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1.  EARLY-IN-LIFE BLADDER INFLAMMATION ALTERS U50,488H- BUT NOT MORPHINE-INDUCED INHIBITION OF VISCEROMOTOR RESPONSES TO URINARY BLADDER DISTENSION 
Neuroscience letters  2012;534:150-154.
Previous research has suggested that early-in-life (EIL) exposure to bladder inflammation impairs the function of endogenous opioid inhibitory system(s) and may contribute to the development of chronic bladder pain. This study examined how acute adult and/or prior EIL exposure to bladder inflammation altered the inhibitory effects of systemic κ- and μ-opioid agonists on the visceromotor reflex (VMR) to urinary bladder distension (UBD). Female rats were exposed intravesically EIL (P14–P16) to either the inflammatory agent zymosan or anesthesia-alone, and then rechallenged as adults (12–17 weeks) with either anesthesia-alone or zymosan. The VMR to 60 mmHg UBD was measured after cumulative intravenous (i.v.) administration of 1 mg/kg and 4 mg/kg of either the κ-opioid agonist U50,488H or the μ-opioid agonist morphine. Morphine produced dose-dependent inhibition of the VMR to UBD in all groups, and U50,488H produced dose-dependent inhibition of the VMR to UBD in all but one group. Animals that received bladder inflammation both EIL and as adults showed significantly augmented VMRs to UBD (>100% baseline values) following 1 mg/kg of U50,488H and diminished inhibition of VMRs following 4 mg/kg of U50,488H when compared with other groups. In contrast, neither EIL nor adult bladder inflammation markedly altered the inhibition of the VMR to UBD produced by either 1 or 4 mg/kg of i.v. morphine. These data suggest EIL and adult exposure to bladder inflammation selectively decreases the inhibitory effects of κ-opioids and thereby may enhance bladder hypersensitivity in patients with painful bladder syndromes.
doi:10.1016/j.neulet.2012.11.035
PMCID: PMC3558537  PMID: 23201636
Opioid; Visceral Pain; Inflammation; Painful Bladder Syndrome; Animals; Newborn
2.  Estrous Cycle Dependent Fluctuations of Regulatory Neuropeptides in the Lower Urinary Tract of Female Rats upon Colon-Bladder Cross-Sensitization 
PLoS ONE  2014;9(5):e94872.
Co-morbidity of bladder, bowel, and non-specific pelvic pain symptoms is highly prevalent in women. Little evidence is present on modulation of pelvic pain syndromes by sex hormones, therefore, the objective of this study was to clarify the effects of hormonal fluctuations within the estrous cycle on regulatory neuropeptides in female rats using a model of neurogenic bladder dysfunction. The estrous cycle in female rats (Sprague-Dawley, 230–250 g) was assessed by vaginal smears and weight of uterine horns. Neurogenic bladder dysfunction was induced by a single inflammatory insult to the distal colon. Protein expression of calcitonin gene related peptide (CGRP), substance P (SP), nerve growth factor (NGF), and brain derived neurotrophic factor (BDNF) in the pelvic organs, sensory ganglia and lumbosacral spinal cord was compared in rats in proestrus (high estrogen) vs diestrus (low estrogen). Under normal physiological conditions, concentration of SP and CGRP was similar in the distal colon and urinary bladder during all phases of the estrous cycle, however, acute colitis induced a significant up-regulation of CGRP content in the colon (by 63%) and urinary bladder (by 54%, p≤0.05 to control) of rats in proestrus. These changes were accompanied by a significant diminution of CGRP content in L6-S2 DRG after colonic treatment, likely associated with its release in the periphery. In rats with high estrogen at the time of testing (proestrus), experimental colitis caused a significant up-regulation of BDNF colonic content from 26.1±8.5 pg/ml to 83.4±32.5 pg/ml (N = 7, p≤0.05 to control) and also induced similar effects on BDNF in the urinary bladder which was also up-regulated by 5-fold in rats in proestrus (p≤0.05 to respective control). Our results demonstrate estrous cycle dependent fluctuations of regulatory neuropeptides in the lower urinary tract upon colon-bladder cross-sensitization, which may contribute to pain fluctuations in female patients with neurogenic bladder pain.
doi:10.1371/journal.pone.0094872
PMCID: PMC4006778  PMID: 24788240
3.  Neonatal Bladder Inflammation Produces Functional Changes and Alters Neuropeptide Content in Bladders of Adult Female Rats 
Neonatal bladder inflammation has been demonstrated to produce hypersensitivity to bladder re-inflammation as an adult. The purpose of this study was to investigate the effects of neonatal urinary bladder inflammation on adult bladder function and structure. Female Sprague-Dawley rats were treated on postnatal days 14-16 with intravesical zymosan or anesthesia alone. At 12-16 weeks of age, micturition frequency and cystometrograms were measured. Similarly treated rats had their bladders removed for measurement of plasma extravasation following intravesical mustard oil, for neuropeptide analysis (CGRP or SubP), or for detailed histological examination. Rats treated with zymosan as neonates exhibited increased micturition frequency, reduced micturition volume thresholds, greater extravasation of Evan's Blue following intravesical mustard oil administration, and greater total bladder content of CGRP and SubP. In contrast, there were no quantitative histological changes in the thickness, fibrosis or mast cells of bladder tissue due to neonatal zymosan treatments. Functional changes in urologic systems observed in adulthood, coupled with the increased neuropeptide content and neurogenic plasma extravasation in adult bladders, suggest that the neonatal bladder inflammation treatment enhanced the number, function and/or neurochemical content of primary afferent neurons. These data support the hypothesis that insults to the urologic system in infancy may contribute to the development of adult bladder hypersensitivity.
Perspective
Inflammation of the bladder early in life in the rat has multiple sequelae including laboratory measures that suggest an alteration of the neurophysiological substrates related to the bladder. Some painful bladder syndromes in humans have similar characteristics and so may be due to similar mechanisms.
doi:10.1016/j.jpain.2009.07.010
PMCID: PMC2835826  PMID: 19945355
developmental; visceral nociception; hyperalgesia; interstitial cystitis
4.  Exogenous overexpression of nerve growth factor in the urinary bladder produces bladder overactivity and altered micturition circuitry in the lumbosacral spinal cord 
BMC Physiology  2007;7:9.
Background
Exogenous NGF or saline was delivered to the detrusor smooth muscle of female rats for a two-week period using osmotic mini-pumps. We then determined: (1) bladder function using conscious cystometry; (2) organization of micturition reflexes using Fos protein expression in lumbosacral (L5-S1) spinal cord neurons; (3) calcitonin gene-related peptide (CGRP)-immunoreactivity (IR) in lumbosacral spinal cord segments.
Methods
An osmotic pump infused 0.9% NaCl (n = 6) or NGF (n = 6)(2.5 μg/μl solution; 0.5 μl/hr) for two weeks into the bladder wall. NGF bladder content was determined by enzyme-linked immunoassays. Bladder function was assessed with conscious cystometry. Immunohistochemical and imaging techniques were used to determine the distribution of Fos-IR cells and CGRP expression in the L5-S1 spinal cord in saline and NGF-treated rats two hours after intravesical saline distention. Fos expression and CGRP-IR in NGF-treated rats with bladder distention was compared to that observed in cyclophosphamide (CYP; 75 mg/kg; i.p.) treated rats with bladder distention.
Results
Two-week infusion of NGF into the bladder wall increased bladder weight, reduced bladder capacity (60%), reduced the intercontraction interval (60%) and increased the amplitude of non-voiding contractions. NGF treatment and intravesical saline distention (2 hr) increased expression of Fos protein in L6-S1 spinal cord and altered the distribution pattern of Fos-IR cells. CGRP-IR in the lumbosacral spinal cord was also increased after NGF treatment.
Conclusion
These data suggest that NGF infusion into the bladder wall induces bladder overactivity, can reveal a "nociceptive" Fos expression pattern in the spinal cord in response to a non-noxious bladder stimulus and increases CGRP-IR in the lumbosacral spinal cord.
doi:10.1186/1472-6793-7-9
PMCID: PMC2000875  PMID: 17725832
5.  Neonatal Bladder Inflammation Alters Activity of Adult Rat Spinal Visceral Nociceptive Neurons 
Neuroscience letters  2010;472(3):210-214.
Purpose
This investigation examined the effect of inflammation produced by intravesical zymosan during the neonatal period on spinal dorsal horn neuronal responses to urinary bladder distension (UBD) as adults.
Methods
Female rat pups (P14-P16) were treated with intravesical zymosan or with anesthesia-only. These groups of rats were subdivided forming four groups: half received intravesical zymosan as adults and half received anesthesia-only. One day later, rats were anesthetized, the spinal cord transected at a cervical level and extracellular single-unit recordings of L6-S1 dorsal horn neurons obtained. Neurons were classified as Type I - inhibited by heterotopic noxious conditioning stimuli (HNCS) or as Type II - not inhibited by HNCS - and were characterized for spontaneous activity and responses to graded UBD (20–60 mm Hg).
Results
227 spinal dorsal horn neurons excited by UBD were characterized. In rats treated as neonates with anesthesia-only, Type II neurons demonstrated increased spontaneous and UBD-evoked activity following adult intravesical zymosan treatment whereas Type I neurons demonstrated decreased spontaneous and UBD-evoked activity relative to controls. In rats treated as neonates with intravesical zymosan, the spontaneous and UBD-evoked activity of both Type I and Type II neurons increased following adult intravesical zymosan treatment relative to controls.
Conclusions
Neonatal bladder inflammation alters subsequent effects of acute bladder inflammation on spinal dorsal horn neurons excited by UBD such that overall there is greater sensory neuron activation. This may explain the visceral hypersensitivity noted in this model system and suggest that impaired inhibitory systems may be responsible.
doi:10.1016/j.neulet.2010.02.007
PMCID: PMC2842906  PMID: 20149841
visceral; urinary bladder; cystitis; zymosan; spinal
6.  Neonatal Cystitis-Induced Colonic Hypersensitivity in Adult Rats: A Model of Viscero-Visceral Convergence 
Background
The objective of this study was to determine if neonatal cystitis alters colonic sensitivity later in life and to investigate the role of peripheral mechanisms.
Methods
Neonatal rats received intravesical zymosan, normal saline, or anesthesia only for three consecutive days (postnatal days 14th–16th). The estrous cycle phase was determined prior to recording the visceromotor response (VMR) to colorectal distension (CRD) in adult rats. Eosinophils and mast cells were examined from colon and bladder tissue. CRD or urinary bladder distension (UBD)-sensitive pelvic nerve afferents (PNAs) were identified and their responses to distension were examined. The relative expression of N-methyl-D-aspartic acid (NMDA) NR1 subunit in the L6-S1 spinal cord was examined using Western blot.
Results
The VMR to CRD (≥10mmHg) in the neonatal zymosan group was significantly higher than control in both the diestrus, estrus phase and in all phases combined. There was no difference in the total number of eosinophils, mast cells or number of degranulated mast cells between groups. The spontaneous firing of UBD, but not CRD-sensitive PNAs from the zymosan rats was significantly higher than the control. However, the mechanosensitive properties of PNAs to CRD or UBD were no different between groups (p > 0.05). The expression of spinal NR1 subunit was significantly higher in zymosan-treated rats compared to saline treated rats (p <0.05).
Conclusion
Neonatal cystitis results in colonic hypersensitivity in adult rats without changing tissue histology or the mechanosensitive properties of CRD-sensitive PNAs. Neonatal cystitis does results in overexpression of spinal NR1 subunit in adult rats.
doi:10.1111/j.1365-2982.2011.01724.x
PMCID: PMC3117950  PMID: 21592255
cystitis; visceral hyperalgesia; neonatal; viscero-visceral convergence
7.  Inflammation-Induced Enhancement of the Visceromotor Reflex to Urinary Bladder Distention: Modulation by Endogenous Opioids and the Effects of Early-In-Life Experience with Bladder Inflammation 
Abdominal electromyographic (EMG) responses to noxious intensities of urinary bladder distention (UBD) are significantly enhanced 24 hrs following zymosan-induced bladder inflammation in adult female rats. This inflammation-induced hypersensitivity is concomitantly inhibited by endogenous opioids because intraperitoneal (i.p.) naloxone administration before testing significantly increases EMG response magnitude to UBD. This inhibitory mechanism is not tonically active since naloxone does not alter EMG response magnitude to UBD in rats without inflammation. At the dose tested, naloxone does not affect bladder compliance in rats with or without inflammation. The effects of i.p. naloxone likely result from blockade of a spinal mechanism, because intrathecal (i.t.) naloxone also significantly enhances EMG responses to UBD in rats with inflammation. Rats exposed to bladder inflammation from P90-P92 prior to re-inflammation at P120 show similar hypersensitivity and concomitant opioid inhibition, with response magnitudes being no different from that produced by inflammation at P120 alone. In contrast, rats exposed to bladder inflammation from P14-P16 prior to re-inflammation at P120 show markedly enhanced hypersensitivity and no evidence of concomitant opioid inhibition. These data indicate that bladder inflammation in adult rats induces bladder hypersensitivity that is inhibited by an endogenous opioidergic mechanism. This mechanism can be disrupted by neonatal bladder inflammation.
doi:10.1016/j.jpain.2007.06.011
PMCID: PMC4012257  PMID: 17704007
bladder; opioids; inflammation; neonatal; visceromotor reflex; pain
8.  Attenuation of cocaine-seeking by progesterone treatment in female rats 
Psychoneuroendocrinology  2008;34(3):343-352.
Summary
Clinical research suggests that gender differences exist in cocaine dependence. Similarly, preclinical studies have shown that female rats exhibit higher response rates during cocaine self-administration, early extinction, and cocaine-primed reinstatement of drug-seeking. These effects are also estrous cycle dependent and inversely related to plasma progesterone, in that proestrus females (high progesterone) exhibit less cocaine-seeking, while estrous females (low progesterone) show the greatest cocaine-seeking. Based on these findings, we hypothesized that progesterone would attenuate cocaine-seeking behavior in intact, freely cycling animals. The role of the estrous cycle on cocaine-seeking behavior during early (first acquisition day) versus late (last maintenance day) cocaine self-administration was also examined. Female, Sprague-Dawley rats self-administered cocaine (0.5 mg/kg/infusion, IV) along a FR1 schedule, followed by daily extinction sessions in the absence of cocaine reinforcement. Once responding was extinguished, rats received an injection of cocaine (10 mg/kg, IP) immediately prior to reinstatement testing. Progesterone (2 mg/kg, SC) or vehicle was administered 20 and 2 h prior to the first day of extinction (early cocaine withdrawal) and the reinstatement trials. To determine estrous cycle phase, we assessed vaginal cytology prior to the first acquisition and last maintenance days of cocaine self-administration, the first day of extinction training, and each reinstatement test. During early and late cocaine self-administration, proestrus and estrous females exhibited the greatest levels of active lever responding, respectively. A significant increase in responding also occurred during cocaine-primed reinstatement for estrous versus nonestrous females, an effect that was selectively attenuated by progesterone. However, progesterone was not effective at reducing cocaine-primed reinstatement for females in other phases of the estrous cycle, nor was it effective at reducing cocaine-seeking during early withdrawal. Taken together, these results suggest that progesterone may be a useful therapeutic for preventing relapse in abstinent female cocaine users, especially when the likelihood of relapse is greatest.
doi:10.1016/j.psyneuen.2008.09.014
PMCID: PMC2675282  PMID: 18977603
progesterone; female; estrous cycle; cocaine; self-administration; reinstatement
9.  Up-Regulation of Calcitonin Gene-Related Peptide and Receptor Tyrosine Kinase TrkB in Rat Bladder Afferent Neurons following TNBS Colitis 
Experimental neurology  2007;204(2):667-679.
Colonic inflammation has profound effects on the urinary bladder physiology and produces hypersensitivity of bladder afferent neurons and neurogenic bladder overactivity. Calcitonin gene-related peptide (CGRP) expressed in dorsal root ganglia (DRG) plays an important role in mediating sensory perception following visceral inflammation. In the present study, we determined that the expression of CGRP was increased in bladder afferent neurons in lumbosacral DRG following tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in rat. After colitis, the percentage of bladder afferent neurons expressing CGRP was increased in L1 (61.2 ± 2.9 % in colitis vs 37.7 ± 5.1 % in controls; p<0.05) and S1 DRG (26.3 ± 2.3 % in colitis vs 15.5 ± 1.9 % in controls; p<0.01). We also demonstrated that the expression of tyrosine kinase receptor TrkB was increased in L1 (39.7 ± 2.9 % in colitis vs 25.2 ± 4.3 % in controls; p<0.05) and S1 DRG (45.6 ± 3.8 % in colitis vs 38.3 ± 3.6 % in controls; p<0.01) following colitis. CGRP and TrkB were co-stored in a subpopulation of DRG neurons in control and colitic animals and the number of DRG cells co-expressing CGRP and TrkB was significantly increased in L1 (2.7-fold, p< 0.01) and S1 DRG (2.4-fold, p<0.01) following colitis. In cultured DRG, exogenous BDNF application significantly increased CGRP expression, which was blocked by TrkB selective inhibitor K252a. These results suggest that up-regulation of CGRP and TrkB in bladder afferent neurons may play a role in colon-to-bladder cross-sensitization following colitis.
doi:10.1016/j.expneurol.2006.12.024
PMCID: PMC1906719  PMID: 17303123
visceral inflammation; CGRP; neurotrophins; primary afferent neuron; convergence; cross-sensitization
10.  Portable Bladder Ultrasound 
Executive Summary
Objective
The aim of this review was to assess the clinical utility of portable bladder ultrasound.
Clinical Need: Target Population and Condition
Data from the National Population Health Survey indicate prevalence rates of urinary incontinence are 2.5% in women and 1.4 % in men in the general population. Prevalence of urinary incontinence is higher in women than men and prevalence increases with age.
Identified risk factors for urinary incontinence include female gender, increasing age, urinary tract infections (UTI), poor mobility, dementia, smoking, obesity, consuming alcohol and caffeine beverages, physical activity, pregnancy, childbirth, forceps and vacuum-assisted births, episiotomy, abdominal resection for colorectal cancer, and hormone replacement therapy.
For the purposes of this review, incontinence populations will be stratified into the following; the elderly, urology patients, postoperative patients, rehabilitation settings, and neurogenic bladder populations.
Urinary incontinence is defined as any involuntary leakage of urine. Incontinence can be classified into diagnostic clinical types that are useful in planning evaluation and treatment. The major types of incontinence are stress (physical exertion), urge (overactive bladder), mixed (combined urge and stress urinary incontinence), reflex (neurological impairment of the central nervous system), overflow (leakage due to full bladder), continuous (urinary tract abnormalities), congenital incontinence, and transient incontinence (temporary incontinence).
Postvoid residual (PVR) urine volume, which is the amount of urine in the bladder immediately after urination, represents an important component in continence assessment and bladder management to provide quantitative feedback to the patient and continence care team regarding the effectiveness of the voiding technique. Although there is no standardized definition of normal PVR urine volume, measurements greater than 100 mL to 150 mL are considered an indication for urinary retention, requiring intermittent catheterization, whereas a PVR urine volume of 100 mL to 150 mL or less is generally considered an acceptable result of bladder training.
Urinary retention has been associated with poor outcomes including UTI, bladder overdistension, and higher hospital mortality rates. The standard method of determining PVR urine volumes is intermittent catheterization, which is associated with increased risk of UTI, urethral trauma and discomfort.
The Technology Being Reviewed
Portable bladder ultrasound products are transportable ultrasound devices that use automated technology to register bladder volume digitally, including PVR volume, and provide three-dimensional images of the bladder. The main clinical use of portable bladder ultrasound is as a diagnostic aid. Health care professionals (primarily nurses) administer the device to measure PVR volume and prevent unnecessary catheterization. An adjunctive use of the bladder ultrasound device is to visualize the placement and removal of catheters. Also, portable bladder ultrasound products may improve the diagnosis and differentiation of urological problems and their management and treatment, including the establishment of voiding schedules, study of bladder biofeedback, fewer UTIs, and monitoring of potential urinary incontinence after surgery or trauma.
Review Strategy
To determine the effectiveness and clinical utility of portable bladder ultrasound as reported in the published literature, the Medical Advisory Secretariat used its standard search strategy to retrieve international health technology assessments and English-language journal articles from selected databases. Nonsystematic reviews, nonhuman studies, case reports, letters, editorials, and comments were excluded.
Summary of Findings
Of the 4 included studies that examined the clinical utility of portable bladder ultrasound in the elderly population, all found the device to be acceptable. One study reported that the device underestimated catheterized bladder volume
In patients with urology problems, 2 of the 3 studies concerning portable bladder ultrasound found the device acceptable to use. However, one study did not find the device as accurate for small PVR volume as for catheterization and another found that the device overestimated catheterized bladder volume. In the remaining study, the authors reported that when the device’s hand-held ultrasound transducers (scanheads) were aimed improperly, bladders were missed, or lateral borders of bladders were missed resulting in partial bladder volume measurements and underestimation of PVR measurements. They concluded that caution should be used in interpreting PVR volume measured by portable bladder ultrasound machines and that catheterization may be the preferred assessment modality if an accurate PVR measurement is necessary.
All 3 studies with post-operative populations found portable bladder ultrasound use to be reasonably acceptable. Two studies reported that the device overestimated catheter-derived bladder volumes, one by 7% and the other by 21 mL. The third study reported the opposite, that the device underestimated catheter bladder volume by 39 mL but that the results remained acceptable
In rehabilitation settings, 2 studies found portable bladder ultrasound to underestimate catheter-derived bladder volumes; yet, both authors concluded that the mean errors were within acceptable limits.
In patients with neurogenic bladder problems, 2 studies found portable bladder ultrasound to be an acceptable alternative to catheterization despite the fact that it was not as accurate as catheterization for obtaining bladder volumes.
Lastly, examinations concerning avoidance of negative health outcomes showed that, after use of the portable bladder ultrasound, unnecessary catheterizations and UTIs were decreased. Unnecessary catheterizations avoided ranged from 16% to 47% in the selected articles. Reductions in UTI ranged from 38% to 72%.
In sum, all but one study advocated the use of portable bladder ultrasound as an alternative to catheterization.
Economic Analysis
An economic analysis estimating the budget-impact of BladderScan in complex continuing care facilities was completed. The analysis results indicated a $192,499 (Cdn) cost-savings per year per facility and a cost-savings of $2,887,485 (Cdn) for all 15 CCC facilities. No economic analysis was completed for long-term care and acute care facilities due to lack of data.
Considerations for Policy Development
Rapid diffusion of portable bladder ultrasound technology is expected. Recently, the IC5 project on improving continence care in Ontario’s complex continuing care centres piloted portable bladder ultrasound at 12 sites. Preliminary results were promising.
Many physicians and health care facilities already have portable bladder ultrasound devices. However, portable bladder ultrasound devices for PVR measurement are not in use at most health care facilities in Ontario and Canada. The Verathon Corporation (Bothell, Wisconsin, United States), which patents BladderScan, is the sole licensed manufacturer of the portable bladder ultrasound in Canada. Field monopoly may influence the rising costs of portable bladder ultrasound, particularly when faced with rapid expansion of the technology.
Several thousand residents of Ontario would benefit from portable bladder ultrasound. The number of residents of Ontario that would benefit from the technology is difficult to quantify, because the incidence and prevalence of incontinence are grossly under-reported. However, long-term care and complex continuing care institutions would benefit greatly from portable bladder ultrasound, as would numerous rehabilitation units, postsurgical care units, and urology clinics.
The cost of the portable bladder ultrasound devices ranges from $17,698.90 to $19,565.95 (Cdn) (total purchase price per unit as quoted by the manufacturer). Additional training packages, batteries and battery chargers, software, gel pads, and yearly warranties are additional costs. Studies indicate that portable bladder ultrasound is a cost-effective technology, because it avoids costs associated with catheterization equipment, saves nursing time, and reduces catheter-related complications and UTIs.
The use of portable bladder ultrasound device will affect the patient directly in terms of health outcomes. Its use avoids the trauma related to the urinary tract that catheterization inflicts, and does not result in UTIs. In addition, patients prefer it, because it preserves dignity and reduces discomfort.
PMCID: PMC3379524  PMID: 23074481
11.  Endogenous neurotensin is involved in estrous cycle related alterations in prepulse inhibition of the acoustic startle reflex in female rats 
Psychoneuroendocrinology  2007;33(2):178-187.
Summary
Ovarian hormones regulate prepulse inhibition (PPI) of the acoustic startle reflex. Results from studies in intact female rodents investigating sex, estrous cycle and ovarian hormone regulation of PPI are inconsistent. In experiment #1, we investigated whether PPI in female rats is influenced by the time of day of testing and the estrous cycle stage of the rat. PPI was examined across the day of proestrus (P) and diestrus 1 (D1) in female rats and compared to males. PPI in males and P females was significantly higher than in D1 females. PPI in males and D1 females was significantly affected by the time of day of testing with PPI being reduced in the afternoon and evening compared to morning. PPI in P females was not significantly affected by the time of day of testing. Previous studies have demonstrated estrous cycle regulation of central nervous system neurotensin (NT) neurons and peripherally administered NT receptor agonists regulate PPI in a manner similar to antipsychotic drugs. Experiment #2 of this study was designed to examine whether endogenous NT is involved in estrous cycle regulation of PPI. The NT receptor antagonist SR 142948A reduced the high levels of PPI during D1 and P. In contrast, when tested at a time of day in which PPI was low in D1 females, administration of both the typical antipsychotic drug haloperidol and the NT receptor antagonist significantly increased PPI. These data support an effect of time of day and estrous cycle stage on PPI in female rats. The estrous cycle variations in PPI are mediated in part by endogenous NT.
doi:10.1016/j.psyneuen.2007.11.005
PMCID: PMC2254501  PMID: 18155361
sensorimotor gating; schizophrenia; ovarian hormones; proestrus; diestrus; antipsychotic drug
12.  Inflammation and enhanced nociceptive responses to bladder distension produced by intravesical zymosan in the rat 
BMC Urology  2006;6:2.
Background
Mycotic infections of the bladder produce pain and inflammatory changes. The present study examined the inflammatory and nociceptive effects of the yeast cell wall component, zymosan, when admininstered into the urinary bladder in order to characterize this form of bladder sensitization.
Methods
Parametric analyses of the time-course (0–48 hr) and concentration (0–2% solutions) variables associated with intravesical zymosan-induced bladder inflammation were performed in female rats. Plasma extravasation of Evan's Blue dye was used as a measure of tissue inflammation. Cardiovascular and visceromotor responses to urinary bladder distension were used as measures of nociception.
Results
Zymosan-induced bladder inflammation, as indexed by plasma extravasation of Evan's Blue, was significantly greater in rats treated with either 1 or 2% solutions as compared to either 0.1 or 0.5% zymosan solutions. In time-course studies (1 – 48 hr post-treatment), 1% zymosan-induced inflammation progressively increased with time following administration, was greatest at 24 hr and began to normalize by 48 hr. In the studies of inflammation-induced changes in nociception, arterial blood pressure (ABP) and visceromotor responses to graded distension of the urinary bladder were significantly increased relative to controls 24 hr after zymosan administration.
Conclusion
These studies provide important time-course and solution concentration parameters for studies of zymosan-induced inflammation of the bladder and suggest utility of this model for the study of bladder-related pain.
doi:10.1186/1471-2490-6-2
PMCID: PMC1395324  PMID: 16469099
13.  Urodynamic Findings in an Awake Chemical Cystitis Rat Model Observed by Simultaneous Registrations of Intravesical and Intraabdominal Pressures 
Purpose
The aim of this study was to investigate the effect of urinary bladder inflammation on bladder function in a rat chemical cystitis model. We also histologically confirmed the effects of inflammation in the detrusor on chronically inflamed bladder in rats.
Materials and Methods
A total of 13 female Sprague-Dawley rats were used in this study. In seven rats, intravesical instillation of HCl induced chemical cystitis, and the other rats with intravesical instillation of saline were used as the sham. After 2 weeks, cystometrograms were obtained with additional intraabdominal pressure measurements in all unanesthetized, unrestrained rats in metabolic cages. The rats were killed just after cystometry. The bladders were removed and examined histologically for mast cells and inflammatory changes.
Results
The rats with acute injury by HCl showed no differences in pressure parameters, including basal pressure, threshold pressure, and maximum bladder pressure, compared with the sham rats. They showed significantly increased bladder capacity, micturition volume, residual volume, and micturition interval compared with the sham group. They also showed an increased frequency of detrusor overactivity compared with the sham group. The percent of detrusor overactivity was 56.3% among the total intravesical pressure rises above 2 cmH2O. The histological findings of the rats with acute injury by HCl were consistent with chemical cystitis.
Conclusions
Overlapping patterns of lower urinary tract symptoms and pelvic pain are common disease characteristics among interstitial cystitis patients. The situation in an animal model of interstitial cystitis is similar, as observed in this study by the histologic and awake cystometric examinations. However, the interstitial cystitis model showed detrusor overactivity during the filling phase without a decrease in bladder capacity and micturition intervals, which differs from the characteristics of overactive bladder patients.
doi:10.5213/inj.2010.14.1.54
PMCID: PMC2989476  PMID: 21120177
Cystitis, interstitial; Urinary bladder; Cystometry; Rat
14.  Enhancement of cue-induced reinstatement of cocaine-seeking in rats by yohimbine: sex differences and the role of the estrous cycle 
Psychopharmacology  2011;216(1):53-62.
Rationale
Previous studies have shown that female rats exhibit enhanced cocaine-seeking across several phases of the addiction cycle when compared to males. Drug-seeking in females is also estrous cycle dependent and inversely associated with plasma progesterone. Although sex and estrous cycle-dependent differences have been reported in the reinstatement of cocaine-seeking triggered by cocaine injections or drug-paired cues, it is not yet known what role the estrous cycle may have on stress-induced reinstatement, either alone or in combination with drug-paired cues.
Objectives
Here, we examined male and female rats for reinstatement of extinguished cocaine-seeking produced by cocaine-paired cues or the stress-activating drug, yohimbine.
Methods
Male and female Sprague-Dawley rats self-administered intravenous cocaine (0.5 mg/kg/infusion) paired with a light+tone stimulus for 10–14 days. Lever responding was then allowed to extinguish, with subsequent reinstatement testing occurring 30 min following an injection of yohimbine (1.25 or 2.5 mg/kg, intraperitoneal) or vehicle either in the presence or absence of the conditioned stimulus.
Results
While males and females showed similar cue- and yohimbine-induced reinstatement (3–4 times over “No Cue”-vehicle responding), combining these stimuli resulted in a robust enhancement in cocaine-seeking in both groups, with a greater increase in females (10–12 vs 14–15 times over “No Cue”-vehicle responding for the males and females, respectively). When examined as a function of the estrous cycle, females in proestrus demonstrated higher levels of responding during yohimbine + cues reinstatement.
Conclusions
This cycle-dependent enhanced sensitivity to stress enhancement of cocaine-paired cues may generalize to greater relapse susceptibility under stressful conditions.
doi:10.1007/s00213-011-2187-6
PMCID: PMC3195378  PMID: 21308466
sex differences; cocaine; relapse; reinstatement; stress; yohimbine; cues; estrous cycle
15.  Experimental colitis triggers the release of substance P and calcitonin gene-related peptide in the urinary bladder via TRPV1 signaling pathways 
Experimental neurology  2010;225(2):262-273.
Clinical data provides evidence of high level of co-morbidity among genitourinary and gastrointestinal disorders characterized by chronic pelvic pain. The objective of this study was to test the hypothesis that colonic inflammation can impact the function of the urinary bladder via activation of TRPV1 signaling pathways followed by alterations in gene and protein expression of Substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons and in the bladder. Inflammation was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS, 12.5 mg/kg) and desensitization of TRPV1 receptors was evoked by intracolonic resiniferatoxin (RTX, 10−7 M). mRNA and protein concentrations of CGRP and SP were measured at 3, 5 and 30 days. RTX instillation in the colon caused 3-fold up-regulation of SP mRNA in the urinary bladder at day 5 (n=7, p≤0.05) followed by 35-fold increase at day 30 (n=5, p≤0.05). Likewise, TNBS colitis triggered 15.8-fold up-regulation of SP mRNA one month after TNBS (n=5, p≤0.05). Desensitization of colonic TRPV1 receptors prior to TNBS abolished SP increase in the urinary bladder. RTX led to 4.3-fold increase of CGRP mRNA at day 5 (n=7, p≤0.05 to control) in the bladder followed by 28-fold increase at day 30 post-RTX (n=4, p≤0.05). Colitis did not alter CGRP concentration during acute phase, however, at day 30 mRNA level was increased by 17.8±6.9 fold (n=5, p≤0.05) in parallel with 4-fold increase in CGRP protein (n=5, p≤0.01) in the detrusor. Protein concentration of CGRP in the spinal cord was diminished by 45–65% (p≤0.05) during colitis. RTX pretreatment did not affect CGRP concentration in the urinary bladder, however, caused a reduction in CGRP release from lumbosacral DRG neurons during acute phase (3 and 5 days post-TNBS). Our results clearly demonstrate that colonic inflammation triggers the release of pro-inflammatory neuropeptides SP and CGRP in the urinary bladder via activation of TRPV1 signaling mechanisms enunciating the neurogenic nature of pelvic organ cross-sensitization.
doi:10.1016/j.expneurol.2010.05.012
PMCID: PMC2939259  PMID: 20501335
inflammation; cross-sensitization; neuropeptides; pelvic pain
16.  Colitis Induces Calcitonin Gene-related Peptide Expression and Akt Activation in Rat Primary Afferent Pathways 
Experimental neurology  2009;219(1):93-103.
Previous study has shown that colitis-induced increases in calcitonin gene-related peptide (CGRP) immunoreactivity in bladder afferent neurons result in sensory cross-sensitization. To further determine the effects of colitis on CGRP expression in neurons other than bladder afferents, we examined and compared the levels of CGRP mRNA and immunoreactivity in the lumbosacral dorsal root ganglia (DRG) and spinal cord before and during colitis in rats. We also examined the changes in CGRP immunoreactivity in colonic afferent neurons during colitis. Results showed increases in CGRP mRNA levels in L1 (2.5-fold, p<0.05) and S1 DRG (1.9-2.4-fold, p<0.05). However, there were no changes in CGRP mRNA levels in L1 and S1 spinal cord during colitis. CGRP protein was significantly increased in L1 (2.5-fold increase, p<0.05) but decreased in S1 (50% decrease, p<0.05) colonic afferent neurons, which may reflect CGRP release from these neurons during colitis. In L1 spinal cord, colitis caused increases in the number of CGRP nerve fibers in the deep lamina region extending to the gray commissure where the number of phospho-Akt neurons was also increased. In S1 spinal cord, colitis caused the increases in the intensity of CGRP fibers in the regions of dorsolateral tract, and caused the increases in the level of phospho-Akt in the superficial dorsal horn of the spinal cord. In spinal cord slice culture, exogenous CGRP increased the phosphorylation level of Akt but not the phosphorylation level of extracellular-signal regulated kinase ERK1/2 even though our previously studies showed that colitis increased the phosphorylation level of ERK1/2 in L1 and S1 spinal cord. These results suggest that CGRP is synthesized in the DRG and may transport to the spinal cord where initiates signal transduction during colitis.
doi:10.1016/j.expneurol.2009.04.026
PMCID: PMC2728778  PMID: 19422825
colitis; CGRP; Akt; DRG; spinal cord
17.  Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons 
Molecular Pain  2012;8:48.
Background
Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated.
Results
In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and phospho-CREB, and reverses cystitis-induced increases in micturition frequency.
Conclusions
A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.
doi:10.1186/1744-8069-8-48
PMCID: PMC3502118  PMID: 22742729
ERK5; Akt; NGF; CGRP; DRG
18.  Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery 
British Journal of Pharmacology  1998;123(8):1645-1654.
Effects of adrenomedullin and α-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca2+ concentrations ([Ca2+]i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and α-CGRP were also investigated.Adrenomedullin and α-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC50 values for adrenomedullin and α-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (L-NMMA, 100 μM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 μM).Adrenomedullin and α-CGRP increased the endothelial [Ca2+]i in the rat aorta with endothelium, whereas they did not change [Ca2+]i in the smooth muscle.An antagonist of the CGRP1 receptor, CGRP (8–37), antagonized the relaxant effects of α-CGRP and the β-isoform of CGRP (β-CGRP) but not those of adrenomedullin in the rat aorta.In the porcine coronary artery contracted with U46619, adrenomedullin and α-CGRP caused a concentration-dependent relaxation with an IC50 of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC50 values nor the maximal relaxations induced by adrenomedullin or α-CGRP. When the artery was contracted with high K+ solution (72.7 mM), these peptides caused a small relaxation.Adrenomedullin and α-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca2+]i in the porcine coronary artery.CGRP (8–37) significantly antagonized the relaxant effects of adrenomedullin and α-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the β-isoform of CGRP (β-CGRP).These results suggest that in the rat aorta, adrenomedullin and α-CGRP increase the endothelial [Ca2+]i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and α-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca2+]i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to α-CGRP, β-CGRP and CGRP (8–37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to α-CGRP, CGRP (8–37) and adrenomedullin and the other is sensitive only to β-CGRP.
doi:10.1038/sj.bjp.0701805
PMCID: PMC1565343  PMID: 9605572
Adrenomedullin; α-calcitonin gene-related peptide (α-CGRP): β-CGRP; CGRP (8–37); cyclic AMP; endothelium; smooth muscle; calcium
19.  Down-Regulation of Nerve Growth Factor Expression in the Bladder by Antisense Oligonucleotides as New Treatment for Overactive Bladder 
The Journal of urology  2013;190(2):757-764.
Purpose
Nerve growth factor over expression in the bladder has a role in overactive bladder symptoms via the mediation of functional changes in bladder afferent pathways. We studied whether blocking nerve growth factor over expression in bladder urothelium by a sequence specific gene silencing mechanism would suppress bladder overactivity and chemokine expression induced by acetic acid.
Materials and Methods
Female Sprague-Dawley® rats anesthetized with isoflurane were instilled with 0.5 ml saline, scrambled or TYE™ 563 labeled antisense oligonucleotide targeting nerve growth factor (12 μM) alone or complexed with cationic liposomes for 30 minutes. The efficacy of nerve growth factor antisense treatments for acetic acid induced bladder overactivity was assessed by cystometry. Bladder nerve growth factor expression levels and cellular distribution were quantified by immunofluorescence staining and enzyme-linked immunosorbent assay. Effects on bladder chemokine expression were measured by Luminex® xMAP® analysis.
Results
Liposomes were needed for bladder uptake of oligonucleotide, as seen by the absence of bright red TYE 563 fluorescence in rats instilled with oligonucleotide alone. At 24 hours after liposome-oligonucleotide treatment baseline bladder activity during saline infusion was indistinct in the sham and antisense treated groups with a mean ± SEM intercontraction interval of 348 ± 55 and 390 ± 120 seconds, respectively. Acetic acid induced bladder overactivity was shown by a decrease in the intercontraction interval to a mean of 33.2% ± 4.0% of baseline in sham treated rats. However, the reduction was blunted to a mean of 75.8% ± 3.4% of baseline in rats treated with liposomal antisense oligonucleotide (p <0.05). Acetic acid induced increased nerve growth factor in the urothelium of sham treated rats, which was decreased by antisense treatment, as shown by enzyme-linked immunosorbent assay and reduced nerve growth factor immunoreactivity in the urothelium. Increased nerve growth factor in bladder tissue was associated with sICAM-1, sE-selectin, CXCL-10 and 1, leptin, MCP-1 and vascular endothelial growth factor over expression, which was significantly decreased by nerve growth factor antisense treatment (p <0.01).
Conclusions
Acetic acid induced bladder overactivity is associated with nerve growth factor over expression in the urothelium and with chemokine up-regulation. Treatment with liposomal antisense suppresses bladder overactivity, and nerve growth factor and chemokine expression. Local suppression of nerve growth factor in the bladder could be an attractive approach for overactive bladder. It would avoid the systemic side effects that may be associated with nonspecific blockade of nerve growth factor expression.
doi:10.1016/j.juro.2013.02.090
PMCID: PMC3734554  PMID: 23454160
urinary bladder; overactive; nerve growth factor; urothelium; chemokines; liposomes
20.  Acute Bladder Inflammation Differentially Affects Rat Spinal Visceral Nociceptive Neurons 
Neuroscience letters  2009;467(2):150-154.
Purpose
The present investigation examined the effect of inflammation produced by intravesical zymosan on spinal dorsal horn neuronal responses to urinary bladder distension (UBD).
Methods
Extracellular single-unit recordings of neurons excited by UBD were obtained in spinalized female Sprague-Dawley rats. Neurons were classified as Type I - inhibited by heterotopic noxious conditioning stimuli (HNCS) or as Type II - not inhibited by a HNCS. In Experiment 1 - following neuronal characterization, 1% zymosan was infused into the bladder and after two hours spinal units were recharacterized. Control rats received intravesical saline or subcutaneous zymosan. In Experiment 2 – rats were pretreated with intravesical zymosan 24 hours prior to surgical preparation. Control rats only received anesthesia.
Results
137 spinal dorsal horn neurons excited by UBD were characterized. In comparison with controls, Type II neurons demonstrated increased spontaneous and UBD-evoked activity following intravesical zymosan treatment (both Experiments 1 & 2) whereas Type I neurons demonstrated either no change (Experiment 1) or decreased activity (Experiment 2) following bladder inflammation. No significant changes were noted in neuronal activity in control experiments.
Conclusions
Inflammation differentially affects subpopulations of spinal dorsal horn neurons excited by UBD that can be differentiated according to the effect of HNCS. This results in an altered pattern of spinal sensory transmission that may serve as the mechanism for the generation of visceral nociception.
doi:10.1016/j.neulet.2009.10.027
PMCID: PMC2783714  PMID: 19822190
visceral; urinary bladder; cystitis; zymosan; spinal
21.  Role of c-Jun N-terminal Kinase (JNK) activation in micturition reflexes in cyclophosphamide (CYP)-induced cystitis in female rats 
c-Jun N-terminal Kinase (JNK) is member of the Mitogen-Activated Protein Kinase (MAPK) family, activated through phosphorylation following cytokine exposure and stress. In this study, phosphorylation of JNK was examined in the urinary bladder with CYP-induced cystitis and the effects of SP600125, a selective inhibitor of phosphorylation of JNK, on urinary bladder function were assessed using conscious, open outlet, cystometry with continuous instillation of intravesical saline. We induced bladder inflammation in adult female Wistar rats by injecting CYP intraperitoneally to produce acute (150 mg/kg; 4 hr), intermediate (150 mg/kg; 48 hr) and chronic (75 mg/kg; every third day for 10 days) treatments. Western blotting of urinary bladder demonstrated a significant (p ≤ 0.01) increase (i.e., phosphorylation) in JNK activation with 4 hr and 48 hr CYP-induced cystitis. Immunohistochemistry and image analyses demonstrated a significant (p ≤ 0.01) increase in JNK activation in the urothelium with 4 hr and 48 hr CYP-induced cystitis. Blockade of JNK phosphorylation significantly (p ≤ 0.01) increased bladder capacity and intercontraction void intervals in CYP-treated rats (4 hr and 48 hr). Furthermore, blockade of JNK phosphorylation reduced (p ≤ 0.01) neuropeptide (substance P, calcitonin gene-related peptide) expression in the urinary bladder with CYP-induced cystitis (4 hr and 48 hr). In contrast, blockade of JNK phosphorylation was without effect on bladder function or neuropeptide expression in urinary bladder in control (no inflammation) rats. Blockade of JNK phosphorylation may represent a novel target for improving urinary bladder function with CYP-induced cystitis.
doi:10.1007/s12031-014-0308-5
PMCID: PMC4209338  PMID: 24763745
micturition; western blot; cystometry; phosphorylation; IHC; neuropeptides
22.  Preclinical assessment of potential interactions between botulinum toxin and neuromodulation for bladder micturition reflex 
BMC Urology  2015;15:50.
Background
While botulinum toxin A (BoNT-A) has become a more commonly used second-line treatment for patients with detrusor overactivity, it remains unknown whether the impacts of this therapy may persist to influence other therapies such as sacral neuromodulation. In this preclinical study we have evaluated urodynamic functions to intradetrusor injection of BoNT-A and the bladder inhibitory effects of spinal nerve stimulation (SNS) following BoNT-A treatment.
Methods
Female rats were anesthetized with 3 % isoflurane. BoNT-A (2 units, 0.2 ml) or saline were injected into the detrusor. Rats then were housed for 2 days to 1 month before neuromodulation study. Monopolar electrodes were placed under each of the L6 spinal nerve bilaterally under urethane anesthesia. A bladder cannula was inserted via the urethra for saline infusion and intravesical pressure recording.
Results
Intradetrusor injection of BoNT-A for 1–2 weeks or 1 month significantly increased bladder capacity compared with saline injection (p < 0.05, two-way ANOVA). Following BoNT-A, SNS attenuated the frequency of bladder contractions, either eliminating bladder contractions or reducing the contraction frequency during electrical stimulation. Inhibition of the contraction frequency by SNS following BoNT-A treated rats was not different from that measured following saline injection.
Conclusions
BoNT-A increased the bladder capacity, but compensating for additional saline infusion to the enlarged urinary bladder in BoNT-A pretreated rats, the bladder contractions induced by bladder filling were attenuated by SNS. BoNT-A did not alter the ability of SNS to inhibit bladder contraction following intradetrusor injection of BoNT-A for 2 days, 1–2 weeks or 1 month. These results support further pre-clinical and clinical studies to evaluate potential interactions or combination therapy with neuromodulation and intradetrusor BoNT-A therapeutic approaches.
doi:10.1186/s12894-015-0048-z
PMCID: PMC4460855  PMID: 26055982
Electrical stimulation; Micturition; Spinal nerve; Botulinum toxin
23.  Urodynamic effects of oxybutynin and tolterodine in conscious and anesthetized rats under different cystometrographic conditions 
BMC Pharmacology  2005;5:14.
Background
Antimuscarinic agents are the most popular treatment for overactive bladder and their efficacy in man is well documented, producing decreased urinary frequency and an increase in bladder capacity. During cystometry in rats, however, the main effect reported after acute treatment with antimuscarinics is a decrease in peak micturition pressure together with little or no effect on bladder capacity. In the present experiments we studied the effects, in rats, of the two most widely used antimuscarinic drugs, namely oxybutynin and tolterodine, utilising several different cystometrographic conditions. The aim was to determine the experimental conditions required to reproduce the clinical pharmacological effects of antimuscarinic agents, as seen in humans, in particular their ability to increase bladder capacity.
Results
Intravenous or oral administration of tolterodine or oxybutynin in conscious rats utilized 1 day after catheter implantation and with saline infusion at constant rate of 0.1 ml/min, gave a dose-dependent decrease of micturition pressure (MP) with no significant change in bladder volume capacity (BVC). When the saline infusion rate into the bladder was decreased to 0.025 ml/min, the effect of oral oxybutynin was similar to that obtained with the higher infusion rate. Also, experiments were performed in rats in which bladders were infused with suramin (3 and 10 μM) in order to block the non-adrenergic, non-cholinergic component of bladder contraction. Under these conditions, oral administration of oxybutynin significantly reduced MP (as observed previously), but again BVC was not significantly changed.
In conscious rats with bladders infused with diluted acetic acid, both tolterodine and oxybutynin administered at the same doses as in animals infused with saline, reduced MP, although the reduction appeared less marked, with no effect on BVC.
In conscious rats utilized 5 days after catheter implantation, a situation where inflammation due to surgery is reduced, the effect of tolterodine (i.v.) and oxybutynin (p.o.) on MP was smaller and similar, respectively, to that observed in rats utilized 1 day after catheter implantation, but the increase of BVC was not statistically significant.
In anesthetized rats, i.v. administration of oxybutynin again induced a significant decrease in MP, although it was of questionable relevance. Both BVC and threshold pressure were not significantly reduced. The number and amplitude of high frequency oscillations in MP were unmodified by treatment.
Finally, in conscious obstructed rats, intravenous oxybutynin did not modify frequency and amplitude of non-voiding contractions or bladder capacity and micturition volume.
Conclusion
Despite the different experimental conditions used, the only effect on cystometrographic parameters of oxybutynin and tolterodine in anesthetized and conscious rats was a decrease in MP, whereas BVC was hardly and non-significantly affected. Therefore, it is difficult to reproduce in rats the cystometrographic increase in BVC as observed in humans after chronic administration of antimuscarinic agents, whereas the acute effects seem more similar.
doi:10.1186/1471-2210-5-14
PMCID: PMC1274333  PMID: 16216132
24.  Intermedin in rat uterus: changes in gene expression and peptide levels across the estrous cycle and its effects on uterine contraction 
Background
The present study demonstrates the expression of intermedin (IMD) and its receptor components in the uterus of the female rat during the estrous cycle and its effect on uterine contraction.
Methods
The gene expression level of intermedin and its receptor components and the peptide level of intermedin were studied by real-time RT-PCR and enzyme immunoassay (EIA) respectively. The separation of precursor and mature IMD was studied by gel filtration chromatography and EIA. The localization of IMD in the uterus was investigated by immunohistochemistry. The effect of IMD on in vitro uterine contraction was studied by organ bath technique.
Results
Uterine mRNAs of Imd and its receptor components and IMD levels displayed cyclic changes across the estrous cycle. Imd mRNA level was the highest at proestrus while the IMD level was the highest at diestrus. IMD was found in the luminal and glandular epithelia and IMD treatment significantly reduced the amplitude and frequency of uterine contraction but not the basal tone. Both calcitonin gene-related peptide (CGRP) receptor antagonist hCGRP8-37 and adrenomedullin (ADM) receptor antagonist hADM22-52 partially abolished the inhibitory effect of IMD on uterine contraction while the specific IMD receptor antagonist hIMD17-47 completely blocked the actions. The enzyme inhibitors of NO (L-NAME) and PI3K (Wortmannin) pathways diminished the IMD effects on uterine contraction while the cAMP/PKA blocker, KT5720, had no effect, indicating an involvement of NO and PI3K/Akt but not PKA.
Conclusions
IMD and the gene expression of its receptor components are differentially regulated in the uterus during the estrous cycle and IMD inhibits uterine contraction by decreasing the amplitude and frequency.
doi:10.1186/1477-7827-11-13
PMCID: PMC3598482  PMID: 23442365
Intermedin; Estrous cycle; Uterine contraction
25.  Maternal-infant separation impedes changes in feeding behavior during estrous cycle of rats 
Experimental Animals  2015;64(4):383-390.
Traumatic and stressful events during childhood are associated with the development of eating disorders. We conducted an animal study to test if association stress in childhood affects ingestive behavior later in life by using female rats that have an adjusted estrous cycle. First, electrical impedance of the vagina was conducted to test estrous cycle adjustment. Second, the effects of 6 h per day maternal separation from birth to weaning, which models a psychologically stressful experience in childhood, was used to test feeding behavior during an ovarian cycle in female adult rats with matched estrous cycles. Food and water intake in maternal separated and non-separated rats was measured in each estrous phase. Non-separated rats showed periodical changes, but maternal separated rats showed no significant changes in food and water intake during an estrous cycle. An opposing tendency for food and water intake was seen between maternal separated and non-separated rats. These observations suggest that electrical impedance of the vagina showed the highest value in the estrous phase of rats housed in a reversed light-dark cycle, and maternal separation was found to disturb changes in feeding behavior during the estrous cycle.
doi:10.1538/expanim.15-0020
PMCID: PMC4637375  PMID: 26119792
eating disorders; electrical impedance of the vagina; estrous cycle; female rat; maternal separation

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