Mast Cell-Derived Histamine Mediates Cystitis Pain
Charles N. Rudick1, Paul J. Bryce3, Laura A. Guichelaar1, Ruth E. Berry1, David J. Klumpp1,2*
1Department of Urology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America, 2Department of Microbiology-Immunology,
Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America, 3Division of Allergy-Immunology, Feinberg School of Medicine,
Northwestern University, Chicago, Illinois, United States of America
Background: Mast cells trigger inflammation that is associated with local pain, but the mechanisms mediating pain are
unclear. Interstitial cystitis (IC) is a bladder disease that causes debilitating pelvic pain of unknown origin and without
consistent inflammation, but IC symptoms correlate with elevated bladder lamina propria mast cell counts. We
hypothesized that mast cells mediate pelvic pain directly and examined pain behavior using a murine model that
recapitulates key aspects of IC.
Methods and Findings: Infection of mice with pseudorabies virus (PRV) induces a neurogenic cystitis associated with lamina
propria mast cell accumulation dependent upon tumor necrosis factor alpha (TNF), TNF-mediated bladder barrier
dysfunction, and pelvic pain behavior, but the molecular basis for pelvic pain is unknown. In this study, both PRV-induced
pelvic pain and bladder pathophysiology were abrogated in mast cell-deficient mice but were restored by reconstitution
with wild type bone marrow. Pelvic pain developed normally in TNF- and TNF receptor-deficient mice, while bladder
pathophysiology was abrogated. Conversely, genetic or pharmacologic disruption of histamine receptor H1R or H2R
attenuated pelvic pain without altering pathophysiology.
Conclusions: These data demonstrate that mast cells promote cystitis pain and bladder pathophysiology through the
separable actions of histamine and TNF, respectively. Therefore, pain is independent of pathology and inflammation, and
histamine receptors represent direct therapeutic targets for pain in IC and other chronic pain conditions.
Citation: Rudick CN, Bryce PJ, Guichelaar LA, Berry RE, Klumpp DJ (2008) Mast Cell-Derived Histamine Mediates Cystitis Pain. PLoS ONE 3(5): e2096. doi:10.1371/
Editor: Jacques Zimmer, Centre de Recherche Public-Sante ´, Luxembourg
Received March 12, 2008; Accepted March 18, 2008; Published May 7, 2008
Copyright: ? 2008 Rudick et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIDDK R01DK066112 (D.J.K.) and T32DK062716-05 (C.N.R.).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
IC is a chronic bladder inflammatory disease with unknown
etiology that afflicts as many as 1 million patients in the United
States. IC is associated with severe pelvic pain and voiding
dysfunction that includes urinary frequency, urgency, and nocturia
[1,2,3]. Clinical studies demonstrate elevated mast cell numbers in
the lamina propria of IC bladder biopsies, and the partial efficacy
of neuromodulatory therapies suggests neural-immune interac-
tions play a role in IC pathogenesis ([4,5] and reviewed in ).
Mast cells contain preformed stores of immune mediators, such as
histamine and TNF, and can be activated to release these stores by
neurotransmitters such as substance P. These observations have
suggested a central role for mast cells in IC pathogenesis whereby
activation of bladder-associated circuits in the central nervous
system (CNS) initiates substance P release by peripheral nerves in
the bladder that then promotes substance P-mediated mast cell
activation . This mast cell activation, in turn, is postulated to
induce bladder inflammation by acting on urothelium, the
epithelium that lines the bladder. In support of this hypothesis,
murine cystitis models have demonstrated a requirement for mast
cells and substance P receptors in bladder inflammation [7,8].
Also, increased levels of urinary histamine metabolites have been
detected in IC patient urines, and accumulation of lamina propria
mast cells is correlated with IC symptoms [4,5,9,10]. However,
other inflammatory markers are inconsistent features of IC, and
the basis of pelvic pain is unclear. Here we examine mechanisms
of pelvic pain in an established murine model that recapitulates
key aspects of IC, including lamina propria mast cell accumulation
and pelvic pain [11,12,13].
The attenuated Bartha’s strain of pseudorabies virus (PRV) is an
a-herpesvirus that is taken up by neurons and undergoes
retrograde transport and viral replication within the CNS. PRV
was originally shown to cause cystitis in rats when injected into the
tailbase abductor caudalis dorsalis muscle and taken up by motor
neurons. PRV-induced cystitis is a neurally mediated event
triggered by viral action in the CNS, and the cystitis is associated
with bladder mast cell activation, even though Bartha’s PRV is
[11,13,14,15,16]. In mice, PRV causes urothelial expression of
RANTES (Re-Activated in Normal, T cell-Expressed, Secreted), a
chemokine known to promote mast cell trafficking [11,12].
RANTES drives mast cell accumulation in the lamina propria
that juxtaposes activated mast cells with the urothelium, and the
proximity of mast cells to the urothelium induces mast cell-
dependent, TNF-dependent bladder pathophysiology. This blad-
der pathophysiology includes the formation of apoptotic lesions in
the urothelium and a marked loss of trans-epithelial resistance
(TER) that is normally characteristic of urothelial barrier function
in the intact bladder [12,17]. Thus, the pathophysiology of murine
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PRV cystitis is consistent with human IC where the presence of
urothelial lesions in patient biopsies correlates with IC symptoms,
and many IC patients are exquisitely sensitive to instillation of
nerve-depolarizing concentrations of KCl into the bladder, a
finding that suggests a loss of barrier function .
Murine PRV cystitis was also recently shown to induce bladder-
associated pain specific to the pelvic region in female C57BL6/J
mice . Although inflammation was restricted to the bladder, the
bladder-associated pelvic pain was blocked by instillation of 2%
lidocaine into either the bladder or colon, indicating modulation of
pelvic pain responses through organ crosstalk by visceral inputs
that mast cells mediate the bladder pathophysiology of murine PRV
cystitis, we hypothesized that pelvic pain is also mediated by mast
cells. In this study, both pelvic pain and bladder pathophysiology
were dependent upon mast cells. TNF mediated cystitis pathophys-
iology, while pelvic pain developed independent of TNF. Converse-
ly, genetic or pharmacologic disruption of histamine receptors H1R
or H2R attenuated pelvic pain without altering pathophysiology.
These data demonstrate that cystitis pain and bladder pathophys-
iology are dependent on mast cells through the independent actions
of histamine and TNF, respectively, and identify histamine receptors
as therapeutic targets for direct intervention in pelvic pain of IC.
PRV induces pelvic pain
Pain originating from a visceral organ is typically referred to a
corresponding ‘‘dermatome’’ on the skin that shares spinal
innervation with the given visceral organ . Therefore, to
quantify bladder-associated pelvic pain, we assessed tactile
sensitivity of the pelvic region in response to stimulation by von
Frey filaments [13,20]. Mechanical stimulation of the pelvic area
of female C57BL/6J (B6) mice evoked baseline responses (pelvic
withdrawal, jumping, or pelvic licking/scratching) where the
percentage of responses increased as a function of force applied
using graded filaments (Fig. 1A). Following PRV infection, mice
exhibited a progressively enhanced sensitivity to pelvic stimulation
consistent with the development of pelvic pain that became
significant by post-infection day 2 (PID 2; Fig. 1C, P,0.05), but
pain responses did not develop in sham-infected mice (Fig. 1B).
These data indicate PRV-induced pelvic tactile hypersensitivity, or
‘‘allodynia.’’ To assess the specificity of PRV-induced tactile
allodynia, we also quantified the 50% threshold sensitivity in the
paw [21,22]. PRV induced no changes in tactile sensitivity of the
plantar region of the hind paw of any groups tested (Table S1).
These data indicate that PRV induces a progressive pain specific
to the pelvic region, consistent with previous observations .
To understand the basis of bladder-associated pain, we next
examined nerve fibers in the mouse bladder. Since C fibers are the
nociceptors associated with chronic pain, bladder sections were
stained with isolectin B4, a marker for C fibers that stains most
bladder nociceptor afferents [23,24,25]. In addition to labeling of
the urothelium, a tissue layer heavily decorated with carbohydrate
structures, isolectin B4 also labeled fibers that were evident in the
lamina propria (Figs. 1D and E and Movie S1). Image analysis of
isolectin B4-positive fibers within the lamina propria revealed a
mean diameter of 1.08960.095 mm with a range of 0.728–
1.361 mm (n=7), consistent with the morphology of bladder C
fiber nociceptor axons in dorsal root ganglia . Since we
previously found that PRV infection induces mast cell accumu-
lation in the lamina propria [11,12,17], the location of C fibers
within the bladder lamina propria raises the possibility that mast
cell-C fiber interactions contribute to PRV-induced pelvic pain.
Pelvic pain requires mast cells
Since clinical studies suggest mast cell involvement in the
pathophysiology of IC, we examined PRV-induced tactile allodynia
in mast cell-deficient mice to directly investigate the role of mast cells
in pelvic pain. Mast-cell deficient KitW-sh/KitW-shmice have a non-
coding mutation in the gene encoding stem cell factor, c-kit, and
have proven valuable for examining the role of mast cells in disease
[26,27].Incontrasttothe development of pelvicpaininwildtypeB6
mice, PRV infection of mast cell-deficient KitW-sh/KitW-shmice failed
to induce enhanced pelvic responsiveness above baseline, suggesting
a role for mast cells in the development of pelvic pain (Fig. 2A).
Induction of pelvic pain by PRV was fully restored in KitW-sh/KitW-sh
mice where bladder mast cells were reconstituted by transfusion with
wild type B6 bone marrow (P,0.05) but not in KitW-sh/KitW-shmice
reconstituted with KitW-sh/KitW-shbone marrow (Figs. 2B and C,
respectively). Bladder mast cells were then quantified in toluidine
responses was indeed associated with reconstitution of bladder mast
cells (Fig. 2D).
Mast cells are not likely required for general pain responses
because KitW-sh/KitW-shmice exhibited normal sensitivity to
capsaicin administered to the colon (colonic capsaicin induced a
226.0666.2% increase in pelvic responses relative to baseline in
wild type B6 mice, and capsaicin induced a 235.7688.6% increase
in responses in KitW-sh/KitW-shmice; P.0.05). To exclude the
possibility that mast cell-deficiency altered pelvic pain responses by
acting on general physiology or behavior, we also quantified
normal behaviors during free roaming (Table S2). No significant
increase in response to PRV was detected in grooming, cage
crossing, or rearing in any treatment group or genetic background
throughout the observation period, suggesting that the role of mast
cells in pelvic pain is not due to generalized alterations in behavior
that might be manifested as enhanced responsiveness to pelvic
tactile stimuli. Similarly, an absence of weight changes or altered
development of end-stage CNS disease in any experimental group
indicates that mast cell status is not associated with dramatic
changes in gross physiology or susceptibility to PRV infection
(Table S3 and data not shown). Therefore, these data suggest a
specific requirement for mast cells in the development of bladder-
associated pelvic pain.
TNF does not modulate pelvic pain
Our previous studies demonstrated that PRV-induced bladder
pathophysiology was dependent upon TNF [12,17], so we
examined the role of TNF in pelvic pain responses by quantifying
pelvic tactile sensitivity in TNF2/2and TNFR1/22/2mice
following PRV infection. Responses to mechanical stimulation of
the pelvic area were significantly greater at PID 2-4 than baseline
responses in both TNF2/2and TNFR1/22/2mice (Figs. 3A and
B, respectively; P,0.05). Furthermore, the increase from baseline
response of either TNF2/2or TNFR1/22/2mice infected with
PRV was indistinguishable from PRV-infected wild-type mice
(Fig. 3C). These data suggest that a non-TNF mast cell factor(s)
mediates pelvic pain induced by PRV.
Histamine receptors 1 and 2 modulate pelvic pain
A role for histamine and histamine receptors in pain responses
has been documented in both humans and animal models
[28,29,30,31]. However, histamine can be derived from various
cell types, including mast cells, basophils, and histaminergic
neurons. More recently, inflammatory cells such as neutrophils
and dendritic cells have been shown to produce histamine [32,33].
The specific cell type regulating histamine-mediated pain is
unknown. To determine whether mast cell-derived histamine
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Figure 1. PRV induces pelvic pain. A) Tactile sensitivity was assessed by stimulating the suprapubic and hindpaw skin with von Frey filaments
(left panel) of a mouse placed into a chamber with a mesh floor (right panel). Referred visceral hyperalgesia was measured in female mice as
responses to mechanical stimulation of the pelvic region using von Frey filaments of 5 calibrated forces. Data represent the mean response to ten
successive applications for each fiber 6SEM at baseline and at PID 1, 2, 3, and 4 following infection with 2.36106pfu Bartha’s PRV in the tailbase
muscle. B) Responses to pelvic stimulation of C57BL/6J mice sham-infected with inactivated PRV (n=12). The symbol key shown in (B) also applies to
panel (C). C) Responses to pelvic stimulation of C57BL/6J mice infected with PRV reveal enhanced sensitivity to suprapubic stimuli (n=12). ANOVA
indicated a significant increase in responses from baseline at all filaments tested in PRV-treated mice at PID 2, 3 and 4 (P,0.05). D) Fluorescein-
isolectin B4 staining (green) of bladder tissue reveals processes in the lamina (arrowheads). E) A single isolectin-stained fiber (open arrowhead in D) is
shown at higher magnification. The mean diameter is 1.31 mm along its length of 1071 mm. Scale bars represent 16 mm and 10 mm for D and E,
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mediates pelvic pain, we examined pelvic sensitivity in KitW-sh/
KitW-shmice reconstituted with bone marrow of mice deficient in
histadine decarboxylase (HDC), the rate-limiting enzyme in
histamine biosynthesis. KitW-sh/KitW-shmice reconstituted with
HDC2/2bone marrow exhibited attenuated pelvic pain responses
such that HDC2/2-reconstituted mice did not develop significant
pain until PID3 (P,0.05), whereas +/+-reconstituted mice
developed significant pain by PID2 (compare Figs. 4A and 1C).
We next examined pain responses in mice deficient for histamine
receptor H1R or H2R to define the mechanisms by which mast
cell-derived histamine induces pelvic pain (Figs. 4B & C). H1R2/2
mice failed to develop significant pelvic pain until PID4 (P,0.05),
whereas PRV-induced pain was completely abrogated in H2R2/2
mice. Comparing overall pain development, HDC2/2-reconsti-
tuted mice developed pelvic pain that was diminished 55.7%
relative to B6 wild type mice, and H1R2/2and H2R2/2mice
developed only 34.3% and 17.6% of normal pelvic pain,
respectively (Fig. 4D). Therefore, both H1R and H2R play roles
in bladder-associated pelvic pain.
Antihistamines attenuate PRV-induced bladder pain
Pilot clinical studies suggest that antihistamine therapy improves
IC-related pelvic pain, and the mechanism for this effect is
unknown . Quantitative RT-PCR revealed that histamine
receptors 1, 2, 3 and 4 are expressed in the bladder and the colon
(Fig. S1). To examine histamine receptors as therapeutic targets
for pelvic pain, we examined the effects of pharmacologic
inhibitors on the development of PRV-induced pain responses.
PRV-infected C57BL6/J mice were treated by oral gavage with
saline or with antihistamines specific for H1R (diphenhydramine),
Bladder mast cells
Figure 2. Mast cells mediate pelvic pain. A) Pelvic stimulation of mast cell-deficient KitW-sh/KitW-shmice infected with PRV reveals no progressive
tactile sensitivity (n=15). B) KitW-sh/KitW-shmice reconstituted with wild type B6 bone marrow exhibited progressive pelvic sensitivity (n=15). ANOVA
indicated a significant increase in response from baseline at all filaments tested in PRV-treated mice at PID 2, 3 and 4 (P,0.05). C) KitW-sh/KitW-sh
reconstituted with KitW-sh/KitW-shbone marrow did not develop progressive pelvic sensitivity (n=10). D) Bone marrow reconstitution of KitW-sh/KitW-sh
mice with B6 bone marrow restored bladder mast cells, whereas reconstitution with KitW-sh/KitW-shbone marrow did not (data represent the mean of
bladder sections from 4 (sham), 5 (PRV), 5 (KitW-sh/KitW-sh), or 4 (KitW-sh/KitW-shmice reconstituted B6 bone marrow) mice). In all panels, error bars depict
SEM. Data indicate values for sham-infected (white bar) and PRV-infected mice (black bars).
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H2R (ranitidine), or H3R/H4R (thioperamide) by gavage. Mice
treated with saline alone exhibited responses to pelvic stimuli that
were significantly greater than baseline by PID 2, 3 & 4 (Fig. 5A;
P,0.05) and were similar to wild-type mice infected with PRV
(compare with Fig. 1C). In contrast, diphenhydramine-treated
mice failed to develop significant pain until PID 3 and 4 and
developed only 49.9% of the total pain exhibited by saline-treated
mice (Fig. 5B, P,0.05, and Fig. 5F), consistent with the reduced
pelvic pain in H1R2/2mice. Similar to the findings in H2R2/2
mice, ranitidine-treated wild type mice showed no significant pain
responses relative to baseline at any timepoint and developed only
7.1% of the pain exhibited by saline-treated mice (Figs. 5C and
5F). Mice treated with a diphenhydramine/ranitidine cocktail
showed similarly reduced pain compared to mice treated with
ranitidine alone, with total pain score of 9.3% of pain exhibited by
saline-treated mice (Figs. 5E and F). The effects of anti-histamine
therapy were specific for H1R and H2R, for thioperamine-treated
mice developed significant pelvic pain that was 90.2% of pain in
mice receiving saline (Fig. 5D, P.0.05, and Fig. 5F). The efficacy
of anti-H1R and anti-H2R therapies was not likely due to non-
specific effects of these drugs on pelvic sensitivity or behavior
because no significant change in baseline tactile sensitivity or free-
roaming behaviors were detected (Fig. 3 and Tables S1 and S2).
Therefore, these data suggest that mast cell-dependent pelvic pain
is mediated by histamine acting via H1R and H2R.
Histamine does not contribute to bladder
We postulated that histamine contributes to bladder patho-
physiology and thus induces pain only indirectly via an
inflammatory cascade associated with tissue damage. To address
this possibility, we examined bladder pathophysiology at the level
of urothelial integrity by quantifying bladder TER ex vivo in an
U¨ssing chamber (Fig. 6A), a physiologic parameter that correlates
with mast cell-dependent bladder pathology in PRV cystitis .
PRV infection of wild type mice resulted in significantly
compromised TER relative to untreated mice (P,0.05), whereas
TER was unchanged in bladders of PRV-infected KitW-sh/KitW-sh
mice. Stable TER following PRV infection was associated only
with mice lacking mast cells or functional TNF signaling,
confirming roles for mast cells and TNF in PRV-induced bladder
pathophysiology. However, significantly compromised TER was
observed in H1R2/2and H2R2/2mice, KitW-sh/KitW-shmice
reconstitiuted with HDC2/2bone marrow (Fig. 6A, P,0.05),
role for histamine in bladder pathophysiology and instead suggest
that mast cell histamine mediates pelvic pain directly.
Histamine-associated pain is not coupled with
To exclude the possibility that histamine mediates pain and
inflammation independent of pathophysiology, we assessed PRV-
induced bladder inflammation using Evan’s blue dye extravasation
as a measure of edema [11,13] (Fig. 6B). PRV infection of wild
type mice resulted in significantly enhanced dye extravazation,
whereas dye extravazation was largely abrogated in TNF2/2mice
(83.5% decrease, Fig. 6B). Since TNF2/2mice exhibited normal
pain responses (Fig. 2), these data suggest that pelvic pain is
distinct from bladder inflammation and pathophysiology. In
support of this possibility, the H2R antagonist ranitidine partially
reduced edema (45.5%), had no protective effect on TER, but
disproportionately relieved pain 92.9% (Figs. 6B, 6C, and 5,
respectively). Finally, the H1R antagonist diphenhydramine
reduced edema 95.0%, whereas PRV-induced loss of TER was
unchanged, and pain was reduced 50.1%. Thus, pain is
dissociated from the inflammatory effects of histamine.
Mast cells and histamine are known mediators of inflammatory
responses, and it is assumed that inflammation is central to the
local pain response. In PRV-induced cystitis, both genetic and
pharmacologic data presented here suggest that mast cells mediate
cystitis pain via H1R and H2R, whereas genetic data exclude TNF
as a significant mediator of acute mast cell-dependent pelvic pain.
These findings are further supported by the observations that pain
precedes TNF-dependent pathophysiology by at least one day in
murine PRV cystitis and that efficacy of antihistamines for pain is
dissociated from effects on edema. The absence of pain responses
in KitW-sh/KitW-shmice indicates that mast cells are required for
histamine-mediated pelvic pain, but KitW-sh/KitW-shmice reconsti-
1.0 2.03.0 4.0
Change (% increase)
Figure 3. TNF does not mediate cystitis pain. A) TNF2/2mice exhibit normal, progressive pelvic sensitivity following PRV infection (n=9). B)
TNFR1/22/2mice exhibit normal, progressive pelvic sensitivity following PRV infection (n=8). In (A) and (B), error bars depict SEM. C) TNF2/2and
TNFR1/22/2mice exhibit overall responses to pelvic stimulation that are indistinguishable from wild type B6 mice (B6). Percent responses at each PID
were calculated as total responses to all fibers relative to baseline responses. The symbol key shown in (A) applies to panels A–B.
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tuted with HDC2/2bone marrow exhibited diminished, albeit
significant, pain. This raises the possibility that histamine from
non-mast cell sources also contributes to pain, probably as a
consequence of TNF-mediated inflammation, consistent with
similar observations in airway models [32,33]. Nonetheless, this
study suggests that mast cells contribute to both pain and bladder
inflammation and pathophysiology through the divergent actions
of histamine and TNF, respectively. Finally, this study indicates
that histamine receptors represent therapeutic targets for direct
intervention in pain.
We observed isolectin B4-positive processes within the lamina
propria, where mast cells accumulate in IC and PRV cystitis
[4,5,11,12,17], suggesting that interactions between mast cell
histamine and C fiber nociceptors mediate pelvic pain in this IC
model. This possibility of C fiber involvement in IC pain is
bolstered by the recent report that pain correlated with sensory
nerve fiber density in patients with painful bladder syndrome
(PBS, a clinical diagnosis for patients presenting with IC-like
symptoms in the absence of cystoscopic evaluation) [34,35].
Histamine may act on such bladder sensory nerves in IC/PBS
because histamine metabolites are increased in IC urine, and mast
cell histamine stimulates C fiber activity via H1R in an esophagitis
model [10,36]. Irritable bowel syndrome (IBS) is another chronic
condition that resembles IC in that abdominal/pelvic pain occurs
in the absence of overt pathology. In IBS, pain is correlated with
activated colonic mucosal mast cells in proximity to nerves, and
IBS colonic tissue extracts that contained elevated histamine
excited rat nociceptors [37,38]. These findings support the idea
that mast cell histamine may mediate pain in multiple pelvic pain
2.0 3.0 4.0
Change (% increase)
infection (n=12). Enhanced pelvic sensitivity relative to baseline (P,0.05) was delayed until PID3. B) H1R2/2mice exhibit reduced pelvic sensitivity
following PRV infection (n=12). Enhanced pelvic sensitivity relative to baseline (P,0.05) was delayed until PID4. (n=5). C) H2R2/2mice did not develop
enhanced pelvic sensitivity following PRV infection (n=4). D) Mice deficient for HDC, H1R, or H2R exhibit diminished pelvic sensitivity at PID 4 relative to
WTmice.PercentresponsesatPID4calculatedastotal responses toallfibers relativetobaselineresponses forsham-infected(whitebar)andPRV-infected
mice (black bars). The symbol key shown in (A) applies to panels A–C. Error bars depict SEM, and significance was determined by ANOVA.
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While this study does not preclude a role for chronic TNF-
mediated pathophysiology in IC symptoms, our evidence for
histamine-mediated pelvic pain provides a mechanistic under-
standing of previous clinical findings. The H2R antagonist
cimetidine produced significant improvement in pain and nocturia
in a limited trial of PBS patients . Similar to our findings that
PRV-induced pain mediated histamine is independent of TNF,
symptom relief from cimetidine therapy was not associated with
improved bladder pathology . Pilot clinical studies yielded
modest pain relief in IC patients receiving the old-line H1R
antagonist hydroxyzine , but studies have not yet been
conducted with newer generation H1R antagonists. In IBS, while
clinical studies are lacking, anecdotal evidence (i.e., Internet
searches) indicates that antihistamines are widely used by IBS
patients. The colon modulates PRV-induced pelvic pain, pelvic
organs generally exhibit neural-mediated crosstalk, and IBS is a
co-morbidity of IC [13,41] [42,43,44]. Therefore, these epidemi-
ologic and animal model findings together suggest that H1R and
H2R antagonists may have general application for the treatment
of pelvic pain.
In summary, we find that the pelvic pain in an IC model is
mediated by mast cell histamine acting on H1R and H2R. These
findings support a general model for IC pelvic pain due to mast
cell-sensory nerve interactions, and similar interactions may drive
pelvic pain in IBS. Thus, antagonists of H1R and H2R are
candidates for expanded clinical trials in the treatment of chronic
Adult female C57BL/6J mice, mice with targeted deletion of
TNF (TNFKO, B6;129S6-Tnftm1Gkl/J) or TNFR1 & TNFR2
(TNFRKO, B6;129S-Tnfrsflatm1ImxTnfrsflbtm1Imx/J) mice (10–14
weeks old) were purchased from Jackson Laboratory (Bar Harbor,
ME). Mast cell-deficient KitW-sh/KitW-shmice (B6.Cg-KitW-sh/
Change (% increase)
Figure 5. Antihistamines attenuate cystitis pain. A) Wild type B6 mice treated by oral gavage with saline exhibited significantly increased pelvic
sensitivity relative to baseline at PID 2, 3 and 4 for all filaments tested (n=8, P,0.05). B) B6 mice treated by oral gavage with the H1R antagonist
diphenhydramine showed reduced pelvic sensitivity that was not significantly increased above baseline until PID 3 (n=7, P,0.05). C) B6 mice treated
by oral gavage with H2R antagonist ranitidine did not develop significant pelvic sensitivity (n=7). D) B6 mice treated by oral gavage with the H3R/
H4R antagonist thioperamine developed pelvic pain similar to saline treated mice that was significant at PID2-4 (n=5, P.0.05). E) Treatment of B6
mice with a diphenhydramine/ranitidine dual therapy reduced pelvic pain responses to a similar extent as in mice treated with ranitidine alone (n=8).
F) The percent increase from baseline in response to pelvic stimulation at PID4 for sham-infected (white bar) and PRV-infected mice (black bars). The
symbol key shown in (A) applies to panels A–E. Error bars depict SEM, and significance was determined by ANOVA.
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HNihrJaeBsmJ), mice targeted deletion of histamine receptor 1
(H1RKO, backcrossed 10 times onto C57BL/6) or histamine
receptor 2 (H2RKO, backcrossed 8 times onto C57BL/6) mice
(10–14 weeks old) were maintained in our breeding colony at
Northwestern University. All experiments were performed using
protocols approved by Northwestern University Animal Care and
Use Committee. Mice were housed in containment facilities of the
Center for Comparative Medicine and maintained on a regular
12:12 hour light:dark cycle with food and water ad libidum.
Induction of neurogenic cystitis
Pseudorabies virus (PRV) was prepared and titrated as
previously reported in Chen et al. Neurogenic cystitis was induced
by injection of 2.296106pfu Bartha’s PRV in the abductor
caudalis dorsalis (ACD) muscle with a 26 gauge Hamilton syringe
while maintaining the animals under Isoflurane anesthesia.
Ultraviolet-irradiated/heat inactivated PRV stocks were employed
as negative control inocula in sham-treated mice. Both sham and
PRV-infected mice were hydrated daily by subcutaneous injection
of 3 ml saline in the shoulder region because previous experiments
indicated that PRV altered fluid intake (Chen and Klumpp,
Mice were tested prior to PRV administration (baseline), 1, 2, 3
and 4 post infection days (PID) after PRV inoculation. Referred
Extravasation (µg/mg tissue)
Figure 6. Pathophysiology and inflammation are separable from cystitis pain. A) Bladder TER was quantified ex vivo for tissues of sham-
infected B6 mice (white bar) or PRV-infected mice (black bars). PRV infection significantly decreased TER in B6 mice (WT, n=8, *P,0.05), KitW-sh/KitW-sh
mice reconstituted with wild type (Sh:WT, n=11, *P,0.05) or HDC2/2bone marrow (Sh:HDC, n=12, *P,0.05), H1R2/2mice (n=5, *P,0.05), or
H2R2/2mice (n=4, *P,0.05). TER was not significantly changed in response to PRV infection in KitW-sh/KitW-shmice (Sh:–, n=9, P.0.05), KitW-sh/KitW-sh
mice reconstituted with KitW-sh/KitW-shbone marrow (Sh:Sh, n=7, P.0.05), TNF2/2mice (n=9, P.0.05), or TNFR1/22/2mice(n=8, P.0.05). B) Evan’s
blue dye was administered i.v. to assess edema by quantifying dye extravasation into bladder tissues of sham-infected (white bar) and PRV-infected
mice (black bars). Dye extravasation in B6 mice not receiving drug therapy (–) was significantly induced by PRV infection relative to sham infection
(*P,0.05), and extravasation was significantly inhibited in TNF2/2 mice (**P,0.05) or in mice treated with diphenhydramine (DHP, **P,0.05) but
not inhibited in mice treated with ranitidine (RTD, P.0.05). C) Bladder TER was not influenced by antihistamine therapy. TER was quantified ex vivo
for bladder of untreated mice (–) and mice receiving oral gavage therapy with diphenhydramine (H1) or ranitidine (H2). All mice infected with PRV
(black bars) exhibited TER significantly reduced TER (*P,0.05) relative to sham-infected mice (white bar). Error bars depict SEM, and significance was
determined by ANOVA.
Histamine Mediates Pelvic Pain
PLoS ONE | www.plosone.org8 May 2008 | Volume 3 | Issue 5 | e2096
hyperalgesia and tactile allodynia was tested using von Frey
filaments applied to the abdomen [20,45] and the plantar region
of the hind paw. Mice were tested in individual Plexiglas chambers
(6 cm610 cm612 cm) with a stainless steal wire grid floor (mouse
acclimation period of ,10 min prior to testing). Frequency of
withdrawal responses to the application of von Frey filaments to
the abdomen was tested using five individual fibers with forces of
0.04, 0.16, 0.4, 1 and 4 grams (Stoelting, USA). Each filament was
applied for ,1 second with an inter-stimulus interval of 2–5 s for a
total 10 times, and the hairs were tested in ascending order of
force. Stimulation was confined to the lower abdominal area in the
general vicinity of the bladder and care was taken to stimulate
different areas within this region to avoid desensitization or ‘‘wind
up’’ effects. Three types of behaviors were considered as positive
responses to filament stimulation: (1) sharp retraction of the
abdomen; (2) immediate licking or scratching of the area of
filament stimulation; or (3) jumping.
Tactile allodynia was tested on the plantar region of the hind paw
The median 50% withdrawal threshold was assessed using the up-
down method where testing was started with 0.04 g filament applied
perpendicularly to the plantar surface of the hind paw until the
filament bent slightly. Filaments were tested in ascending order until
a positive response was observed. A positive response to the filament
was defined as either a sharp withdrawal of the paw or licking of the
test paw. When a positive response was recorded the next weaker
filament was applied, and if a negative response was observed, then
the next stronger filament was applied.
Spontaneous behavior was recorded (Sony VAIO USB camera)
for five minutes in a clear plastic open field chamber
(18629612 cm) at 96 hours after PRV infection and scored for
rearing, grooming and cage crossing to assess general activity.
Capsaicin (50 ml) was administered as 0.3% solution (dissolved in
10% ethanol, 10%Tween 80 and 80% saline) instilled into the colon
via a Hamilton syringe catheter (rounded-tip needle 3.8 cm long)
while the mouse was maintained under Isoflurane anesthesia. All
mice were tested for referred hyperalgesia and tactile allodynia using
von Frey filaments before and 20 min after capsaicin treatment.
Antihistamine treatment (10 mg/kg) was initiated 1 hour prior
to PRV inoculation and was continued every 24 hours until PID 4.
Diphenhydramine, ranitidine, thioperamide (Sigma, St. Louis,
MO) or a combination of diphenhydramine/ranitidine was
administered by oral gavage (Hamiltion syringe with a rounded
tip needle 2.5 cm long). PRV-infected mice and sham controls
were gavaged with saline until PID4. All mice were tested for
referred hyperalgesia and tactile allodynia using von Frey
filaments before PRV (baseline) and PID1-4.
Mast cell reconstitution
Bone marrow was collected from the femurs of either wild-type
female C57BL/6J or KitW-sh/KitW-shmice. Cell suspensions were
generated by trituration, red blood cells were lysed, and the
remaining whole bone marrow cells were harvested by centrifuga-
tion at 1200 rpm for 5 min. The resulting bone marrow cells were
then resuspended in 1x phosphate buffered saline for injection.
Female 4-6 week old sash mice were injected retro-orbitally with
200 ml PBS containing 16107whole bone marrow cells from
C57BL/6J mice, KitW-sh/KitW-shmice, or saline alone. All mice were
tested 8 weeks after reconstitution for referred hyperalgesia and
tactile allodynia using von Frey filaments before PRV (baseline) and
PID1-4. Bladders were excised, and mast cells were quantified in
paraffin sections following toluidine blue staining.
Acidified toluidine blue staining
Tissue sections were deparaffinized, rehydrated, and stained
with acidified toluidine blue to visualize mast cells, as described.
Briefly, tissue sections were immersed in 0.5% potassium
permanganate. After rinsing in distilled water, sections were
immersed in 2% potassium metabisulfite, incubated in tap water,
and washed with distilled water. Lastly, sections were immersed in
0.02% toluidine blue in 0.25% glacial acetic acid, pH 3.2. To
quantify mast cells, toluidine blue-stained cells were counted in
two non-adjacent tissue sections and reported as the mean of
sections for each animal.
Isolectin B4 staining and image analysis
C57BL/6 mouse bladders were fixed in 4% paraformaldehyde
for 10 minutes and cryoprotected in 10% sucrose for 1 hour and
30% sucrose overnight. Bladders were mounted in OCT, cut into
20 mm sections, and dried at 37uC for 1.5 hours. Sections were
incubated with 10 mg/ml fluorescein-conjugated Griffonia(Bandeir-
aea) Simplicifolia Lectin I, isolectin B4 (Vector Laboratories), for 1
hour at room temperature. As a negative control, additional
sections were incubated with isolectin B4 that was pre-incubated
with 500 mM D-galactose for 1 hour prior to staining. Sections
were washed and counterstained with 49,6-diamidino-2-phenylin-
dole (DAPI). Images were collected using a Leica DMIRE2
inverted microscope using Volocity 4.0 software (Improvision) for
acquisition and analysis. A Z-stack of 30 mm total thickness was
taken at 0.1 mm intervals and was deconvolved using the Volocity
Deconvolution module (25 iterations). The C fiber was identified
by pixel intensity and its skeletal length was measured using the
Volocity Measurements module.
Whole bladders and sections of the colon were removed from
five wildtype female C57BL/6 mice. Total RNA was extracted
from the samples using Trizol (Invitrogen) extraction according to
manufacturer’s directions. RNA was reverse transcribed using an
iScript cDNA Synthesis Kit (Biorad) according to manufacturer’s
directions. Quantitative Real Time PCR analysis was performed
using iQ SYBR Green Supermix (Bio-Rad) in a MJ Research
Chromo 4 thermocycler using the following primer sequences.
Hrh1 (Forward) 59-TCG TCA TGC CCA TGA ACA TCC TCT-
39 and (Reverse) 59-TGA AGC ACG GGT CTT GGT TCG
ATA-39; Hrh2 (Forward) 59-CGC GTT GCC ATC TCT TTG
GTC TTT-39 and (Reverse) 59-TCG TTG ACC TGC ACT
TTG CAC TTG-39; Hrh3 (Forward) 59-TGG TGT CCT CCC
TAA TGC AAA CCT-39 and (Reverse) 59-ATT AAG GAA GAG
ACA GCG GCA GCA-39; Hrh4 (Forward) 59-AGG ACT GTG
AGC CTG GCT TTG TTA-39 and (Reverse) 59-AAG CCA
CAG AGA TGA CAG GAA GCA-39; L19 (Forward) 59-CCA
TGA GTA TGC TCA GGC TTC AGA-39 and (Reverse) 59-
TAC AGG CTG TGA TAC ATG TGG CGA-39. Histamine
receptor mRNA levels were normalized to ribosomal protein L19
Bladders were harvested, rinsed with PBS, and dissected
exposing the luminal surface. The tissue was carefully secured in
an U¨ssing chamber (model CHM8; World Precision Instruments
Inc., Sarasota, FL) and filled with 0.1 M KCl. Using electrodes
filled with 0.1 M KCl solution, TER measurements were obtained
Histamine Mediates Pelvic Pain
PLoS ONE | www.plosone.org9 May 2008 | Volume 3 | Issue 5 | e2096
with a EVOMX epithelial voltohmeter (WPI Inc.). Bladder TER
was determined based upon resistance after stabilazation (,5 min)
and reported as the product of the stabilized TER and the tissue
area (0.126 cm2) and reported as Vcm2. All measurements were
performed at room temperature.
Evans blue extravasation
Vascular permeability was assessed by measuring Evans blue dye
extravasation. Evans blue (30 mg/kg in phosphate buffered saline)
was administered intravenously via the tail vein and allowed to
circulate for 30 minutes prior to sacrifice. Individual bladders or
and incubated for24 hoursat60uC.Extravasationwasquantified by
measuring absorbance (A620) relative to a standard curve of dye.
Results were expressed as mean6SEM and analyzed for
statistical significance by a one-way ANOVA followed by a post-
hoc test comparison using Dunnet’s multiple comparison. A value
of p,0.05 was considered statistically significant.
Histamine receptor mRNAs were quantified by real-time RT-
PCR of total RNA prepared from bladder (A) or colon (B) of wild
type B6 mice (n = 5). Primers were specific for mouse ribosomal
protein L19 or receptors H1R, H2R, H3R, or H4R.
Found at: doi:10.1371/journal.pone.0002096.s001 (0.36 MB EPS)
Bladder and colon express histamine receptors.
Found at: doi:10.1371/journal.pone.0002096.s002 (0.05 MB
Found at: doi:10.1371/journal.pone.0002096.s003 (0.05 MB
Found at: doi:10.1371/journal.pone.0002096.s004 (0.05 MB
a bladder nociceptor labeled by staining with isolectin B4.
Found at: doi:10.1371/journal.pone.0002096.s005 (0.67 MB
Deconvolution microscopy and 3D reconstruction of
We thank Dr. Hiroshi Ohtsu for providing HDC2/2mice, Dr. Takeshi
Watanabe for providing H1R2/2and H2R2/2mice, Ryan Yaggie and
Dr. Anne Mongiu for technical assistance, and Drs. Praveen Thumbikat
and Anthony Schaeffer for many helpful discussions. We also are very
grateful to Drs. Melissa Brown and Bradley Undem for careful reading of
the manuscript and helpful suggestions.
Conceived and designed the experiments: DK CR PB. Performed the
experiments: CR LG. Analyzed the data: CR. Contributed reagents/
materials/analysis tools: PB RB. Wrote the paper: DK CR PB.
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