Download A Corticotropin-releasing Factor Receptor Antagonist Improves

Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 3, 2013). doi:10.1152/ajpregu.00257.2012
A Corticotropin-releasing Factor Receptor Antagonist Improves Urodynamic
Dysfunction Produced by Social Stress or Partial Bladder Outlet Obstruction in Male
Rats
Susan K. Wood1*, Kile McFadden1*, Tagan Griffin2, John H. Wolfe1, Stephen Zderic1
and Rita J. Valentino1
1
The Children's Hospital of Philadelphia, Philadelphia, PA 19104
2
The University of Pennsylvania, Philadelphia, PA 19104
*Authors contributed equally to this work
Running title: CRF receptor antagonists and urodynamic function
Rita J. Valentino
Dept. of Anesthesiology and Critical Care Medicine
The Children's Hospital of Philadelphia
402D Abramson Pediatric Res. Center
Civic Center Blvd.
Philadelphia, PA 19104
215-590-0650
215-590-3364
[email protected]
Copyright © 2013 by the American Physiological Society.
Abstract
Barrington’s nucleus, in the pons, regulates micturition through spinal projections to
preganglionic parasympathetic neurons. The stress neuropeptide, corticotropinreleasing factor (CRF) is prominent in these projections and has an inhibitory influence.
Social stress in rats causes urinary retention and abnormal urodynamics resembling
those produced by partial bladder outlet obstruction (pBOO) and this is associated with
CRF upregulation in Barrington’s nucleus. Here we examined the role of CRF in social
stress- and pBOO-induced urodynamic dysfunction by assessing the ability of a CRF1
receptor antagonist to alter these effects. Male rats exposed to repeated residentintruder stress were administered vehicle or a CRF1 antagonist (NBI-30775) daily prior
to the stress. Urodynamic function was recorded in the unanesthetized state 72 h after
the final stress. NBI-30775 prevented the increased intermicturition interval, micturition
volume and bladder capacity produced by social stress, but not the increase in CRF
expression in Barrington’s nucleus neurons. The urinary dysfunction was also partly
prevented by shRNA targeting of CRF in Barrington’s nucleus suggesting that stressinduced urinary dysfunction results in part from CRF upregulation in Barrington’s
nucleus and enhanced postsynaptic effects in the spinal cord. Finally, NBI-30775
improved urodynamic function of rats that had pBOO of 2 week duration when
administered daily during the second week, but did not block the increase in CRF
expression in Barrington's nucleus neurons. These findings implicate a role for
Barrington’s nucleus CRF in stress- and pBOO-induced urodynamic changes and
suggest that CRF1 antagonists may be useful therapeutic agents for the treatment of
urinary dysfunction.
Keywords: cystometry, resident-intruder, Barrington’s nucleus, urinary
Introduction
Barrington’s nucleus in the pons regulates the descending limb of the micturition
reflex through its axonal projections to preganglionic neurons of the lumbosacral spinal
cord that provide the parasympathetic input to the detrusor muscle (21). Electrical and
chemical stimulation of Barrington’s nucleus elicits detrusor contraction, and conversely,
lesions disrupt the micturition reflex (1, 27, 29). More recent anatomical and
physiological evidence suggests that Barrington’s nucleus plays a key role in
coordinating central and visceral responses during micturition (39)(for review see (41).
In addition to its connections to the bladder, Barrington’s nucleus neurons are
transsynaptically linked to the distal colon and other pelvic viscera suggesting a broader
role for this nucleus in the regulation of pelvic visceral functions (23, 24, 33, 40, 44).
Elucidating the function of neuromodulators expressed by Barrington’s nucleus neurons
could lead to a better understanding of central control of pelvic visceral functions and
how these can be modulated by pharmacological interventions to treat pelvic visceral
disorders.
The stress-related neuropeptide, corticotropin-releasing factor (CRF) is
prominently expressed in Barrington’s nucleus neurons and in its spinal projections (36,
37, 43). CRF is the major neurohormone that initiates adrenocorticotropin release from
the anterior pituitary in response to stress (38). Additionally, CRF is present in
extrahypophyseal brain circuits involved in autonomic and behavioral responses to
stress (37). CRF release in different brain regions is hypothesized to coordinate the
many aspects of the stress response. In vivo cystometry studies examining the effects
of CRF agonists and/or antagonists have suggested conflicting roles for CRF in the
regulation of micturition (16, 17). However, a study of the specific effect of CRF in
Barrington’s nucleus spinal projections suggested an inhibitory influence on
parasympathetic input to the bladder (29). Thus, discrete chemical activation of
Barrington’s nucleus neurons elicited bladder contractions that were increased by
intrathecal administration of a CRF antagonist, and conversely, decreased by
intrathecal CRF. An inhibitory role for CRF in Barrington’s nucleus regulation of the
bladder is consistent with reports that social stress in rodents leads to urinary retention,
abnormal urodynamics and bladder hypertrophy (4, 6, 12, 13, 45) and this is associated
with increased expression of CRF in Barrington’s nucleus neurons (45). These findings
suggest that pharmacological manipulation of CRF may improve bladder dysfunctions
associated with stress or other conditions.
The present study used in vivo cystometry in unanesthetized rats to determine
whether treatment with a CRF antagonist, NBI-30775, could prevent social stressinduced changes in urodynamics and/or CRF expression in Barrington’s nucleus
neurons. Because some of the urodynamic changes produced by social stress mimic
those seen in rats with partial bladder outlet obstruction (pBOO), the effects of NBI30775 on the bladder dysfunction associated with a 2-week pBOO were also
investigated.
Materials and Methods
Animals. The experimental subjects were male Sprague-Dawley rats (Charles River,
approximately 300 g). Male Long-Evans retired breeders (Charles River, 650 g-850 g)
were used as residents in the social stress study. All rats were singly housed in a 12-h
light-dark cycle (lights on at 7:00 AM), climate controlled room and given free access to
food and water. All studies were approved by the Children’s Hospital of Philadelphia’s
Institutional Animal Care and Use Committee and conformed to the Principles of
Laboratory Animal Care.
Social Stress. The social stressor used in these studies was modified from the residentintruder model originally described by Miczek and colleagues (26). Rats were randomly
assigned to either social stress or control exposure for 30 min on seven consecutive
days. During each social stress exposure an intruder rat was placed into the home cage
territory of an unfamiliar Long-Evans resident previously screened for high aggression
(2, 3, 47). A typical social stress exposure resulted in intruder subordination, termed
defeat, and was operationally defined by the Sprague Dawley intruder assuming a
supine posture. Following defeat, a wire mesh partition was placed in the cage to
prevent physical contact between the resident and intruder, but allowing auditory,
olfactory and visual contact to continue for the remainder of the 30 min social stress
session. Control rats were placed in a novel cage behind a wire partition for 30 minutes
daily. Rats were returned to their home cage after each session.
Partial bladder outlet obstruction. Surgery for pBOO was identical to that previously
described (31). Rats were anesthetized with isofluorane and an incision was made
along the midline of the lower abdomen to access the bladder and prostates.
Connective tissue was dissected between the bladder and prostates to provide a clear
view of the bladder neck and ureters. An 18 gauge needle was placed parallel to the
urethra and suture was laced inside of the ureters around the bladder neck and needle.
The needle was removed leaving the ligature around the bladder neck and the
abdominal muscles and skin were then sutured closed. This ligature remained
throughout the experiment. Sham rats had the same surgery up to the point of exposing
the bladder neck and base of the urethra.
Drug treatment. One hour prior to each of the 7 social stress or control exposures rats
were treated with vehicle (10% solution of 0.1 M tartaric acid in sterile water, s.c.) or
NBI-30775 (10 mg/kg, s.c.). For the partial bladder outlet obstruction (PBOO) study
PBOO rats were treated with vehicle or NBI-30775 (10 mg/kg., s.c.) on 7 consecutive
days (days 7-14 post surgery). Sham rats were administered vehicle (1 ml/kg, s.c.)
daily 7-14 days post surgery. This dose of NBI-30775 has a half life of 130 minutes in
vivo and has been shown to result in 75% occupancy of brain CRF1 (8, 9, 11).
Additionally, this has been demonstrated to prevent stress-induced ACTH release and
behavioral and cardiovascular consequences of social stress (14, 46).
shRNA vector design and construction. Adeno-associated viral vectors (AAV2/1)
containing short-hairpin RNAs were produced in order to knockdown CRF expression.
shRNAs were targeted against the 3’ coding region of CRF mRNA or a scrambled
control sequence. The CRF shRNA (gift from Dr. Alon Chen, Weizmann Institute of
Science, Rehovot, Israel) was previously shown to dramatically reduce expression in
293T cells (30). The scrambled shRNA sequence was generated using siRNA Wizard
V3.1 and synthesized de novo. shRNA sequences (sense and antisense in italics,
hairpin in bold): shRNA-CRF: 5’AGATTATCGGGAAATGAAATCTCTTGAATTTCATTTCCCGATAATCT.
shRNA-CRFscramble: 5’GTGAAATATCGAAGGTAAATCTCTTGAATTTACCTTCGATATTTCAC. The CRF and
scrambled shRNAs were ligated into the AAV2 vector plasmid pZAC2.1 under the
control of the H1 promoter. The packaging and purification of vector was performed by
the University of Pennsylvania Vector Core, as previously described (28). Viral titers
were determined by the Vector Core by running vector preps on an SDS-PAGE gel and
comparing the intensity of VP3 bands to a reference standard. Viral titers were as
follows; AAV-shRNA-CRF: 4x10^12 and AAV-shRNA-CRFscramble: 7.5x10^12.
Surgery and Vector Injection. Surgery and injection of AAV vectors into Barrington’s
nucleus were as previously described (25). Rats were anesthetized with a mixture of
isofluorane-in-air, positioned in a stereotaxic and surgically prepared for placement of a
double barrel glass micropipette into Barrington’s nucleus. The center barrel was filled
with 2% pontamine sky blue in 0.5 M sodium acetate and served as the recording
pipette. The ejection barrel was filled with a solution containing a mixture (1:1) of AAV
containing green fluorescent protein (GFP) cDNA and either AAV-shRNA-CRF or AAV-
shRNA-CRFscramble. The AAV-GFP served to help visualize the injections in sections
and verify the localization in Barrington’s nucleus. Barrington’s nucleus was localized
through electrophysiological recordings as previously described (34). Once the pipette
was positioned into the region considered to be Barrington’s nucleus 200 nl of the
vector solution was microinfused by application of pressure pulses (20-40 psi, 30 msec)
using a picospritzer. The ejection pipette was calibrated to deliver known volumes.
Bilateral injections were done in every animal. After the skin over the skull was sutured,
rats were observed until ambulatory and placed back into their home cage. One cohort
of rats was exposed to social stress on days 8-14 after AAV-shRNA infusion. Twentyfour hours after the final social stress catheters were surgically implanted for
quantification of urodynamics as described below. Rats were decapitated after
cystometry (17 days after AAV injection) and brains removed for in situ hybridization of
CRF mRNA at this time after injection. Another cohort of unstressed rats was just
decapitated and the brain removed 9 days following AAV-shRNA injection for in situ
hybridization of CRF mRNA in Barrington’s nucleus.
Quantification of urodynamics. Twenty-four hours after the last stress or control
manipulation, a catheter (5-French umbilical artery catheter) was surgically inserted into
the bladder dome and tunneled subcutaneously from the bladder to the scapulae to an
incision between the scapulae as in our previous study (16). Forty-eight hours after this
surgery rats were placed into a cystometry chamber (Medical Associates, St. Albans,
VT), the catheter was connected to a swivel device and urodynamic function was
recorded for 1 h in the unanesthetized, unrestrained (no jacket) state using cystometry
equipment and software (Medical Associates, St. Albans, VT) as previously described
(16). Sterile saline was continuously infused into the bladder (100 μl/min) through a
closed circuit system to monitor intermicturition interval, bladder capacity and voided
volume. Figure 1 shows how data were calculated from the cystometry records. The
top trace shows the pressure recording and the points at which resting pressure (RT),
micturition threshold (MT) and micturition pressure (MP) were determined. Urine was
collected in a pan situated on a scale under the cage. The micturition volume was
derived from the weight of the urine that fell into the pan during each cycle.
Intermicturition interval was defined as the time between the end of one micturition cycle
and beginning of another (Fig. 1, bottom panel). The recording pen reset for each trace
after each micturition event. This allows for an estimate of bladder capacity as the
volume of saline infused during one micturition cycle (Fig. 1, middle panel). This is only
an estimate because it assumes no residual volume. However, there was no reason to
assume residual volumes in these cases. Values were averaged for at least 3 stable
micturition cycles. Rats were sacrificed by decapitation, the brains were dissected and
flash frozen for CRF mRNA or immunohistochemistry.
CRF mRNA quantification. Brain sections collected on slides were postfixed by
incubation in 10% formalin and processed to quantify CRF mRNA as previously
described (42). Sections containing Barrington’s nucleus were hybridized with an
antisense riboprobe to CRF mRNA (Dr. Audrey F. Seasholtz, University of Michigan)
and coated with Kodak NTB2 liquid autoradiographic emulsion (7-day exposure at 4°C)
identical to that previously published (45). The tissue was lightly stained with cresyl
violet and brightfield images were captured and magnified using Open Lab software to
identify cresyl violet-stained cells containing silver grains greater than 2-4 times
background. The number of CRF-expressing cells from two Barrington’s nucleus
sections were averaged for each rat by an observer blinded to the treatment conditions.
CRF immunofluoresence. Brains were cut into 14 μm-thick sections and collected on
charged slides (ProbeOnPlus, Fisher Scientific). Sections were postfixed in 4%
paraformaldahyde for one hour then rinsed in PBS. Sections were then incubated with
rabbit anti-CRF (C-70, 1:2,000, Wylie Vale, Salk Institute) for 48 h at 4°C. Following
rinses, slides were incubated with rhodamine-conjugated donkey anti-rabbit IgG
antibody (1:200; Jackson Laboratories) for 90 minutes. Sections from rats of all
experimental groups were processed at the same time using the same solutions. CRFimmunoreactivity was visualized with a Leica DMRX fluorescent microscope. Images
were captured using a Hamamatsu ORCA-ER digital camera and Open Lab software
(Perkin-Elmer) using the identical exposure times for all sections so that
immunoreactivity could be compared between groups. For quantification of CRFimmunoreactive cells in Barrington’s nucleus, sections were photographed at the same
exposure and the grayscale images were inverted using Image J (rsbweb.nih.gov/ij/) as
previously reported (45). Images were magnified and inverted grayscale
photomicrographs resulted in black cells on a white background allowing for
unambiguous distinction between staining within a cell and background. An oval region
of interest was drawn to encompass Barrington’s nucleus at its largest extent and this
was superimposed on all images. CRF-immunoreactive cells within this region of
interest were quantified by an individual blinded to experimental groups. CRF cells
were counted in 2-3 non-serial sections from an individual rat and averaged as the
value for that rat. The mean determined from all individual rats was averaged for the
group mean.
Statistical Analysis. All data are presented as means ± SEM. Two-way ANOVAs
followed by Holm-Sidak’s multiple comparison method were used within the social
stress-CRF antagonist study to identify the effects of drug on stress-induced changes in
urodynamic parameters. Separate one-way ANOVAs followed by Student’s-NewmanKeuls method were used to compare bladder parameters between stressed rats
injected with AAV-shRNAs and rats in the stress-CRF antagonist study. Likewise,
separate one-way ANOVAs followed by Student’s-Newman-Keuls method were used to
identify differences in urodynamic parameters between sham/vehicle, pBOO/vehicle,
and pBOO/NBI-30775 rats. A one-way ANOVA was also used to identify differences in
the number of CRF-expressing cells within Barrington’s nucleus between
control/vehicle, stress/vehicle, and stress/NBI-30775 rats as well as in CRF mRNAexpressing cell count between sham, pBOO/vehicle and pBOO/NBI-30775. A Student’s
t-test was used to identify differences in the number of CRF mRNA-expressing cells in
Barrington’s nucleus in defeated rats treated with AAV-shRNA-CRFscramble versus
AAV-shRNA-CRF. For all analyses, two-tailed p values of <0.05 were considered
significant. All post-hoc significance is reported in the figure legends.
Results
NBI-30775 improves stress-induced urodynamic dysfunction. Consistent with
previous reports (45) social stress resulted in an abnormal urodynamic profile. Figure 2
shows representative examples of cystometry traces from control and stressed rats
treated with vehicle or NBI-30775 before each manipulation. Intermicturition interval,
bladder capacity and micturition volume were all elevated in social stressed rats
administered vehicle compared to control rats administered vehicle (Figs. 2A,C, 3A-C).
Notably, pretreatment with NBI-30775 prior to each stress prevented the effects of
stress on all urodynamic measures but had no effect of its own in control rats (n=5)
(Figs. 2B,D, 3A-C). Micturition threshold, micturition pressure and resting pressure were
unaffected by social stress (Fig. 3D). A two-way ANOVA for intermicturition interval (IMI)
revealed significant effects of stress (F(1,29)=4.9, p<0.05), treatment (F(1,29)=4.5,
p<0.05) and a stress x treatment interaction (IMI; F(1,29)=4.6; p=0.041<0.05). In stressed
rats administered vehicle (n=9) the IMI was greater compared with control rats
administered vehicle (n=10; p<0.005). The effect of stress on IMI was significantly
prevented by treatment with NBI-30775 (n=9; p<0.005). For bladder capacity (BC) there
was a significant effect of stress (F(1,29)=p<0.05), treatment (F(1,29)=p<0.05 and a
stress x treatment interaction (F(1,29)=4.6; p<0.05). Bladder capacity was significantly
greater in social stressed rats pretreated with vehicle compared with control rats
administered vehicle (p<0.005). Stress-induced increases in BC were significantly
prevented by treatment with NBI-30775 (p<0.005). For micturition volume (MV) there
was a trend towards a significant effect of treatment (p=0.051) and a significant stress x
treatment interaction (F(1,29)=4.2; p<0.05). Stressed rats administered vehicle had a
larger mean MV compared with control rats administered vehicle (p<0.01). NBI-30775
significantly prevented the stress-induced increases in MV (p<0.005).
Social stress-induced bladder hypertrophy was also observed in vehicle-treated
social stress rats, consistent with previous findings (45). The bladder to body weight
ratio in stressed rats treated with vehicle (stress/vehicle; 0.60±0.02, n=9) was greater
than control rats treated with vehicle (0.48±0.02, n=10). This effect was prevented in
stressed rats treated with NBI-30775 (stress/NBI-30775; 0.50±0.02, n=9) which had no
effect on bladder to body weight ratio in control rats (0.52±0.03, n=5). A two-way
ANOVA revealed a significant stress x treatment interaction (F(1,27)=7.4; p<0.05). Posthoc analysis revealed a significant effect of stress within vehicle treated rats (p<0.001;
control/vehicle vs stress/vehicle). There was also a significant effect of treatment within
stressed rats (p<0.01; stress/vehicle vs. stress/NBI-30775). There was no effect of
stress (p=0.2) or treatment (p=0.4) on body weight (mean body weight (g) ±SEM;
control/vehicle: 331±8, control/NBI: 346±10, stress/vehicle: 329±8, stress/NBI: 328±8).
Role of CRF upregulation in Barrington’s nucleus in stress-induced urodynamic
dysfunction. We previously reported that social stress increases CRF mRNA and the
number of CRF-immunolabeled neurons in Barrington’s nucleus (45). Figure 4A shows
representative photomicrographs of CRF immunolabeling in the core of Barrington’s
nucleus in vehicle treated control (top) and social stressed (bottom) rats. Consistent
with our previous report, social stress increased the mean number of CRF
immunolabeled Barrington’s nucleus neurons (n=9; p<0.05) compared to vehicle-treated
control rats (n=8) and NBI-30775 pretreatment did not prevent this effect (n=7; p<0.05)
(Figure 4B; F(2,21)=5.5; p<0.05).
To evaluate the role of CRF upregulation in Barrington’s nucleus neurons in the
urodynamic consequences of social stress, local injections of AAV-shRNA-CRF were
used to inhibit CRF expression in Barrington’s nucleus neurons. By 17 days after
injection (72 h after the last stress exposure) the mean number of CRF immunoreactive
Barrington’s nucleus neurons was less in rats administered AAV-shRNA-CRF (n=27+3,
n=7; p=0.03) compared to those administered AAV-shRNA-CRFscramble (40+4, n=7).
Notably the number of CRF-immunolabeled Barrington’s nucleus neurons in rats that
were stressed and received AAV-shRNA-CRFscramble was comparable to those that
were stressed and received vehicle or NBI-30775, whereas stressed rats that received
AAV-shRNA-CRF were similar to non-stressed controls (Fig. 4B). In contrast to the
immunohistochemical findings, there were no statistically significant differences in the
number of Barrington’s nucleus neurons expressing CRF mRNA in AAV-shRNACRFscramble (57+6) compared to AAV-shRNA-CRF (45+6) groups (p=0.18). Because
recovery of mRNA may precede protein, the number of Barrington’s nucleus neurons
expressing CRF mRNA was examined at an earlier time after injection that would
correspond to an early point in the stress procedure (9 days after injection). Figure 5A
shows representative darkfield photomicrographs of CRF mRNA expression in
Barrington’s nucleus of rats treated with AAV-shRNA-CRFscramble or AAV-shRNACRF 9 days after injection. shRNA targeting of CRF (n=6) significantly reduced the
number of CRF mRNA-expressing cells compared with the scrambled RNA at this time
(n=4) (treatment (t(8)=2.7; p=0.029, Figure 5B).
The urodynamic parameters of stressed rats injected with AAV-shRNA-CRF or
AAV-shRNA-CRFscramble were compared to those of control and stressed rats
administered vehicle or NBI-30775 using a one-way ANOVA (Fig. 2). For intermicturition
interval (IMI) a one-way ANOVA revealed a significant difference between groups
(F(5,41)=4.3, p=0.003). Posthoc analysis confirmed that social stress similarly
increased IMI in rats treated with vehicle or AAV-shRNA-CRFscramble and these
effects were prevented by NBI-30775 and shRNA targeting of CRF in Barrington’s
nucleus resulted in a trend towards a decrease (p=0.06) in IMI. For bladder capacity
(BC) there were also significant differences between groups (F(5,41)=4.2; p=0.003.
Bladder capacity was significantly greater in social stressed rats treated with vehicle or
AAV-shRNA-CRFscramble compared with control rats (Fig. 2B). Stress-induced
increases in BC were significantly prevented by treatment with NBI-30775 and there
was a trend for shRNA targeting of CRF in Barrington’s nucleus to block increase in BC.
For micturition volume (MV) there was also a significant difference between groups
(F(5,31)=3.8; p=0.006). Stressed rats administered vehicle or AAV-shRNACRFscramble had a larger mean MV compared with control rats administered vehicle
(Fig. 2C). Both NBI-30775 treatment and AAV-shRNA-CRF infusion significantly
prevented the stress-induced increases in MV (Figure 2C). One-way ANOVA analysis
revealed an overall significant difference between groups for micturition threshold
(F(5,41)=3.5; p=0.011), micturition pressure (F(5,41)=3.4; p=0.011) and resting
pressure (F(5,41)=4.4; p=0.003). These effects reflected lower pressures, for the AAV
injected rats and were not an effect of stress or treatment. The shRNA studies were
conducted separately from the NBI studies, possibly contributing to the differences
observed in MT, MP, and RP.
NBI-30775 improves pBOO-induced urodynamic dysfunction. Fourteen days of
partial bladder outlet obstruction resulted in abnormal urodynamics that were mitigated
in part by daily treatment with NBI-30775 from day 7-14. Figure 6 shows representative
cystometry records from a vehicle-treated sham rat and pBOO rats treated with vehicle
or NBI-30775. Quantification of mean urodynamic parameters demonstrated a
significant increase in intermicturition interval in pBOO rats treated with vehicle (n=9)
compared with pBOO rats administered NBI-30775 (n=10; F(2,22)=4.0; p<0.05) (Fig. 7A).
Bladder capacity was also significantly greater in pBOO rats treated with vehicle
compared with pBOO rats treated with NBI-30775 (BC; F(2,22)=4.0; p<0.05) (Fig. 7B).
Micturition volume was also increased in pBOO rats treated with vehicle compared with
sham rats treated with vehicle (n=6) (F(2,22)=5.4; p<0.05) and this was prevented by NBI30775 (p<0.05) (Fig. 7C). In contrast, NBI-30775 treatment did not affect the increased
micturition pressure produced by pBOO (F(2,21)=3.6; p<0.05) (Figure 7D). Micturition
threshold and resting pressure were similar between groups.
The bladder:body weight ratio tended to increase in the 2 week pBOO rats
regardless of pretreatment (F(2,21)=2.4, p=0.1). Thus, in pBOO rats treated with vehicle
(n=9) and NBI-30775 (n=10) the ratios were 0.72±0.05 and 0.73±0.07, respectively
compared with 0.54±0.03 in sham rats treated with vehicle (n=6) (p=0.07 and p=0.05,
respectively). There was no difference in body weight between groups (mean body
weight (g) ±SEM; sham/vehicle: 366±15, pBOO/vehicle: 371±7, pBOO/NBI: 367±11;
p=0.9).
pBOO increases CRF mRNA-expressing neurons in Barrington’s nucleus. Given
the inhibitory influence of CRF in Barrington’s nucleus and the effects of NBI-30775, the
effect of 2-week pBOO on CRF mRNA was examined. Figure 8A shows representative
darkfield photomicrographs of the hybridization signal for CRF mRNA in Barrington’s
nucleus of a sham rat (top panel) and a pBOO rat treated with vehicle (bottom panel).
Two-week pBOO (vehicle treated, n=9) increased the number of CRF mRNA positive
neurons in Barrington’s nucleus compared with sham (n=7) and this was unaffected by
NBI-30775 treatment (n=6; F(2,19)=0.1; p=0.028; Figure 8B).
Discussion
The present results confirmed previous studies showing that repeated social
stress increases bladder mass and alters urodynamics in a manner that is consistent
with urinary retention (45). The results also supported the previous finding that
repeated social stress upregulates CRF in Barrington’s nucleus neurons (45). The
present demonstration that pretreatment with a CRF1 receptor antagonist prior to the
stress prevents the abnormal urodynamic profile implicates CRF in the social stressinduced bladder pathology. That the urodynamic dysfunction produced by stress can
also be attenuated by shRNA targeted against Barrington’s nucleus CRF supports a
role for CRF upregulation in Barringtons’ nucleus neurons in these effects. The inability
of the antagonist to alter stress-induced CRF upregulation in Barrington’s nucleus
neurons implies an action downstream from these neurons at the level of the spinal cord
where their axons terminate (21). By preventing the inhibitory influence of excess CRF
at the level of the spinal cord, the CRF antagonist can prevent the development of the
urinary dysfunction associated with social stress. The CRF antagonist was also
effective in reversing some of the same urodynamic consequences associated with
pBOO. Together, the results support a role for CRF in urinary retention in certain
bladder dysfunctions and suggest that CRF receptor antagonists may improve function
in these conditions.
Social stress-induced bladder dysfunction. Although fear has been anecdotally
associated with micturition, numerous studies have documented that rodents that
become subordinate by exposure to social stress develop urinary retention that is
sufficient to increase bladder mass (4, 6, 12, 22, 45). In some cases this can be
sufficiently severe as to result in death due to nephritis (12). Cystometry studies in mice
and rats exposed to repeated social stress demonstrated that the voiding dysfunction
was characterized by increased intermicturition interval, bladder capacity and micturition
volume (4, 45). Importantly, it was not associated with increased micturition pressure,
consistent with decreased central drive to the detrusor as opposed to increased outlet
resistance as occurs in pBOO. Notably, social stress can induce some of the same
molecular changes as produced by pBOO and that are thought to lead to bladder
remodeling (4). The urodynamic effects of social stress have been documented in
males only because rodent models of confrontational social stress are generally limited
to males. Special conditions (exposure to lactating females) are required to produce a
comparable confrontational stress for female rats so that the effects of this stress on
urodynamic dysfuntion in females have yet to be studied (10).
Role of CRF. CRF in Barrington’s nucleus neurons was hypothesized to be a
mediator of social stress-induced voiding dysfunction based on evidence that it has an
inhibitory influence in Barrington’s nucleus spinal projections. For example localized
chemical activation of Barrington’s nucleus elicits bladder contractions that are
increased by intrathecal administration of CRF antagonists and conversely, intrathecal
CRF administration decreased bladder contractions elicited by Barrington’s nucleus
stimulation (29). Moreover, social stress upregulates CRF mRNA and protein in
Barrington’s nucleus neurons (45). Interestingly, repeated restraint stress, which does
not alter CRF expression in Barrington’s nucleus neurons, has no effect on urodynamics
(45). These results led to the hypothesis that social stress increases CRF expression in
Barrington’s nucleus neurons and its spinal projections resulting in decreased central
drive to the detrusor and urinary retention. This may be an adaptive response to being
in the presence of a dominant conspecific. However, if it persists, the voiding
postponement and abnormal urodynamics can progress to bladder remodeling similar to
that seen with pBOO.
In a recent study of the effects of NBI-30775 on cardiovascular consequences of
social stress, the antagonist was demonstrated to prevent stress-induced increases in
bladder mass (46). The present study confirmed that finding and further showed that
repeated administration of NBI-30775 prevented the urodynamic consequences of
repeated social stress. Acute administration of NBI-30775 and antalarmin, another
CRF1 antagonist, have been reported to increase micturition frequency and decrease
bladder capacity in control rats (16). However, the effects of repeated NBI-30775 in the
present study were unrelated to these acute effects because cystometry was performed
72 h after the last dose and repeated NBI-30775 had no effect on urodynamics in
unstressed rats as seen in control subjects of the social stress study (Fig. 3).
The receptor-ligand binding kinetics of CRF1 receptor antagonists at
physiological temperatures have been extensively studied (8). NBI-30775 is a highly
potent CRF1 receptor antagonist, possessing a kd of 0.36 nM and a half life of 130
minutes. Importantly, NBI-30775 is highly efficacious in vivo at the dose used in the
present study (10 mg/kg). At this dose NBI-30775 selectively occupied 75% of CRF1
receptors in the cortex 3 hrs after treatment and suppressed ACTH in adrenalectomized
rats for more than 6 hours (8, 9, 46). When administered daily, as in the present study,
10 mg/kg NBI-30775 blocked the behavioral, cardiovascular and bladder hypertrophic
effects of repeated social defeat stress (46).
Systemic NBI-30775 could act at multiple sites. However, given that CRF is a
major neurotransmitter in the pontine micturition circuit extending from Barrington’s
nucleus to the preganglionic parasympathetic neurons of the lumbosacral spinal cord,
this is its most likely site of action in affecting urodynamics (43). Other brain circuits in
which CRF is prominent have not been directly associated with bladder regulation.
Moreover, it is unlikely that the site of action of NBI-30775 is at the level of the bladder
because neither CRF1 receptor or CRF1 immunoreactivity are present in bladder (19).
The finding that NBI-30775 prevented the urodynamic consequences of social stress
without affecting CRF upregulation in Barrington’s nucleus neurons indicates that it was
acting at the level of the spinal cord where Barrington’s nucleus neurons terminate so
that the social stress-induced bladder dysfunction failed to occur in spite of elevated
levels of CRF in this pathway.
Social stress had similar urodynamic consequences in rats injected with AAVshRNA-CRFscrambled as in vehicle injected rats in the drug study and the mean
numbers of CRF-immunoreactive Barrington’s nucleus neurons were similar in both
groups. AAV-shRNACRF was effective in reducing the number of CRF-immunoreactive
Barrington’s nucleus neurons in stressed rats and alleviated some of the urodynamic
consequences. For these experiments the timing between the shRNA injection and
examination of the urodynamic endpoints is critical to observe optimal effects of CRF
knockdown. The number of CRF-immunoreactive Barrington’s nucleus neurons (i.e.,
protein expression) was reduced 17 days after shRNA injection, consistent with the
improved urodynamics. However, CRF mRNA recovered at this time. CRF mRNA was
significantly decreased by 9 days after injection, which would correspond to the second
day of social stress. Given that urodynamic endpoints are examined 3 days after 7 days
of social stress, this suggests that the optimum timing of the injection may be closer to
the beginning of the stress. However, having a short recovery duration between
surgical craniotomy and exposure to social stress is not optimal for animal welfare and
so was not tested here. Nonetheless, the results of these experiments remain
consistent with the idea of a role for CRF upregulation in Barrington’s nucleus neurons
in social stress-induced bladder dysfunction.
Together the CRF1 antagonist and shRNA results support a role for central CRF
in social stress-induced bladder dysfunction and suggest that pharmacological
manipulation of CRF actions may be a useful line of therapy in stress-induced voiding
dysfunctions. Notably, CRF1 antagonists such as NBI-30775 are being developed for
treating diverse stress-related disorders and some of these have been found to have a
profile of ideal pharmacokinetic properties and minor side effects (15, 18).
pBOO induced bladder dysfunction. The abnormal urodynamic profile produced
by repeated social stress has many similarities with that seen in rats with pBOO in
relatively early stages (1-2 weeks). A major difference between the two models is the
increased micturition pressure with pBOO as a result of increased outlet resistance.
Repeated administration of NBI-30775 during the second week of pBOO could not
reverse the increased resistance due to the obstruction and therefore did not prevent
increases in micturition pressure or resulting, bladder hypertrophy. Nonetheless, the
intermicturition interval, bladder capacity and micturition volume were normalized by
administration of the CRF antagonist during the second week. Interestingly, like social
stress, pBOO was found to increase CRF expression in Barrington’s nucleus neurons.
It is tempting to speculate that this contributes to the initial urodynamic profile and that
by removing the inhibitory influence of CRF in Barrington’s nucleus spinal projections
and increasing micturition frequency, chronic antagonism of CRF receptors may
improve function in early stages of pBOO.
Summary. Although the treatment of urologic disorders has focused on the bladder, the
present results underscore the potential role of the brain in these disorders and that
neuromodulators within circuits linking the brain and bladder are potential therapeutic
targets. Here we provided evidence for a role of CRF, one neuromodulator expressed
by Barrington’s nucleus neurons, in social stress induced urinary retention. In humans
voiding postponement has been linked with psychiatric comorbidity and childhood
sexual or physical abuse (5, 20, 32). Traumatic social events in adulthood such as loss
of a loved one or broken marriages have also been associated with urinary retention (7).
Notably, Barrington’s neurons in human brain also exhibit CRF immunoreactivity (35).
Manipulation of CRF actions may be a rational therapeutic avenue for the treatment of
these and other urinary disorders.
Acknowledgements
The authors wish to thank Dr. Dimitri Grigoriadis (Neurocrine Biosciences Inc., La Jolla,
CA) for the gift of NBI-30775. The authors also acknowledge Ms. Sandra Luz for
technical assistance in situ hybridization studies.
Grants
This work was supported by a George O’Brien Center Grant P50 DK52620, R01-NS38690 and T32-DK007748.
Author Contribution
Susan K. Wood directed and performed the social stress studies, analyzed data, helped
to prepare figures and write the manuscript.
Kile McFadden performed the partial bladder outlet obstruction, AAV injections into
Barrington’s nucleus and all cystometries, analyzed cystometry and in situ hybridization
data and helped to write the manuscript.
Tagan Griffin produced the AAV2/1 vectors and helped to write the manuscript.
John Wolfe designed the AAV2/1 vector studies, consulted on data analysis and
interpretation and the manuscript.
Stephen Zderic designed the partial bladder outlet obstruction studies, consulted on
cystometry analysis and the manuscript.
Rita Valentino designed and supervised the overall study and wrote the manuscript.
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Figure Legends
Figure 1. Sample cystometry indicating how endpoints were determined. The top,
middle and bottom panels show bladder pressure (BP, mm Hg), bladder capacity (BC,
μl) and micturition volume (MV, μl). The arrowheads in the top panel show the points at
which resting pressure (RP), micturition threshold (MT) and micturition pressure (MP)
were determined. The vertical line labeled “BC (μl)” indicates how bladder capacity was
determined. The horizontal line labeled “IMI (s)” indicates how intermicturition was
derived. See Methods for details.
Figure 2. Representative cystometry traces from different experimental groups. For
each panel A-D and all traces the abscissae indicate Time from 0-500 s. The top trace
of each panel shows bladder pressure (BP, mmHg), the middle trace shows bladder
capacity (BC, μl) as defined by the volume of saline infused into the bladder between
micturition cycles and the bottom trace shows micturition volume (MV, μl). For
comparison the ordinates were scaled to be equivalent for all groups.
Figure 3. Quantification of the effects of different treatments on urodynamics of control
and stressed rats. A-C show micturition interval, bladder capacity and micturition
volume, respectively and D shows micturition threshold (MT), micturition pressure (MP
and resting pressure (RP) for all groups in one graph. Results of a two-way ANOVA
comparing stress and drug treatment are presented in the text. A one-way ANOVA was
also performed comparing all groups including the AAV-shRNA groups and symbols in
the figure refer to results of post-hoc tests from this analysis. *p<0.05, **p<0.01,
+p<0.06. Control/vehicle (n=10), control/NBI (n=5), stress/vehicle (n=9), stress/NBI
(n=9), stress/shRNA (n=8), stress/control RNA (n=7).
Figure 4. NBI-30775 had no effect on the stress-induced increase in the number of
CRF-immunoreactive neurons in Barrington’s nucleus. A) Fluorescent
photomicrographs of CRF-immunoreactive Barrington’s nucleus neurons of
representative control (top) and stressed (bottom) rats treated with vehicle. Top is
dorsal and medial is to the left in all photomicrographs. B) Bars represent the mean
number of CRF-immunoreactive Barrington’s nucleus neurons for controls treated with
vehicle and stressed rats treated with vehicle or NBI-30775. Newman-Keuls post-hocs:
*p<0.05 vs. Control/vehicle.
Figure 5. CRF mRNA expression in Barrington’s nucleus is reduced in stressed rats
with infusion of AAV-shRNA-CRF into Barrington’s nucleus. A) Darkfield
photomicrographs showing the CRF mRNA hybridization signal in Barrington’s nucleus
of stressed rats that were infused with AAV-shRNA-CRFscrambled (top) and AAVshRNA-CRF (bottom). The two photomicrographs were taken using identical exposure
times and brightness and contrast of the composite photograph were adjusted after the
composite was flattened so adjustments were identical for both examples. B) The bar
graph indicates the mean number of CRF mRNA expressing Barrington’s nucleus
neurons for rats infused with shRNA-CRF scrambled (n=4) and shRNA-CRF (n=6).
*p<0.05.
Figure 6. Representative cystometrograms from sham and pBOO rats. Shown are
traces from (A) a vehicle-treated sham rat, (B) a vehicle-treated pBOO rat and (C) a
pBOO rat treated with NBI-30775. For each panel A-C and all traces the abscissae
indicate Time from 0-500 s. The top trace of each panel shows bladder pressure (BP,
mmHg), the middle trace shows bladder capacity (BC, μl) as defined by the volume of
saline infused into the bladder between micturition cycles and the bottom trace shows
micturition volume (MV, μl). For comparison the ordinates were scaled to be equivalent
for all groups.
Figure 7. NBI-30775 attenuates the urodynamic effects of pBOO. Bars represent the
mean values for sham rats treated with vehicle (n=6) and pBOO rats treated with
vehicle (n=9) or NBI-30775 (n=10) for intermicturition interval (IMI, s), bladder capacity
(BC, μl), micturition volume (MV, μ), micturition threshold (MT, mm Hg), micturition
pressure (MP, mm Hg), and resting bladder pressure (RP, mm Hg). Student’s-NewmanKeuls post-hoc: *p<0.05 vs. pBOO/NBI, †p<0.05 vs. sham/vehicle, ‡p=0.06 vs.
sham/vehicle.
Figure 8. CRF mRNA expression in Barrington’s nucleus is increased in pBOO rats. A)
Darkfield photomicrographs showing the CRF mRNA hybridization signal in Barrington’s
nucleus of a sham (top) and a pBOO rat (bottom). The two photomicrographs were
taken using identical exposure times and brightness and contrast of the composite
photograph was done after the composite was flattened so adjustments were identical
for both examples. B) The bar graph indicates the mean number of CRF mRNA
expressing Barrington’s nucleus neurons for sham rats administered NBI-30775 (n=7),
pBOO rats administered vehicle (n=9) and pBOO rats administered NBI-30775 (n=6).
*p<0.05.