64 rats, the cannula tip deviated by more than 0.5 mm from the target
structure, and the data from these 5 cases were excluded from the statistical analysis.
Drug administration
Drugs that were administered by intra-uterine, intra-colonic, intra-thecal or intra-venous injections included the following: mustard oil (MO, allyl isothiocyanate; 0.05, 0.1, and 0.5% dissolved in 0.2 ml corn oil, intra-uterine, Sigma-Aldrich, St. Louis, MO), a pungent component causing acute colon inflammation;3 corn oil (CO, 0.2 ml, intra-uterine, Sigma-Aldrich, St. Louis,
MO), a control solution for MO;3 HC-030031 (HC, 30 mg/kg, 0.2 ml,
intra-uterine, Sigma-Aldrich, St. Louis, MO), a TRPA1 receptor antagonist;27 acetic
acid (1%, 3% and 10%, 0.2 ml, intra-colonic, Sigma-Aldrich, St. Louis, MO), an irritant inducing colonic motility;24 Ro 25-6981 (RO, 10 μM, 10 μl,
intra-thecal, Sigma-Aldrich, St. Louis, MO), a selective NMDAR NR2B subunit antagonist;2 ghrelin (1, 3 and 10 μg/kg, 10 μl, intra-thecal, Sigma-Aldrich, St.
Louis, MO), an agent causing rhythmic colonic motility;28 and Evans blue (50
mg/kg; intra-venous, Sigma-Aldrich, St. Louis, MO), a dye to quantify plasma extravasation.29 In all cases, solvent solutions of volume identical to those of
the tested agents served as vehicle controls.
Western blotting
The rats were deeply anesthetized, and the spinal cords were quickly obtained before (pre-injection control) and 10, 30, or 60 min after intra-uterine mustard oil injections. The dorsal horn of the right lumbosacral spinal cord (L6-S1) was dissected for Western blot analysis using methods described previously.30 In brief, protein samples (20 μg) were separated by SDS-PAGE
milk and probed with antibodies against beta actin (actin, 1:8,000, Santa Cruz, Santa Cruz, CA) total and phosphorylated NR2B (tNR2B and pNR2B, respectively, 1:1,000, Millipore , Billerica, MA). The blots were incubated with horseradish peroxidase-conjugated antibody (1:2,000) for 1 hour at room temperature and visualized with an enhanced chemiluminescence solution (5 min) followed by film exposure (2 min). Densitometric analysis of the WB membrane was performed with Science Lab 2003 (LAS-300; Fuji, Kanagawa, Japan).
Quantitation of plasma protein extravasation
A subset of animals was prepared to evaluate the effect of intra-uterine mustard oil on plasma protein extravasation in the uterus and colon.29 Evans
blue (50 mg/kg, Sigma-Aldrich, St. Louis, MO) was injected intravenously 30 min prior to the removal of tissue. At the end of the experiments, the animals were decapitated and exsanguinated. The uterus and the descending colon were removed, blotted on wet filter paper, weighed, and stored in formamide at room temperature for 72 hr to extract Evans blue from the tissue. The amount of extracted Evans blue was quantified by measuring the optical density of the extracted dye at a wavelength of 620 nm with the use of a spectrophotometer, and the value was expressed as micrograms per gram of wet tissue weight.29 The concentration was estimated from a standard
regression curve for Evans blue concentrations ranging from 0.1 to 5 μg/ml.
Immunofluorescence
The rats were deeply anesthetized and perfused with 100 ml of 0.01 M phosphate-buffered saline (pH 7.4), followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The right lumbosacral
spinal cord (L6-S1) was then harvested, post-fixed at 4°C for 4 hours, and cryoprotected in 30% sucrose overnight. For double-labeling immunohistochemistry analyses, the spinal cord sections were incubated overnight at 4°C with a mixture of rabbit anti-phosphorylated NR2B (1:200, Abcam, Cambridge, U nited K ingdom) and mouse monoclonal anti-neuronal nuclear antigen (NeuN, a neuronal marker, 1:1,000, Chemicon, Billerica, MA), mouse anti-glial fibrillary acidic protein (GFAP, a marker of astroglial cells, 1:500, Millipore, Billerica, MA), or mouse anti-integrin αM (OX-42, a marker of microglia, 1:400, Santa Cruz, Santa Cruz, CA). The sections were then incubated with Alexa Flour 488 (1:1,500) and Alexa Flour 594 (1:1,500, Invitrogen, Carlsbad, CA ) for 1 h at 37 °C.
Statistical analysis
A total of 263 rats used in this study, including 142 rats for manometry recording and 121 rats for Western blot (70 rats), immunohistochemistry (26 rats) and plasma protein extravasation (25 rats) analysis. The density of specific bands from Western blot analysis was measured using computer-assisted imaging analysis (LAS-300; Fuji, Kanagawa, Japan) and normalized against corresponding loading control bands. The motility of the descending colon was quantified by counting the ICP contractions and electromyogram
activity was quantified by integrating the area under the rectified
electromyogram signal using programs built in the recording software (Student Lab BSL PRO 3.7, Biopac, Santa Barbara, CA). All data were analyzed using SigmaPlot (version 10.0; Systat Software, Inc., San Jose, CA), and are presented as the mean ± SD. For serial measurements over time, one-way ANOVA was used to assess changes in values, and a post-hoc
Tukey test was used to compare means for groups. In experiments evaluating the effect of different reagents administration, paired, two tailed Student’s t-test
was used to compare means for groups. In all cases, significance was assigned at a p<0.05.
RESULTS
Baseline colonic motility
To test the validity of the modified balloon method, we added normal saline (approximately 0.5 ml) into the balloon, which had been intra-anally inserted into the descending colon of urethane-anesthetized rats, and the intra-colonic pressure (ICP) was then continuously recorded. In all 142 rats tested, balloon distension provoked rhythmic colonic contractions (RCCs) characterized by periodic peaks in the ICP, which gradually subsided during an equilibrium period (typically 90 min; Figure 1C). After the equilibrium period, the ICP maintained a relatively constant level without significant RCCs in 135 out of 142 rat s, and t hese animals were then used for further study. The manometric tracing in Figure 1D demonstrated a stable ICP throughout the recording period, and summarized data showed no difference between the contraction count in the ICP curve measured at 10, 30 and 60 min after equilibrium (Figure 1E, =0.p 631, 0. 642, and 0. 913 in 10 vs. 30, 30 vs. 60, and 10 vs. 60
min respectively, n=7), indicating this modified balloon method reflects baseline colonic motility in routine circumstances.
Effects of intrathecal ghrelin
In experiments tested the reliability of the modified balloon method in response to physiological challenges, cumulative administration of ghrelin (Figure 2A; 1, 3, and 10 μg/kg, 10 μl, intra-thecal), but not of the vehicle solution (veh, 10 μl), enhanced colonic motility as characterized by provoking RCCs in the manometric tracing. Figure 2B shows when compared to the pre-injection control, spinal ghrelin administration at 1, 3 and 10 μg/kg
dose-dependently increased the contraction count in the ICP curve (p =0.0 21, p
<0.001 , and p =0.0 01 vs. control, respectively. n=7), while no difference was found between the pre-injection control and vehicle solution injections (p =0. 962 vs. control, n=7). These results implied the balloon technique used in this study responds to physiological impacts on colonic motility.
Effects of intra-colonic acetic acid
In contrast to the vehicle solution, which failed to affect colonic motility in the experiment testing the reliability of the modified balloon method in response to pathophysiological challenges (Figure 2C veh, 0.2 ml), cumulative acetic acid injections (1%, 3%, and 10%, 0.2 ml, intra-colonic) enhanced colonic motility by provoking RCCs in the manometric tracing. The summarized data showed when compared to the pre-injection controls, intra-colonic acetic acid injections dose-dependently increased the contraction count in the ICP curve (Figure 2D, p =0.0 02, p <0.001 , and p <0.001 vs. control in 1%, 3%, and 10%, respectively. n=7), suggesting this modified balloon technique responds to pathophysiological conditions.
Mustard oil induced painful uterus-colon crosstalk
Next, colon manometry and the electromyogram activity of the rectus abdominis in response to uterine mustard oil (MO, 0.1%, 0.2 ml) or corn oil (CO; 0.2 ml) injections were continuously recorded in anesthetized rats. We observed when compared to the pre-injection control, CO injections into the uterine horn neither induced RCCs in the manometric recording nor triggered firing in the electromyogram tracing (Figure 3A). In contrast, MO
administration (Figure 3B) dramatically provoked RCCs in the manometry and elicited synchronized burst electromyogram discharges. Figures 3C and D summarizes that it was uterine MO injections, but not CO injections,
significantly increased the mean contraction count in the ICP curve (p =0.0 02
and p =0. 778 vs. control, repectively. n=7) and the mean area of integrated
electromyogram (iEMG, p =0.0 03 and p =0. 716 vs. control, respectively. n=7) compared to the pre-injection control. These data imply acute irritation of the uterus provokes painful colonic motility in a cross-organ manner.
Mustard oil induced spinal NR2B phosphorylation
We next tested whether uterine MO administration could also induce spinal NMDAR NR2B subunit phosphorylation using Western blot analysis of spinal dorsal horn samples (L6-S1) obtained at 10, 30, and 60 min after MO injections. In correlated with the colonic hyper-motility, we found MO administration (0.1%, 0.2 ml) time-dependently increased the band intensity labeled by the phosphorylated NR2B-specific antibody (Figure 3E, pNR2B, normalized by the beta-actin; =0.0p 33, 0.0 08, and 0.0 02 , vs. Control in 10, 30, and 60 min , respectively. n=7) when compared to the pre-injection control. In contrast, this treatment failed to affect the band intensity labeled by the total NR2B-specific antibody at these time points (tNR2B, normalized by the beta-actin; p =0. 864, 0. 350, and 0. 925 vs. Control in 10, 30, and 60 min , respectively. n=7), implying spinal NR2B activation play a key role in uterine-colon sensitization. We therefore focused our investigations on dorsal horn NR2B phosphorylation.
Uterus mustard oil instillation induced no colon extravasation
We next tested whether acute intra-uterine MO injections induce inflammation in the uterus or descending colon by measuring the extravasation of Evans blue dye in CO- or MO-treated animals. Figure 3F summarizes when compared to the pre-injection control, it was MO (0.1%, 0.2 ml) but rather than CO (0.2 ml) administration into the uterine horn caused an increase in Evans blue extravasation in the uterus as evidenced by significantly enhancing the optical density of the uterus extraction (p =0. 547 in
CO and p =0.0 01 in MO vs. Control, both n=7). In contrast, there was no difference found in the optical density of the colon extraction of CO- or MO-treated animals compared to the pre-injection control ( =0.p 578 in CO and p
=0. 974 in MO vs. control, both n=7), indicating that acute uterine MO injections provoke extravasation in the uterus itself but rather than the descending colon.
Mustard oil induced NR2B phosphorylation in dorsal horn neurons
We next examined the cellular localization of the MO-induced NR2B phosphorylation using immunohistochemistry analysis. Staining of spinal cord sections (L6-S1; obtained 60 min after reagent instillation) for phosphorylated NR2B immunoreactivity demonstrated when compared with CO (Figure 4A), uterine administration of MO (Figure 4B) increased the immunoreactivity of phosphorylated NR2B in the dorsal horn. Moreover, double staining for phosphorylated NR2B and NeuN (neuron-specific nuclear protein, a neuronal marker), OX-42 (integrin αM, a microglial marker) or GFAP (glial fibrillary acidic protein, an astrocyte marker) showed the MO-enhanced
phosphorylated NR2B immunoreactivity overlapped with NeuN (Figure 4C) but not OX-42 (Figure 4D) or GFAP (Figure 4E), implying the MO-enhanced NR2B phosphorylation occurs in dorsal horn neurons but not in glial cells.
Mustard oil dose-dependently induced uterus-colon crosstalk
Next, MO at concentrations of 0.05, 0.1, and 0.5% (Figure 5A 0.2 ml) or corn oil (CO, 0.2 ml) was instilled into the uterine horn of urethane-anesthetized rats. In vivo manometric recordings demonstrated while CO instillation exhibited no effect, administration of MO dose-dependently induced colonic motility as evidenced by provoking RCCs when compared with the pre-injection control. Figure 5B summarizes that MO administration at concentrations of 0.05, 0.1, and 0.5% dose-dependently increased the contraction count in the ICP curve when compared to the pre-injection control (p =0.0 09, 0.0 02, and 0.0 02 , vs. control, respectively. n=7).
Effects of Ro 25-6981 pretreatment
We next pharmacologically blocked spinal NR2B phosphorylation using a selective antagonist to further elucidate its role in the MO-induced uterus-colon crosstalk. As anticipated, uterine MO injections induced colonic motility (Figure 6A and B MO; 0.1%, 0.2 ml), as shown by significantly enhancing the contraction count in the ICP curve (Figure 6C, p <0.001 and p = 0.001 vs. control in the left and right, respectively. n=7) associated with an increase in the band intensity of the pNR2B in dorsal horn samples (Figure 6D, p <0.001 vs. control, n=7). It is the pre-treated Ro 25-6981 (Ro+MO, a NR2B antagonist. 10 μM, 10 μl, intrathecal), unlike the vehicle solution (veh+MO, 10
μl, intrathecal), attenuated MO-induced hyper-motility as shown by a significant decrease in the contraction count in the ICP curve compared to the MO-treated animals (Figure 6C, p =0.0 07 in Ro+MO and p =0. 506 in veh+MO vs. MO, n=7). Additionally, spinal Ro 25-6981 injections also prevented MO-induced dorsal horn NR2B phosphorylation because it significantly decreased the band intensity of pNR2B when compared with MO-treated animals (Figure 6D, p <0.001 vs. MO, n=7). Moreover, neither the contraction count in the ICP curve nor the intensity of the pNR2B band was affected by the injection of Ro 25-6981 during baseline control before MO instillation (data not shown). These results provide pharmacological support for the pivotal role of phosphorylation of spinal NR2B subunits in the uterus-colon crosstalk.
Effects of HC-030031 pretreatment
A s shown in Figure 6B, uterine MO injections induced colon hyper-motility by a significant increase in contraction count in the ICP curve (Figure 6C, p
=0.0 01 vs. control, n=7) and an increase in the band intensity of pNR2B in the dorsal horn sample (Figure 6D). The administration of HC-030031 (30 mg/kg, 0.2 ml, HC+MO), but not the vehicle solution (veh+MO), into the uterine horn prior to MO injections attenuated MO-induced hyper-motility (Figure 6B) as shown by a significant decrease in the contraction count in the ICP curve when compared to the MO-treated animals (Figure 6C, p =0.0 02 in
HC+MO and p =0. 924 in veh+MO vs. MO, n=7). In addition, pretreatment with HC-030031 prevented MO-elicited spinal NR2B phosphorylation as demonstrated by a significantly decrease in the intensity of the pNR2B band compared to the MO-treated group (Figure 6D, p =0.0 01 vs. MO, n=7).
Conversely, when it was injected during baseline control before MO instillation, HC-030031 per se exhibited no effect on the contraction count in the manometric recording or in the level of dorsal horn pNR2B expression (data not shown). These results provide evidence that the activation of TRPA1
in the uterus could reflexively induce colon hyper-motility via a mechanism involving the phosphorylation of the spinal NMDAR NR2B subunit.
DISCUSSION
Through a complex neural network innervating viscera,8,10,31 pathological
alterations in one pelvic organ could lead to modifications in the function of others in a cross-organ manner. 32-34 In the present study, acute MO dispensing
into the uterus reflexively enhanced colonic motility by provoking RCCs. The possibility that MO directly established an organic injury in the descending colon, and thereby resulted in colon hyper-motility is unlikely because we administered MO into the lumen of the uterus but rather than the descending colon. This proposal is further supported by the fact that uterine MO administration led to Evans blue extravasation in the uterus itself but not in the descending colon, indicating our procedure did not result in colon inflammation. That is, the enhanced colon motility demonstrated in this study is a phenomenon resembling functional bowel disorders, such as IBS, in which colon functions are altered without a known pathological etiology. Moreover, in accompanied with colon hyper-motility, we observed uterine MO injections provoked abdominal electromyogram discharges that were synchronized with the RCCs. Since it is well established that painful colon-rectal distension provokes visceromotor response characterized by enhanced
electromyogram activity of abdominal striated muscle,17 and therefore
abdominal electromyogram firing is used as an index of visceral pain, particularly in colon/rectum case. Our data suggest acute uterus irritation provokes painful colon hyper-motility in a cross-organ manner; a phenomenon could underlie the clinical finding that IBS, which is characterized by hyperactivity in the distal gastrointestinal tract in association with abdominal
pain, often occurs in conjunction with obstetrical/gynecological inflammation. 35-37
The understanding of the TRPA1 in pain-related behavior advances rapidly
in recent years,19,20,38 and TRPA
1activation is currently recognized as
necessary for the somatic pain provoked by intraplantar complete Freund’s adjuvant injection39 as well as the visceral pain caused by trinitrobenzene
sulfonic acid- and dextran sulphate sodium-induced colitis.19,40 Although the
therapeutic doses are still too high for drug development, known TRPA1
antagonist (s) play promising in vivo activity against cystitis-associated hyperalgesia,15 carrageenan-induced inflammatory pain,41 and post-operative
guarding pain.42 By instilling a TRPA
1 agonist, MO, into the lumen of uterine
horn, we provoked painful crosstalk between the uterus and colon in this study. Conversely, pharmacological antagonism of uterine TRPA1 using
HC-030031 abolished MO-induced uterus-colon crosstalk. Our result is the first demonstration that activation of uterine TRPA1 modified the function of pelvic
viscera. This finding implies TRPA1 participates in the uterus
physiology/pathophysiology and suggests the development of pharmacological agents targeting TRPA1 is a possible therapeutic strategy for
the treatment of pelvic visceral pain.
In the present study, we showed that activation of uterine TRPA1 induced
painful colonic motility in a cross-organ manner. Considering our previous investigation demonstrated that activation of uterus TRPV1 sensitized the
urethra reflex activity,1 it would be interesting to test the likely interaction
between these receptors because an electrophysiological study analyzed the TRPA1-mediated current in mouse trigeminal ganglion neurons demonstrated
synergic interactions between TRPA1 and TRPV1.43 Our unpublished data
displayed that pretreated capsazepine, a selective TRPV1 antagonist, partially
attenuated MO-induced uterus-colon crosstalk. However, whether this observation is resulted from TRPA1-TRPV1 interactions or other mechanisms
needs further study to be fully elucidated.
NR2B-containing NMDARs have been proposed to participate in the plasticity underlying hyperalgesia/allodynia44-50 because phosphorylation of
NR2B tyrosine residues modulates NMDAR-mediated currents.51 In addition to
its role in the development of spinal nerve ligation-induced neuropathic pain,49,52 the contribution of spinal NR2B to visceral pain was investigated in
this study. Our results demonstrated that intra-uterine MO instillation provoked colonic motility in associated with NeuN-colocalized NR2B phosphorylation that were both thwarted by intrathecal dispensing of selective NR2B antagonist, Ro 25-6981. These findings suggest activation of NR2B-containing NMDARs in dorsal horn neurons are crucially involved in the pain-related uterus-colon crosstalk. Though spinal NR2B subunits have been demonstrated to participate in the urethra3,21,22,53 and bladder4 hyper-reflexia
caused by acute colon irritation, our results are the first demonstration of a role of spinal NR2B in the painful crosstalk between the uterus and colon. With regard to a complex convergent neural network innervating and integrating the function of pelvic organs, including the urinary bladder, urethra, uterus, and colon,8,10,31 our findings suggest that spinal NR2B phosphorylation
could be a common mechanism shared by the crosstalk between pelvic organs, in which irritation of one pelvic viscera induced spinal NR2B phosphorylation, thus provoking crosstalk with another viscera.
In the present study, a modified balloon manometric analysis was used to record colon motility. We observed that after an equilibrium period of stress-relaxation, a tracing with a relatively constant ICP was recorded before physiological/pathological manipulations, indicating this technique is a valid measurement of colonic motility under baseline physiological conditions. To test the reliability of this method in response to physiological and pathophysiological challenges, we thecally injected ghrelin and intra-colonically infused acetic acid to the animal because in vivo studies demonstrated spinal ghrelin injection elicits propulsive colonic contractions,28
and acute colonic irritation caused by acetic acid triggers colonic motility.24
Our results showed both these treatments dose-dependently provoked RCCs, indicating this modified manometry is a reliable measurement of motility changes in response to physiological and pathological perturbations. Although studies are needed to further verify the reliability and validity of the manometric analysis used in this study, this method provides available technique for investigating colonic motility. Additionally, using this manometry technique, the design of this study offers an in vivo animal model to investigate the interaction between the colon and uterus, which elucidates the mechanism underlying the co-morbidity of painful gynecological problems and IBS.
In summary, our findings not only established the feasibility of a unique model to study the impact of various treatments on colonic motility but also enabled an eventual characterization of the pathophysiological mechanisms involved in the development and co-morbidity of uterus inflammation and IBS. Our data support the notion that the co-morbidity of gynecological/obstetrical
and gastrointestinal problems is not coincidental but rather causal in nature. Via the activation of uterine TPRA1, spinal NMDAR NR2B phosphorylation
triggered by nociceptive afferents may set the foundation for the pathophysiological uterus-colon crosstalk. Perhaps leading to sensitization caused by the release of neurotrophic factors and other mediators,8 long-term
or ongoing stimulation of these pathways may possibly lead to permanent sensory changes in the descending colon. Although further researches are warranted to expand on the current findings, we suggest that clinicians consider gynecological or urological disease in cases of bowel disease with no known pathological etiology.
Grants support:
This research was founded by the Grant from National Science Council , Taipei, Taiwan (NSC 101-2320-B-715-001-MY3 and NSC 102-2628-B-715-001) and Mackay Memorial Hospital, New Taipei, Taiwan (MMH-MM-10206)
to Dr HY Peng , Department of Health, Executive Yuan, Taipei, Taiwan
(PG10107-0052) to Dr CM Yeh, Mackay Memorial Hospital, New Taipei, Taiwan (MMH-10153) and National Science Council , Taipei, Taiwan (NSC 101-2314-B-195-002-MY2) to Dr JK Cheng and National Science Council , Taipei, Taiwan (NSC 101-2320-B-039-013-MY3) to Dr TB Lin .
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What We Already Know About This Topic
Patients with inflammatory gynecological or obstetrical problems often complain of irritable bowel syndrome.
What This Article Tells Us That Is New
Acute irritation of the TRPA1 in uterine horn provoked painful colonic
hyper-motility that is highly dependent on the phsophorylation of NMDAR NR2B subunit in the lumbosacral spinal cord.
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