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GASTROENTEROLOGY 2006;130:1729–1742

he Role of Neurokinin 1 Receptors in the Maintenance ofisceral Hyperalgesia Induced by Repeated Stress in Rats

YLVIE BRADESI,*,‡,§ EFI KOKKOTOU,¶ SIMOS SIMEONIDIS,¶ SIMONA PATIERNO,*,‡,§

ELENA S. ENNES,*,‡ YASH MITTAL,*,‡ JAMES A. MCROBERTS,*,‡ GORDON OHNING,*,‡,§

ETER MCLEAN,# JUAN CARLOS MARVIZON,*,§ CATIA STERNINI,*,‡,§,�,**HARALABOS POTHOULAKIS,¶ and EMERAN A. MAYER*,‡,§,**,††

Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, ‡CURE: Digestive Diseases Research Center,Department of Neurobiology, **Brain Research Institute, and ††Department of Physiology, Psychiatry, and Biobehavioral Sciences, Davideffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California; §VA Greater Los Angeles Healthcareystem, Los Angeles, California; ¶Gastrointestinal Neuropeptide Center, Division of Gastroenterology, Beth Israel Deaconess Medical Center,
arvard Medical School, Boston, Massachusetts; and #GlaxcoSmithKline (Neurology & G1 Centre of Excellence for Drug Discovery), Harlow, UK
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ackground & Aims: The neurokinin 1 receptors (NK1Rs)nd substance P (SP) have been implicated in the stressnd/or pain pathways involved in chronic pain conditions.ere we examined the participation of NK1Rs in sustainedisceral hyperalgesia observed in rats exposed to chronicsychological stress. Methods: Male Wistar rats were ex-osed to daily 1-hour water avoidance stress (WA) or shamA for 10 consecutive days. We tested intraperitoneal or

ntrathecal injection of the NK1R antagonist SR140333 onhe visceromotor reflex to colorectal distention in bothroups at day 11. Real-time reverse-transcription polymer-se chain reaction, Western blot, and immunohistochem-

stry were used to assess the expression of NK1Rs and/orP in samples of colon, spinal cord, and dorsal root gan-lia. Results: Both intraperitoneal and intrathecalR140333 injection diminished the enhanced visceromo-or reflex to colorectal distention at day 11 in stressed ratsut did not affect the response in control animals. Real-ime polymerase chain reaction and Western blotting dem-nstrated stress-induced up-regulation of spinal NK1Rs.mmunohistochemistry showed an increased number ofK1R-expressing neurons in the laminae I of the dorsalorn in stressed rats. The expression of NK1Rs was de-reased in colon from stressed rats compared with control.he expression of SP gene precursor in dorsal root gangliaas unchanged in stressed rats compared with controls.onclusions: Stress-induced increased NK1R expression onpinal neurons and the inhibitory effect of intrathecal NK1Rntagonist on visceral hyperalgesia support the key contri-ution of spinal NK1Rs in the molecular pathways involved

n the maintenance of visceral hyperalgesia observed afterhronic WA.

rritable bowel syndrome belongs to a group of stress-sensitive chronic disorders that shares visceral hyper-

lgesia and altered autonomic nervous system responsive-

ess and commonly overlaps with anxiety disorders suchs posttraumatic stress disorder and panic disorder.1

ased on pathophysiologic models of enhanced respon-iveness of brain-gut interactions2 and of central stressircuits,3 several neuropeptide receptors have been im-licated as plausible targets for novel therapies for irri-able bowel syndrome, including the corticotropin-re-easing factor (CRF)/CRF1 receptor system and theubstance P (SP)/neurokinin 1 receptor (NK1R) sys-em.4,5

Both preclinical and clinical studies suggest thatndogenous release of SP is involved in acute andhronic activation of central stress and/or pain cir-uits.6 – 8 Recent evidence obtained in patients withosttraumatic stress disorder showed increased cere-rospinal fluid levels of SP-like immunoreactivity,uggesting increased release of SP from sources in-olved in central stress circuits.9 Increased cerebrospi-al fluid levels of SP have also been reported inatients with chronic functional pain syndromes suchs fibromyalgia10,11 and in irritable bowel syndrome.12

ven though it is unclear if human SP cerebrospinaluid levels in these conditions reflect increased releaserom spinal and/or supraspinal structures, these find-ngs strongly suggest involvement of the SP/NK1R

Abbreviations used in this paper: ANOVA, analysis of variance; CRD,olorectal distention; CRF, corticotropin-releasing factor; DRG, dorsaloot ganglia; EMG, electromyographic; IR, immunoreactive; NK1R, neu-okinin 1 receptor; PPT-A, preprotachykinin A; RT-PCR, reverse-tran-cription polymerase chain reaction; VMR, visceromotor response; WA,ater avoidance.© 2006 by the American Gastroenterological Association Institute

0016-5085/06/$32.00
doi:10.1053/j.gastro.2006.01.037

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1730 BRADESI ET AL GASTROENTEROLOGY Vol. 130, No. 6

ignaling system within the central nervous system intress-related chronic anxiety and pain conditions.

In rodents, SP and NK1Rs are widely distributedhroughout the central and peripheral nervous system,articularly on spinal neurons in laminae I and X, whereisceral primary afferents terminate,13,14 and in brainreas involved in affective and stress responses.15 In theut, SP and/or NK1Rs are present on enteric neurons,16

eripheral terminals of spinal afferents, capsaicin-sensi-ive sensory neurons,17,18 and immune cells.17,19,20 At thepinal cord level, SP is released from small-diameterensory C fibers into the dorsal horn in response tontense peripheral stimulation, including inflammation,issue irritation, or sustained noxious stimuli.21,22 It isow well established that activation of NK1R neurons onaminae I of the dorsal horn contributes to the develop-ent of somatic hyperalgesia. For example, selective

blation of these neurons using an SP/toxin conjugateroduces a dramatic decrease in hyperalgesia induced byapsaicin, inflammation, and nerve injury,23,24 withoutompromising the response to acute noxious stimulation.imilarly, spinal injections of SP antibodies or NK1Rntagonist block the somatic hyperalgesia induced byepeated cold stress in rats.25,26 Because the great major-ty of lamina I neurons project to central structuresnvolved in descending modulation of nociception, it haseen suggested that they are part of a spinal-bulbo-spinalircuitry that modulates pain signaling in sensitizedonditions.27,28

In terms of visceral nociception, accumulating evi-ence indicates the contribution of the NK1R/SP systemn stress- or colonic irritation–induced visceral hyperal-esia. Both peripheral and spinal sites of action have beenroposed.29–31 The observation that mice with a selectiveeletion of the NK1R gene show a normal visceral no-iceptive response under acute stimulation, but fail toevelop hyperalgesia following colonic inflammation,onfirms a specific role of the SP/NK1R signaling systemn the regulation of visceral nociception in sensitizedtates.32

Taken together, the available data support the in-olvement of the SP/NK1R system in the transmissionnd processing of enhanced nociception from both theomatic and visceral fields. An up-regulation of thisignaling system is seen in human and animal models oftress sensitization6–8 and of inflammation-induced hy-eralgesia.33,34 Yet, the site and mechanism underlyinghe plasticity of this signaling system in the condition ofersistent visceral pain are poorly understood.In the present study, we tested the general hypothesis

hat the maintenance of sustained visceral hyperalgesia
ollowing repeated psychological stress observed in a s
ecently reported rodent model35 involves the up-regu-ation of the SP/NK1R system. Specifically, we testedhether chronic stress-induced visceral hyperalgesia is

bolished by an NK1R antagonist and whether thisyperalgesia is associated with up-regulation of the SP/K1R signaling system on visceral afferent pathways,

ither at the periphery and/or in the spinal cord. Weeport that visceral hyperalgesia in this model is abol-shed by spinal application of an NK1R antagonist andhat this response is associated with a selective up-egulation of the NK1R on superficial dorsal horn neu-ons.

Materials and Methods

Animals

Adult male Wistar rats (250–275 g) were purchasedrom Harlan (Indianapolis, IN). Animals were maintained on aormal light-dark cycle, housed in pairs or singly whenquipped with a chronic intrathecal catheter. They were pro-ided with food and water ad libitum. All protocols werepproved by the Institutional Animal Care and Use Commit-ee at the VA Greater Los Angeles Healthcare System (Losngeles, CA).

Surgery

Implantation of electromyographic electrodes.dult male rats were deeply anesthetized with pentobarbital

odium (45 mg/kg, Nembutal; Abbott Laboratories, Northhicago, IL) administered intraperitoneally (IP). Electrodes

Teflon-coated stainless steel wire; AstraZeneca, Mölndal, Swe-en) were stitched into the external oblique musculature, justuperior to the inguinal ligament, for electromyographicEMG) recordings as previously described.36 Electrode leadsere then tunneled subcutaneously and externalized laterally

or future access. Wounds were closed in layers with 4-0 silk.ollowing surgery, rats were allowed to recover for 5–7 days.ounds were tested for tenderness to ensure complete recov-

ry from surgery before testing.Implantation of chronic intrathecal catheter.

ats were deeply anesthetized with pentobarbital sodium45 mg/kg, Nembutal; Abbott Laboratories) administeredP. Animals were placed in a stereotaxic frame, and a smallncision was made at the back prone of the neck. A smalluncture was made in the atlanto-occipital membrane of theisterna magna, and a 32-gauge polyurethane catheter of.5 cm (ReCathCo, LLC, Allison Park, PA) was inserteduch that the caudal tip reached the lumbar enlargement ofhe spinal cord. The dead volume of the catheter was 10 �L.he rostral end of the catheter was exteriorized at the top of

he head, and sutures were used to secure the placement ofhe catheter and close the wound. The rats were allowed toecover from the surgery for 5–7 days. Rats exhibiting any
ign of neurologic or motor impairment, as evidenced by

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May 2006 CHRONIC STRESS–INDUCED VISCERAL HYPERALGESIA 1731

aralysis, abnormal gait, weight loss, or negligent groom-ng, were excluded from the study. Rats were housed sep-rately to ensure catheter patency. After completion of drugesting, the catheter position was verified in each animal byostmortem examination of the spinal cord.

Assessment of Visceromotor Response toColorectal Distention

The visceral stimulus used was distention of theescending colon and rectum using a well-established andalidated method for the evaluation and quantification ofisceral nociceptive responses.36 Briefly, under light halo-hane anesthesia, a flexible latex balloon (6 cm) was insertedntra-anally (after the distal part of the rectum was gentlyleared by massage) such that its end was 1 cm proximal tohe anus. Once recovered from anesthesia, animals equippedith the balloon were placed in a Plexiglas cylinder for 30inutes before the colorectal distention (CRD) procedureas initiated. The CRD procedure consisted of 2 series ofhasic CRDs to constant pressures of 10, 20, 40, and 60m Hg (20-second duration; 4-minute interstimulus in-

erval). The visceromotor response (VMR) to CRD wasuantified by measuring EMG activity in the externalblique musculature. EMG activity was recorded 20 sec-nds before (baseline), 20 seconds during, and 20 secondsfter termination of CRD. The EMG activity was rectified,nd the increase in the area under the curve of EMGmplitude during CRD over the baseline period beforeRD was recorded as the response. In the following text, wese the term EMG referring to the VMR to CRD. Animalshowing an EMG signal/noise ratio �0.05 were excludedrom the study.

Water Avoidance Stress Protocol

The test apparatus consisted of a Plexiglas tank (25 �5 � 45 cm) with a block (8 � 8 � 10 cm) affixed to theenter of the floor. The tank was filled with fresh water at roomemperature (25°C) to within 1 cm of the top of the block. Thenimals were placed on the block for 1 hour daily for 10onsecutive days corresponding to the chronic stress protocolwater avoidance [WA]). The sham WA stress consisted oflacing the rats similarly for 1 hour daily for 10 days on theame platform in a waterless container. This well-characterizedest represents a potent psychological stressor with large ele-ations of adrenocorticotropic hormone and corticosterone lev-ls within 30 minutes.37

Detection of NK1R and SP Messenger RNAExpression

Semiquantitative polymerase chain reaction foretection of NK1R messenger RNA in the colon. TotalNA was isolated by the TRIzol extraction method (Invitro-en Inc, Carlsbad, CA). RNA integrity was confirmed bylectrophoresis through a 1% agarose gel containing formal-ehyde. Complementary DNA was prepared from 1 �g of total
NA as previously described. Added in a ribonuclease-free w
ube were 2–5 �g RNA, 1 �L random primer (0.125 �g/�L),nd ribonuclease-free water to a final volume of 10 �L. Tubesere heated at 70°C for 2 minutes, put on ice, and then 4 �Lf ribonuclease-free water, 5 �L of 5� RT buffer, 2 �L of 10mol/L deoxynucleoside triphosphates, 2 �L of 0.1 mol/L

ithiothreitol, 1 �L of RNasin (40 U/�L; Promega, Pitts-urgh, PA), and 1 �L of Moloney murine leukemia viruseverse transcriptase (200 U/�L; Invitrogen Inc) were added.he RT reaction was performed at 37°C for 1 hour and 70°C

or 15 minutes, and then tubes were left on ice for polymerasehain reaction (PCR). The primers used for the PCR of theK1R gene were as follows: forward primer 1, 5= GACTC-TCTGACCGCTACCA 3=, reverse primer 2, 5= GGATT-CATTTCCAGCCCCT 3=. The PCR reaction contained9.75 �L of sterile water, 5 �L of 10� PCR buffer, 1 �L of0 mmol/L deoxynucleoside triphosphates, 1 �L of each for-ard and reverse primer, 1 �L of primer 2, 4 �L of 18Srimers/18S competitor (1:9 ratio; Ambion, Austin, TX), 0.25L of Taq DNA polymerase (50 �g/�L; Qiagen, Valencia,A), and 2 �L of the RT reaction mixture. The PCR condition

or coamplification of NK1R and 18S messenger RNAmRNA) was initial denaturation for 5 minutes at 94°C,ollowed by 3-step cycling: denaturation for 0.5 minutes at4°C, annealing for 0.5 minutes at 58°C, and extension for 0.5inutes at 72°C (45 cycles), followed by final extension for 7inutes at 72°C. Products were resolved by 1.2% agarose.uantification of the reverse-transcription (RT)-PCR bandsas performed using a phosphorimager (Quantity one Gel docrogram; Bio-Rad, Hercules, CA) and expressed the amount ofK1R PCR product relative to that of the 18S PCR product.

Real-time RT-PCR for detection of SP mRNA (pre-rotachykinin A) and NK1R mRNA in spinal cord andorsal root ganglia. Immediately after collection, samples oforsal root ganglia (DRG) (corresponding to the spinal level1–L2) and spinal cord (L1–L2) were stored in RNAlaterQiagen) at �80°C until RNA extraction. Total RNA wassolated using the RNeasy Mini Kit along with deoxyribonu-lease treatment (Qiagen). Using the Taq-Man One Step RT-CR reagents (Applied Biosystems, Foster City, CA), gene-pecific primers, and FAM-labeled probe (TaqMan Assay byemand; Applied Biosystems), 50 ng of RNA was subjected

o RT-PCR. The samples were run in duplicate in an ABI700 Sequence Detection System (Applied Biosystems), andhe values obtained (arbitrary mRNA units) were normalizedy TBP expression and compared between the WA group andham WA control. Detection of SP mRNA expression waserformed by measuring the mRNA for the gene PPT-A,rimary transcript for the synthesis of SP.

Detection of NK1R Protein Expression

Western blotting for NK1R in samples of distalolon and spinal cord. Immediately after collection, samplesere frozen on dry ice and stored at �80°C until they wererocessed. Proteins extracted from colonic or spinal cord sam-les of similar weight (200 mg) for each experimental group
ere subjected to sodium dodecyl sulfate/polyacrylamide gel

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lectrophoresis on 3%–8% gels and electrophoretically trans-erred to nitrocellulose membranes (Invitrogen, Inc). A posi-ive control (50-�g load) (catalog SC-2239; Santa Cruz Bio-echnology, Santa Cruz, CA) was processed simultaneously.he membranes were blocked for 1 hour in 5% nonfat dryilk in phosphate-buffered saline (PBS) and were incubatedith NK1R rabbit antiserum (catalog AB5060; Chemicon,emecula, CA) at a final dilution of 1:1000 overnight at 4°C.he membranes were then washed and incubated with horse-

adish peroxidase–conjugated anti-rabbit immunoglobulin Gt 1:10,000 dilution for 1 hour at room temperature. Controlncluded preabsorption of the diluted primary antisera witheceptor fragments (393KTMTESSSFYSNMLA407, 1 mg/mL)vernight at 4°C before incubation with the membrane. Im-unoreactive (IR) bands were detected using enhanced chemi-

uminescence reagents (Amersham Pharmacia, Piscataway,J). Autoradiograms were scanned using the GS-710 Cali-

rated Imaging Densitometer (Bio-Rad), and the labeledands were quantified using the Quantity software programBio-Rad).

Immunohistochemical characterization of NK1Rn samples of distal colon and spinal cord. On the day ofissue collection, rats were anesthetized with halothane anderfused intracardially with cold PBS and subsequently with% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4.he distal part of the colon and the L1–L2 segments of spinalord were removed and postfixed in the same fixative overnightnd were then transferred to 30% sucrose in 0.1 mol/L phos-hate buffer, pH 7.4, for cryoprotection. Cryostat sections onhe coronal plan (5 �m for the colon and 25 �m for the spinalord) were performed and processed for immunohistochemistryor NK1R. Sections were washed twice with PBS, followedwice with a solution of PBS, 0.3% Triton X-100, and 0.001%himerosal (PBS/Triton) containing 5% normal goat serumnd then incubated at room temperature overnight with theK1R primary antibodies. We used an NK1R rabbit anti-

erum (1:1000 dilution for the colon, 1:3000 dilution for thepinal cord) raised against amino acids 393–407 of the ratK1R (catalog AB5060; Chemicon). This antiserum has been

reviously characterized for immunohistochemistry and West-rn blot analysis.16 Sections were then washed 3 times withBS and incubated for 2 hours at room temperature with theecondary antibodies in PBS/Triton. Secondary antibodies wereoat anti-rabbit immunoglobulin G conjugated to Alexa 488uorophore (Molecular Probes, Eugene, OR). Sections werehen washed 4 more times with PBS and mounted in ProlongMolecular Probes).

Microscopy and image processing. Both colonicnd spinal cord preparations were analyzed with a Zeiss Ax-oplan 2 research microscope equipped with fluorescence andxiocam color digital camera system (Carl Zeiss Inc, Thorn-ood, NY). A Zeiss Plan-Apochromat 40� oil-immersionbjective (Carl Zeiss Inc) was used to collect images of colonicnd spinal cord sections. In addition, confocal images wereollected to illustrate NK1R-expressing neurons. Confocal im-
ges were acquired at UCLA’s Carol Moss Spivak Cell Imaging c
acility with a Leica TCS-SP confocal microscope (Wetzlar,ermany) as previously described.38–42 Images obtained with

he 20� objective consist of 2 optical sections 2.53 �m thickfull width half maximum) separated 2.48 �m. Images ob-ained with the 100� objective consist of 2 or 3 opticalections (full width half maximum of 0.62 �m) separated 0.57m. Images were processed with Adobe Photoshop 5.5 (Ken-esaw, GA), using the “curves” feature of the program todjust the contrast. Images were initially acquired at a digitalize of 1024 � 1024 pixels and were later cropped to theelevant part of the field. The pinhole was 1.0 Airy unit.ptical sections were averaged 4 times to reduce noise. Theumber of NK1R IR neurons was quantified using establishedrotocols38–40 with minor modifications.41,42 Briefly, we de-ermined the total number of NK1R IR neurons in the laminaeof the dorsal horn. We counted the number of NK1R IReurons in 5 sections from the L1–L2 segment of the spinalord from each rat. The person counting the neurons wasnaware of the prior treatment given to the animals.

Experimental Design

Visceral nociceptive response to chronic WAtress: effect of peripheral and spinal treatment with theK1R antagonist SR140333. A total of 8 groups of 6 ratsere included in this study. Animals were equipped with

lectrodes in the abdominal muscles for the EMG recording.fter placement of the electrodes, rats were allowed to recover

or 5–7 days. Acclimation to the experimental conditions waserformed for 3 days preceding the start of the experiment.ach day, animals were transported to the testing room andlaced for 30 minutes in the Plexiglas cylinders used for partialestraint during the CRD experiments.

On day 0, a baseline response to CRD was evaluatedCRD#1). From day 1 to day 10, rats were submitted daily toither 1-hour WA for the stress group or to 1-hour sham WAor the control group. The response to CRD was recorded again4 hours after the end of the last WA or sham WA session, onay 11 (CRD#2).The effect of the NK1R antagonist SR140333 (provided by

anofi Aventis, Montpellier, France) on the response to CRD atay 11 was tested according to the following protocol.Four groups of rats were used to test the effect of peripheral

njection of the NK1R antagonist. SR140333 (1 mg/kg) orehicle (2% dimethyl sulfoxide in 0.9% saline, 1 mL/kg) wasnjected IP 1 hour after the end of CRD#2. Then, the responseo CRD was again measured 1 hour after injection of theompound (CRD#3). SR140333 and vehicle were tested inoth chronically stressed and sham stressed animals. The dosef 1 mg/kg was selected based on published reports.29,43

Four different groups of rats were used to test the effect ofpinal injection of NK1R antagonist. At the time of surgicallacement of EMG electrodes, animals were equipped withntrathecal catheters implanted chronically. SR140333 (20g/kg) or vehicle (2% dimethyl sulfoxide in 0.9% saline) was

njected 1 hour after the end of CRD#2 via the intrathecal
atheter. Volume of injection was 6 �L followed by a 10-�L

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ush of vehicle. The response to CRD was measured again 30inutes after injection (CRD#3). SR140333 and vehicle were

ested in both chronically stressed and sham stressed animals.fter completion of drug testing, the catheter position waserified in each animal by postmortem examination of thepinal cord, and only animals with the appropriate catheterosition on the dorsal side of the spinal cord were included inhe study. The dose of 20 �g/kg was selected based on aublished report.44

Collection of colonic, spinal cord, and DRG sam-les for NK1R expression analysis. The different samplesollected for molecular analysis were taken from animals thatad not undergone surgery or CRD to avoid the potential biashat this procedure may induce regarding neurotransmitterroduction or release. This strategy permitted us to study theffect of chronic stress per se on NK1R and SP expression.

Two groups of 8 rats were used for the collection of freshamples to be processed for semiquantitative RT-PCR or real-ime RT-PCR and Western blotting. Animals were subjectedither to WA stress or sham WA 1 hour daily for 10 consec-tive days. On day 11, animals were killed, and samples ofistal colon, spinal cord, and DRG were quickly removed andhen placed either in RNAlater solution for RT-PCR experi-ents or frozen on dry ice for Western blotting analysis. Spinal

ord samples and DRG were collected from the lumbar level1–L2, corresponding to one of the segments that receivesrimary afferents from the colon. This specific segment wasreviously shown to play a role in persistent visceral pain andisceral hyperalgesia.45

Two separate groups of 8 rats were used to generate tissuesor immunohistochemical analysis of the colon and spinal cord.s previously described, samples of distal colon and spinal cordere collected after perfusion with fixative and processed forK1R immunolabeling.

Data Presentation and Statistical Analysis

To examine the pressure-response relationship, EMGmplitudes were normalized as percentage of the baselineesponse for the highest pressure (60 mm Hg) for each rat andveraged for each group of rats. This type of normalization hasenerally been used to account for individual variations of theMG signal.29 The effect of stress and/or pharmacologic treat-ent on the EMG response to CRD within one group of

nimals was analyzed by comparing the poststress or posttreat-ent measurements with the baseline or pretreatment values

t each distention pressure using repeated-measures 2-waynalysis of variance (ANOVA) followed by Bonferroni posttestomparisons. We have presented the data showing the EMGesponse at day 11 for rats treated with compound or vehiclexpressed as the mean change from baseline for differentressures of distention. This method of analysis has beenreviously validated in a similar model of EMG measurementn response to CRD.29 These data were analyzed using Student
test. i
Data for the expression of NK1Rs and SP obtained fromT-PCR, Western blot, and immunohistochemistry between

tressed and control groups were compared using an unpairedtest.

Results

Effect of Pharmacologic Blockade of NK1Rson Chronic WA Stress-Induced VisceralHyperalgesia

IP injection of the NK1R antagonist SR140333.here was a significant increase of the VMR following

epeated exposure to WA stress at day 11 (CRD#2)ompared with baseline (CRD#1). The increase wasignificant for the pressures of 40 and 60 mm Hg (P �05, repeated-measures ANOVA followed by Bonfer-oni posttest). One hour later, the effect of systemicpplication of the NK1R antagonist SR140333 (at thereviously established dose of 1 mg/kg27) or vehicleas tested on the VMR to CRD#3. As shown inigure 1A, SR140333 abolished the stress-inducedncrease of the VMR compared with vehicle (Student

test, comparison between groups treated withR140333 and vehicle). SR140333 application re-uced the EMG response to a level similar to baselinechange in EMG response after SR140333 over base-ine, �2.2 � 8.7 at 40 mm Hg and �8.0 � 10.6 at0 mm Hg), while no significant effect of vehiclepplication was observed (change in EMG responsefter vehicle over baseline, �51.2 � 18.2 at 40 mmg and �49.1 � 16 at 60 mm Hg).To determine the effect of the NK1R antagonist in

ontrol conditions, we tested the response to SR140333r vehicle injection in animals previously subjected toepeated sham WA stress. Compared with baseline, re-eated exposure to sham WA stress had no significantffect on the VMR to CRD. As shown in Figure 1B,njection of vehicle or SR140333 did not change theesponse to CRD.

Finally, there was no difference in the EMG responseo CRD#3 compared with CRD#2 after vehicle injectionn the WA group or after vehicle or SR140333 injectionn the sham WA group, indicating that repeated CRD atay 11 does not affect the VMR.

Intrathecal injection of the NK1R antagonistR140333. Consistent with results from previous ex-eriments, repeated exposure to WA stress resulted in anncreased VMR to CRD for the pressures of 40 and 60m Hg at day 11 compared with baseline (P � .05,

epeated-measures ANOVA followed by Bonferroniosttest). While vehicle injection had no effect on the
ncreased EMG response at day 11 (change in EMG

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esponse after vehicle over baseline, �38.3 � 23.1 at 40m Hg and �113.1 � 37.1 at 60 mm Hg), intrathecal

R140333 (20 �g/kg) abolished the enhanced VMRchange in EMG response after vehicle over baseline,

16.7 � 15.3 at 40 mm Hg and �1.9 � 9.6 at 60 mmg, Student t test, comparison between groups treatedith SR14033 and vehicle; Figure 2A).The change in EMG response at the highest pressure

f distention (60 mm Hg) in stressed rats injected withehicle intrathecally was 113.13 � 37.14, whereas it wasnly 49.14 � 16.10 in stressed rats injected with vehicleP. However, this difference did not reach statisticalignificance (P � .1767) and illustrates the interanimalariability of EMG response after chronic stress as pre-iously discussed in a recent report.35

igure 1. Effect of peripheral administration of the NK1R antagonistR140333 (1 mg/kg IP) on the VMR to CRD (day 11). (A) EMGmplitude expressed as mean change from baseline after treatmentith vehicle or SR140333 in rats previously exposed to repeated WAtress. IP injection of SR140333 abolished the WA stress-inducedncrease in the VMR to CRD at pressures of 40 and 60 mm Hg. (B)MG amplitude expressed as mean change from baseline after treat-ent with vehicle or SR140333 in rats previously exposed to re-eated sham WA stress. SR140333 did not affect the EMG responsefter chronic sham WA stress compared with vehicle. Data are ex-ressed as mean � SE, n � 6 in each group, *P � .05 significantly

hifferent compared with vehicle, Student t test.

As shown in Figure 2B, in sham WA condition,either vehicle nor intrathecal SR140333 affected theMG response to CRD for any pressure of distention.

Effect of Chronic WA Stress on NK1R andSP Expression

Expression of NK1R and SP in the spinal cord. Theffect of chronic WA stress on NK1R and SP expressionn the spinal cord was assessed using real-time quantita-ive RT-PCR, Western blotting analysis, and immuno-

igure 2. Effect of central administration of the NK1R antagonistR140333 (20 �g/kg intrathecally) on the VMR to CRD (day 11). (A)MG amplitude expressed as mean change from baseline after treat-ent with vehicle or SR140333 in rats previously exposed to re-eated WA stress. Intrathecal injection of SR140333 abolished thehronic stress-enhanced VMR to CRD compared with vehicle at theressures of distention of 40 and 60 mm Hg. (B) EMG amplitudexpressed as mean change from baseline after treatment with vehicler SR140333 in rats previously exposed to chronic sham WA stress.R140333 did not affect the EMG response after chronic sham WAtress compared with vehicle. Data are expressed as mean � SE, n

6 in each group, *P � .05 significantly different compared withehicle, Student t test.

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Real-time quantitative RT-PCR for NK1R mRNA in1–L2 spinal cord samples. NK1R mRNA was expressed inhe L1–L2 segment of the spinal cord, and the authenticityf the PCR product was verified by sequencing analysis. Weound enhanced expression for NK1R mRNA in samplesrom WA stressed animals compared with the sham WAroup. Relative expression of NK1R mRNA to glyceralde-yde-3-phosphate dehydrogenase was 2.5 � 0.3 in the

igure 3. Expression of NK1Rs in L1–L2 spinal cord segments follow-ng repeated WA stress. (A) Levels of mRNA for NK1R measured byeal-time quantitative RT-PCR. We observed a significantly higher levelf NK1R mRNA in samples from chronic WA stress rats compared withontrol. Data are expressed as the relative NK1R PCR product to thatf the housekeeping gene GAPDH, mean � SEM, n � 8 in each group,P � .05, significantly different from sham WA, Student t test. (B)evels of NK1R protein measured by Western blotting. A significantncrease in the level of NK1R proteins in samples from chronic WAtress rats compared with control was observed. Data are expresseds normalized optical density, mean � SEM, n � 8 in each group, *P

.05, significantly different from sham WA, Student t test. (C)epresentative Western blotting for NK1R in spinal cord extractshowing a prominent band at �65 kilodaltons.

ham WA group and 11.3 � 2.3 in the WA group (ex- (

ressed as arbitrary units, P � .0053, Student t test; FigureA).

Western blotting of NK1R protein in samples of L1–L2pinal cord. Western blot analysis in WA and controlham groups revealed a �65-kilodalton band corre-ponding to the NK1R protein (Figure 3C). The authen-icity of the NK1R protein was verified by simultaneousigration of a positive control for NK1R protein anti-

erum (cell lysate; Santa Cruz Biotechnology) at the sameevel. The specificity of the antibody immunostainingas confirmed by significant reduction of the intensity of

he band in samples blotted with the antibody pread-orbed with an excess of receptor fragment used forhe antibody production 393KTMTESSSFYSNMLA407.uantitative densitometry of the Western blots showedsignificant increase of 80% in NK1R protein expression

n the WA group compared with the sham WA group (P.0004, Student t test; Figure 3B). A representative

llustration of the reduced signal intensity after preab-orption with peptide fragment in both spinal cord ex-racts and a positive control load is shown in Figure 4.

Immunohistochemical localization of NK1Rs in spinalord samples. In both control and stress conditions, aattern of spinal NK1R immunoreactivity consistentith previous reports on dorsal horn NK1R localizationas observed.41 NK1R IR neurons were localized in

aminae I, V, and VI and around the central canal (lam-nae X), with the densest staining observed in lamina IFigure 5A and B). In all regions, NK1R immunoreac-ivity was predominant on the plasma membrane, ashown in confocal pictures of spinal cord sections fromoth control and stressed animals. In some neurons fromoth groups, a few endosomes were found near the cellurface but did not meet our criteria for NK1R internal-zed neurons. In laminae I, a quantifiable increase in the

igure 4. Representative illustration of Western blots showing theigration of spinal cord extracts incubated either with the primarynti-NK1R serum alone (A1) or the primary anti-NK1R serum preab-orbed with the receptor peptide fragment (B1); preincubation withhe antigenic receptor fragment reduces the intensity of stainingompared with A1. Similar results were observed with a positiveontrol load migrating at the same level (�65 kilodaltons); preabsorp-ion with the receptor peptide fragment attenuated the intensity oftaining (B2) compared with incubation with primary antibody alone
A2).

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umber of NK1R IR neurons in spinal cord samples fromtressed rats compared with control was evident (18.7 �.7 vs 12.6 � 0.58, P � .0052, Student t test; Figure 6).

Real-time quantitative RT-PCR for SP mRNA in1–L2 spinal cord samples. We found detectable expres-ion of preprotachykinin A (PPT-A) mRNA, the primaryranscript for the synthesis of SP, in the spinal cordamples collected from the L1–L2 level (data not shown).here was no significant difference in the relative levelsf PPT-A mRNA to glyceraldehyde-3-phosphate dehy-rogenase, measured in samples from stressed animalsompared with control (9.9 � 1.2 and 9.5 � 0.8 arbi-

igure 5. Representative confocal images of NK1R IR neurons in theorsal horn of the L1–L2 spinal segment of rats subjected to (A)hronic sham WA stress or (B) chronic WA stress. Images in the mainanels were taken with a 20� objective (scale bars � 50 �m) andonsist of 2 optical sections 2.53 �m thick separated 2.48 �m.mages in the insets were taken with a 100� objective (scale bars �0 �m) and consist of 2 or 3 optical sections 0.62 �m thick sepa-ated 0.57 �m. The densest NK1R immunostaining is found in lami-ae I of the dorsal horn. In both sham WA and WA stress conditions,

mmunoreactivity was predominant on the plasma membrane.

rary units, respectively). .

Expression of NK1R and SP in DRG neurons. DRGeurons showed detectable levels of NK1R mRNA ex-ression (data not shown). Quantitative analysis ofK1R mRNA levels in the DRG did not show any

ignificant change in the relative abundance betweentressed and sham-stressed animals (8.9 � 2.8 vs 9.8 �.6 arbitrary units, respectively).There was detectable expression of PPT-A mRNA in

he DRG collected from the L1–L2 level (data nothown). However, there was no significant difference inhe relative levels of PPT-A mRNA measured in samplesrom stressed animals compared with controls (2.6 � 0.6nd 2.5 � 0.2 arbitrary units, respectively). Because ofhe lack of a difference between stress and control con-itions, the proteins were not further quantified.

Expression of NK1R in the distal colon. The effectf chronic WA stress on NK1R expression in full-hickness colon tissues was assessed using semiquan-itative RT-PCR, Western blotting analysis, andmmunohistochemistry.

Semiquantitative RT-PCR for colonic NK1R. NK1RRNA was expressed in the distal colon, and the au-

henticity of the PCR product was verified by sequencingnalysis. We found significantly reduced expression forK1R mRNA in samples from chronically stressed an-

mals compared with control. A semiquantitative analy-is of NK1R mRNA levels showed that WA stress re-ulted in a significant decrease in the relative abundancef colonic NK1R mRNA compared with sham WA stress1.11 � 0.13 in sham WA, 0.75 � 0.08 in WA,

igure 6. Quantification of NK1R IR neurons in the laminae I of theorsal spinal cord from rats previously subjected to chronic WA stressr control sham WA stress. There are an increased number of NK1R-xpressing neurons in the spinal segment from stressed rats com-ared with control. Results are expressed as the mean number ofK1R IR-positive cells in the laminae I of one side of the dorsal horn

rom 5 sections/rat; mean � SEM, n � 8 rats in each group, *P �
05, significantly different from sham WA, Student t test.

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xpressed as normalized arbitrary units, P � .05, Studenttest; Figure 7A).

NK1R protein expression in distal colon. Westernlot analysis in control and WA stress groups revealed a65-kilodalton band corresponding to the NK1R pro-

ein (Figure 7C). As previously described for the spinalord samples, the authenticity of the NK1R protein waserified by simultaneous migration of a positive controload (Santa Cruz Biotechnology, Santa Cruz, CA) at the

igure 7. Expression of NK1Rs in the distal colon. (A) Levels of mRNAor NK1Rs measured by semiquantitative RT-PCR. A significant reduc-ion in the level of NK1R mRNA was observed in samples from chronicA stress rats compared with controls. Data are expressed as themount of NK1R PCR product relative to that of 18S PCR productarbitrary units, mean � SEM, n � 8 in each group, *P � .05,ignificantly different from sham WA, Student t test). (B) Levels ofK1R protein measured by Western blotting. A significant reduction in

he level of NK1R proteins in samples from chronic WA stress ratsompared with controls was observed. Data are expressed as nor-alized optical density, mean � SEM, n � 8 in each group, *P � .05,ignificantly different from sham WA, Student t test. (C) Representa-ive Western blotting for NK1R in colon extracts shows a prominent

jand at �65 kilodaltons.

ame level. The specificity of the antibody immunostain-ng was confirmed by reduction of the intensity of theand in samples blotted with the antibody preadsorbedith an excess of receptor fragment used for the antibodyroduction 393KTMTESSSFYSNMLA407. Optical den-ity analysis of blots showed significantly less expressionf the NK1R in the stress group compared with controlP � .0134, Student t test); a 31% reduction was ob-erved in the WA group compared with control (FigureB).

Immunohistochemical localization of NK1R in colonicamples. The distribution of NK1R in the rat colon wasimilar to previously reported results,16,46 with promi-ent localization in myenteric and submucosal neurons,uscle cells, and epithelial cells. The NK1R immunore-

ctivity was localized to the plasma membrane of neuronsocated in the myenteric plexus in samples from bothontrol and stressed animals, with no evidence for recep-or endocytosis. There was no detectable difference in theistribution and intensity of NK1R in WA and shamA specimens. NK1R immunostaining was abolished

y preadsorption of the antibody with an excess of theeptide antigen, confirming specificity of NK1R immu-oreactivity (not shown).

Discussion

The present study expands on previous observa-ions that repeated psychological stress in male Wistarats results in a phenotype characterized by increasedutonomic responsiveness, anxiety-like behavior, andustained visceral hyperalgesia, consistent with stressensitization.35 Our results point to a major role for theP/NK1R signaling system in mediating the visceralyperalgesia component of this model, because thetress-induced exaggerated VMR to CRD was abolishedy intrathecal administration of a selective NK1R antag-nist and increased expression of NK1Rs was shown athe spinal cord level. The absence of peripheral up-egulation of the SP/NK1R signaling system distin-uishes this model from models of intestinal inflamma-ion where peripheral up-regulation of this system haseen reported.47,48 This, to our knowledge, represents therst evidence of increased expression of NK1R located onpinal neurons of the superficial dorsal horn in a model ofustained visceral hyperalgesia induced by chronic psy-hological stress.

Pharmacologic Characterization of the Roleof NK1R in Stress-Induced VisceralHyperalgesia

The selective NK1R antagonist SR140333, in-
ected via either the intraperitoneal or intrathecal

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outes, normalized the exaggerated VMR to CRDssociated with repeated WA stress. These findings,ogether with the lack of effect on baseline responseso CRD, support a predominant antihyperalgesic ef-ect of the NK1R antagonist in this model and suggest

stress-induced up-regulation of the SP/NK1R sig-aling system either on peripheral and/or central com-onents of visceral afferent pathways mediating vis-eral pain. Previous reports have shown an inhibitoryffect of different NK1R antagonists on visceral hy-eralgesia in several animal models of peripheral co-onic sensitization. For example, Okano et al30 showedhat intraduodenal administration of the NK1R an-agonists TAK-637 or CP99,994 inhibit the enhancedociceptive response to CRD in rabbits previouslyubjected to colonic irritation with acetic acid. Inuinea pigs sensitized by intracolonic administrationf acetic acid, central injection of TAK-637 abolishesisceral hyperalgesia.31 There is also evidence for a rolef NK1Rs in the mediation of visceral hyperalgesiabserved after psychological stressors, such as acuteartial restraint stress in guinea pigs,31 partial re-traint stress in rats,43 or acute WA stress in rats.29

owever, despite this converging evidence for in-olvement of the SP/NK1R system in the expression ofisceral hyperalgesia following physical or psycholog-cal stress, the sites of NK1R modulation have nevereen identified. The available data suggest that, de-ending on the nature of the sensitization paradigm,oth peripheral and central sites may be involved.30,31

The NK1R antagonist SR140333 used in theresent study has been characterized as a highly selec-ive and functional antagonist for the rat NK1R,49,50

hich shows poor ability to penetrate into the centralervous system under basal conditions.50 While theseroperties may suggest that the inhibitory effect ofR140333 injected IP might be mediated via anction on peripheral NK1Rs, several studies havehown increased blood-brain barrier permeability inats following stressful stimulation.51,52 Our findinghat intrathecal administration of SR140333 in chron-cally stressed rats normalized the enhanced responseo CRD supports the critical contribution of spinalK1Rs to the enhanced nociceptive response and is

onsistent with a recent report showing an inhibitoryffect of intrathecally applied TAK-637 on visceralyperalgesia in the guinea pig.31 However, the possi-ility that SR140333 injected into the spinal cord intressed animals may have leaked into the systemicirculation, targeting peripheral receptors, cannot be
xcluded. e
Stress-Induced Modulation of SP/NK1RExpression in the Spinal Cord and Colon

We found increased NK1R mRNA and proteinxpression in the spinal cord in samples from stressed ratsompared with controls. Comparable increased expres-ion of NK1R in the spinal cord has been previouslyeported in models of peripheral inflammation, such asladder irritation,53 or nerve injury associated with per-istent pain.54 However, the mechanism(s) underlyingncreased spinal NK1R expression observed in associationith peripheral inflammation is incompletely under-

tood. In vitro studies in different cell types, includingpinal neurons, have implicated several mediators andelated intracellular signaling molecules, including theensory neuropeptide calcitonin gene-related peptide (viactivation of the adenosine 3=,5=-cyclic monophosphateesponsive element),55 proinflammatory cytokines, inarticular interleukin-1 (via activation of the nuclearactor B pathway),20,54,56 or SP metabolites57 in NK1Rxpression.

The analysis of NK1R immunostaining showed thatnhanced protein expression corresponds to an in-reased number of neurons labeled for NK1Rs in theaminae I of the dorsal horn. In both control andtressed groups, NK1R distribution was more evidentn the cell surface, without evidence for significanteceptor internalization. Based on previous reportshat showed the positive relationship between spinalP release and NK1R internalization,58 the absence ofndosomes labeled for NK1R in the cytoplasm arguesgainst a significant release of spinal SP under base-ine, nondistended conditions in both stressed andonstressed animals. Thus, our data indicate thathronic stress triggers up-regulation of NK1R expres-ion on the superficial layer of dorsal horn neurons androvides indirect evidence that this phenomenon isot related to increased SP release from central termi-als of primary afferents. This hypothesis is supportedy the lack of significant difference in the relativeevels of PPT-A mRNA expression in DRG samplesrom stressed animals compared with controls, sug-esting that increased neuropeptide release from sen-itized primary afferents is not necessary for the ex-ression of visceral hyperalgesia in this model. Thesendings contrast previous studies showing a promi-ent role of spinal release of SP (and glutamate) fromhe central terminals of primary afferent fibers in theevelopment of spinal sensitization (also called centralensitization), associated with peripheral injury or in-ammation and persistent pain.59 However, we cannot
xclude the possibility of a transient spinal SP release

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rom primary afferents that occurs early during theourse of the 10 days of stress and thereby contributeso the development of the central sensitization pro-ess.

In the gut, NK1Rs are located on epithelial cells,mooth muscle cells, myenteric neurons, interstitialells of Cajal, and different types of inflammatoryells.46,48 Up-regulation of NK1Rs on a variety ofhese cell types has been characterized in clinicalnflammatory conditions60 – 62 and in several forms ofxperimental intestinal inflammation.17,47,63 SP andts natural analogue, hemokinin, are produced at theites of inflammation and act through the NK1Rxpressed on T cells, macrophages, and dendritic cells,eading to the production of mediators such as tumorecrosis factor �17,64 and interferon gamma65 and am-lification of the Th1 response.66 Patients with ulcer-tive colitis and Crohn’s disease have increased NK1Rxpression on epithelial cells lining the mucosal sur-ace and on endothelial cells of capillaries, venules,nd colonic lymphoid aggregates.60 – 62 In rats, NK1Rxpression is increased in intestinal epithelial cells andn cells of the lamina propria in a model of enteritisnduced by Clostridium difficile toxin A.47 It is nowell established that NK1R and SP (as well as its

nalogue hemokinin) participate in many aspects ofhe neuroimmune response triggered by intestinal in-ection or inflammation, and increasing evidencehows that proinflammatory cytokines play a signifi-ant role in the regulation of NK1R expression.56 Forxample, it has previously been shown that the proin-ammatory cytokine interleukin-1, via activation ofhe transcription factor nuclear factor B, can stimu-ate NK1R expression in human THP-1 monocytes.56

oreover, we have shown that the model of chronicA stress is associated with signs of mucosal immune

ctivation in the colon characterized by an increasedumber of mucosal mast cells and increased expressionf mRNA for the cytokines interleukin-1 and inter-eron gamma.35 Based on these data, together with theeport by Soderholm et al67 that a similar chronictress paradigm in rats induces changes in gut perme-bility and signs of mucosal immune activation, oneight expect an up-regulation of the SP/NK1 system

n the colon of chronically stressed animals. However,ur semiquantitative RT-PCR and Western blot anal-ses showed decreased NK1R expression at the mRNAnd protein levels in stressed rats compared withontrol. Consistent with prior reports,46 we foundrominent localization of NK1R immunostaining inyenteric and submucosal neurons, muscle cells, and

pithelial cells of the rat colon but no difference in the t

attern of distribution in NK1R-positive cells be-ween stressed and control rats. The mechanism(s)nderlying the observed down-regulation of NK1Rs inhe colon is unclear but may be related to other aspectsf general stress sensitization in this model. For ex-mple, Ihara et al suggested that, in pancreatic acinarells, altered outputs of the autonomic nervous systemr the hypothalamic-pituitary-adrenal axis are likelyo modulate gut immune function, thereby regulatingK1R expression via glucocorticoids.68

Summary and Conclusions

The findings from the present study show anp-regulation of NK1R on spinal neurons in a model ofhronic WA stress associated with sustained visceralyperalgesia. The pharmacologic efficacy of a spinalreatment with an NK1R antagonist to abolish the en-anced visceral response in this model suggests an im-ortant contribution of the spinal NK1R-expressing neu-ons in visceral hyperalgesia. The lack of enhanced SPene expression in DRG or enhanced SP spinal releaseuggests that increased expression of NK1R and theaintenance of sustained visceral hyperalgesia is not

ependent on ongoing input from sensitized peripheralrimary afferents. When viewed together with our pre-ious report35 indicating mild colonic immune activa-ion after chronic WA stress exposure, our findings in-icate that these subtle mucosal changes are notufficient to stimulate increased expression of SP (andresumably calcitonin gene-related peptide) and theirespective receptors in the periphery.

Our results address the role of the SP/NK1R signalingystem in the maintenance of sustained stress-inducedisceral hyperalgesia. We cannot rule out that an earlier,ransient peak of stress-induced immune activation dur-ng the 10-day WA stress paradigm occurs and that thiseak is essential for initiating a long-lasting increasedxpression of the NK1R on dorsal horn cells. However,nce established, the maintenance of the increased ex-ression of spinal NK1R, and thus of the visceral hyper-lgesia, may be driven primarily by descending input tohe dorsal horn from the brainstem.25,26 For example, apinal-bulbo-spinal loop has been proposed in rats fol-owing peripheral tissue irritation by which increasedK1Rs expressed on superficial dorsal horn neurons drive

scending projections to pontine serotonergic regions,hich in turn send descending projections to the spinal

ord.25 These serotonergic projections mediate a facilita-ory effect on neurons in superficial and deeper layers of
he dorsal horn, which is mediated by a 5-HT3 recep-

te

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or.69 Future studies are needed to address these hypoth-ses.

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bacterial adherence and initiates mucosal inflammation in ratintestine. Gastroenterology 2002;123:1099–1108.

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Received June 21, 2005. Accepted January 11, 2006.Address requests for reprints to: Emeran A. Mayer, MD, Center

or Neurovisceral Sciences and Women’s Health, CURE Building 2

15, Room 223, VAGLAHS, 11301 Wilshire Boulevard, Los Angeles,alifornia 90073. e-mail: [email protected]; fax: (310) 794-2864.

Supported by National Institutes of Health grants P50 DK64539to E.A.M.), R24 AT00281 (to E.A.M.), DK47343 (to C.P.), DK41301orphology and Imaging Core (to C.S.) and Antibody Core (to G.O.),01 DK57037 (to C.S.), and DK54155 (to C.S.); a Fellowship Award

rom the Crohn’s and Colitis Foundation (to S.S.); GlaxoSmithKlineNeurology and GI Centre of Excellence for Drug Discovery, Harlow,ngland); and R21 DK071767-01 (to S.B.).Presented in part at the 2005 annual meeting of the American

astroenterological Association in abstract form (Gastroenterology
005;128:A-494).

The Role of Neurokinin 1 Receptors in the Maintenance of Visceral Hyperalgesia Induced by Repeated...

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