Cyclophosphamide Cystitis Research Articles

Corticotropin-Releasing Hormone from the Pontine Micturition Center Plays an Inhibitory Role in Micturition.

Lower urinary tract or voiding disorders are prevalent across all ages and affect >40% of adults over 40 years old, leading to decreased quality of life and high health care costs. The pontine micturition center (PMC; i.e., Barrington's nucleus) contains a large population of neurons that localize the stress-related neuropeptide, corticotropin-releasing hormone (CRH) and project to neurons in the spinal cord to regulate micturition. How the PMC and CRH-expressing neurons in the PMC control volitional micturition is of critical importance for human voiding disorders. To investigate the specific role of CRH in the PMC, neurons in the PMC-expressing CRH were optogenetically activated during in vivo cystometry in unanesthetized mice of either sex. Optogenetic activation of CRH-PMC neurons led to increased intermicturition interval and voided volume, similar to the altered voiding phenotype produced by social stress. Female mice showed a significantly more pronounced phenotype change compared with male mice. These effects were eliminated by CRH-receptor 1 antagonist pretreatment. Optogenetic inhibition of CRH-PMC neurons led to an altered voiding phenotype characterized by more frequent voids and smaller voided volumes. Last, in a cyclophosphamide cystitis model of bladder overactivity, optogenetic activation of CRH-PMC neurons returned the voiding pattern to normal. Collectively, our findings demonstrate that CRH from PMC spinal-projecting neurons has an inhibitory function on micturition and is a potential therapeutic target for human disease states, such as voiding postponement, urinary retention, and underactive or overactive bladder.SIGNIFICANCE STATEMENT The pontine micturition center (PMC), which is a major regulator of volitional micturition, is neurochemically heterogeneous, and excitatory neurotransmission derived from PMC neurons is thought to mediate the micturition reflex. In the present study, using optogenetic manipulation of CRH-containing neurons in double-transgenic mice, we demonstrate that CRH, which is prominent in PMC-spinal projections, has an inhibitory function on volitional micturition. Moreover, engaging this inhibitory function of CRH can ameliorate bladder hyperexcitability induced by cyclophosphamide in a model of cystitis. The data underscore CRH as a novel target for the treatment of voiding dysfunctions, which are highly prevalent disease processes in children and adults.

Corticotropin-Releasing Hormone Expressing Neurons of the Pontine Micturition Center Inhibit Volitional Micturition

Lower urinary tract or voiding disorders are prevalent across all ages and affect over 40% of adults over 40 years old leading to decreased quality of life and high healthcare costs. The pontine micturition center (PMC) contains a large population of neurons that co-localize the stress-related neuropeptide, corticotropin-releasing hormone (CRH) and project to neurons in the spinal cord to regulate micturition. How the PMC and CRH-expressing neurons in the PMC control volitional micturition is of critical importance for human voiding disorders. To investigate the specific role of these CRH-expressing PMC neurons, these neurons were optogenetically stimulated during in vivo cystometry in unanesthetized mice. Optogenetic activation of CRH-PMC neurons led to increased intermicturition interval and voided volume, similar to the altered voiding phenotype produced by social stress. These effects were eliminated by CRH-receptor 1 antagonist pretreatment. In a cyclophosphamide cystitis model of bladder overactivity, optogenetic stimulation of CRH-PMC neurons returned the voiding pattern to normal. Collectively, our findings demonstrate that CRH in PMC spinal projections has an inhibitory function on micturition and may be a potential therapeutic target for human disease states such as voiding postponement, urinary retention, and under- or over-active bladder.

MIF mediates bladder pain, not inflammation, in cyclophosphamide cystitis

Macrophage migration inhibitory factor (MIF), a proinflammatory mediator, is recognized as a player in inflammatory and neuropathic pain. Cyclophosphamide (CYP) results in bladder inflammation and pain and it’s a frequently used animal model of interstitial cystitis/bladder pain syndrome (IC/BPS). Because pretreatment with a MIF inhibitor (ISO-1) prevented both CYP-induced bladder pain and inflammation we used genetic MIF knockout (KO) mice to further investigate MIF’s role in CYP-induced bladder pain and inflammation. Abdominal mechanical threshold measured bladder pain induced by CYP in wild type (WT) and MIF KO mice at several time points (0–48 h). End-point (48 h) changes in micturition parameters and histological signs of bladder inflammation were also evaluated. Abdominal mechanical hypersensitivity developed within 4 h after CYP injection (and lasted for the entire observation period: 48 h) in WT mice. MIF KO mice, on the other hand, did not develop abdominal mechanical hypersensitivity suggesting that MIF is a pivotal molecule in mediating CYP-induced bladder pain. Both WT and MIF KO mice treated with CYP showed histological signs of marked bladder inflammation and showed a significant decrease in micturition volume and increase in frequency. Since both changes were blocked in MIF KO mice by pretreatment with a MIF inhibitor (ISO-1) it is likely these are non-specific effects of ISO-1. MIF mediates CYP-induced bladder pain but not CYP-induced bladder inflammation. The locus of effect (bladder) or central (spinal) for MIF mediation of bladder pain remains to be determined.

Anti-vascular endothelial growth factor treatment decreases bladder pain in cyclophosphamide cystitis: a Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network animal model study.

To investigate whether treatment with anti-vascular endothelial growth factor (VEGF)-neutralizing antibodies can reduce pain and voiding dysfunction in the cyclophosphamide (CYP) cystitis model of bladder pain in mice. Adult female mice received anti-VEGF-neutralizing antibodies (10mg/kg i.p. B20-4.1.1 VEGF mAb) or saline (control) pre-treatment, followed by CYP (150mg/kg i.p.) to induce acute cystitis. Pelvic nociceptive responses were assessed by applying von Frey filaments to the pelvic area. Spontaneous micturition was assessed using the void spot assay. Systemic anti-VEGF-neutralizing antibody treatment significantly reduced the pelvic nociceptive response to CYP cystitis compared with control (saline). In the anti-VEGF pre-treatment group, there was a significant increase in pelvic hypersensitivity, measured by the area under the curve (AUC) using von Frey filaments at 5h post-CYP administration (P = 0.004); however, by 48h and 96h post-CYP administration, pelvic hypersensitivity had reduced by 54% and 47%, respectively, compared with the 5h post-CYP administration time point, and were no longer significantly different from baseline (P = 0.22 and 0.17, respectively). There was no difference in urinary frequency and mean voided volume between the two pre-treatment groups. Systemic blockade of VEGF signalling with anti-VEGF-neutralizing antibodies was effective in reducing pelvic/bladder pain in the CYP cystitis model of bladder pain. Our data support the further investigation of the use of anti-VEGF antibodies to manage bladder pain or visceral pain.

Hyperbaric Oxygen Therapy in Interstitial Cystitis/Painful Bladder Syndrome

Introduction: Hyperbaric oxygen therapy (HBO) is emerging as an alternative technique for treating refractory interstitial cystitis (IC). The theory is that the increased dissolved oxygen in the blood raises the levels of oxygen in the tissues and improves tissue healing. Other urological disorders such as radiation cystitis, Fournier’s gangrene and cyclophosphamide cystitis have also shown a good response to HBO therapy. Method: A literature search with the terms “interstitial cystitis”, “painful bladder syndrome” and “hyperbaric oxygen therapy” found four papers that have trialed HBO therapy in IC patients; three case series and one randomized control trial. Results: A total of 31 patients have been treated with HBO therapy. All four studies show a symptomatic improvement in pain, urgency, bladder capacity and O’Leary-Sant interstitial cystitis index. Urinary frequency only improved in three out of four of the trials. The percentage of patients considered as responders to treatment varied at 25%, 66.7%, 82% and 100%. Conclusion: Preliminary trials of HBO therapy seem to have a good patient response with effective symptomatic relief. The patient response rates varied greatly between the trials. This is due to each trial’s different definition of a “patient responder”. Further work in urology departments at HBO centers is required to further assess the benefits of HBO treatment in IC.

Urinary retention in women: Causes and management

Urinary retention in women is uncommon and there are numerous etiological factors. Most reported studies are from Europe and North America, with few studies from developing countries. The aim of this study was to review the etiology of urinary retention in women referred to our institution, a public sector hospital serving a largely indigent population. We reviewed the clinical records of all patients admitted with urinary retention to the Urology wards in our hospital during the period September 1998 to June 2007. In total there were 589 patients with urinary retention, 558 (94.7%) men and 31 (5.3%) women. The average age of the 31 women was 51.9 years (range 20 to 88 years). The underlying pathology was cervical carcinoma (4 patients), urethral carcinoma (4), transitional cell carcinoma of the bladder (3), eosinophilic cystitis (3), hematuria due to miscellaneous causes (3), antiincontinence surgery (2), cerebral palsy (2), multiple sclerosis (1 patient), diabetes mellitus (1), hypotonic detrusor (1), bladder stone (1), vaginal leiomyoma (1), cyclophosphamide cystitis (1), constipation (1), postpartum (1), blocked indwelling catheter and idiopathic (1). Renal dysfunction was present in 17 (55%) of the patients. The most common causes of urinary retention in women in this study were malignancy in 11 patients (36%) and neuropathic bladder dysfunction in 5 (16%). Eosinophilic cystitis, normally a rare condition, was diagnosed in 3 women (10%). The high incidence of malignancy in this study differs from other reported series, in which neuropathic bladder dysfunction was the most common cause of urinary retention in women.

Antagonism of macrophage migration inhibitory factor decreases cyclophosphamide cystitis in mice

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine found pre-formed in the urothelium. During inflammation, MIF is released into the bladder lumen and bladder MIF mRNA is upregulated. Since MIF also has tautomerase activity and blocking tautomerase activity also blocks MIF's biological activity, we hypothesized that blocking MIF's tautomerase activity would prevent bladder inflammation. Therefore, we examined the effects of a MIF tautomerase inhibitor (ISO-1; also blocks biological activity) on cyclophosphamide (CYP)-induced cystitis in mice. Mice receiving CYP (300 mg/kg; i.p.) to induce cystitis or saline (control) were treated either with ISO-1 (20 mg/kg; i.p.; daily) or vehicle (20% DMSO; i.p.; daily) for 2 days. After 2 days, micturition volume and frequency in awake mice were recorded and also mechanical sensitivity to abdominal stimulation using von Frey monofilaments. Bladders were collected under anesthesia and examined histologically, nerve growth factor levels were assayed in bladder homogenates, and production of inflammatory cytokines in the bladder was determined using a targeted array. CYP treatment resulted in decreased micturition volume, increased frequency, decreased threshold, increased histological signs of cystitis, increased bladder NGF levels and production of inflammatory cytokines when compared to the control group. Treatment with ISO-1 prevented or greatly decreased all these changes. Antagonizing MIF's activity with a systemic MIF tautomerase inhibitor was able to prevent or greatly reduced chemical cystitis in mice, thus indicating the MIF mediates bladder inflammation in this model. MIF represents a novel and important modulator of cystitis.

Sesquiterpene Lactone Parthenolide Ameliorates Bladder Inflammation and Bladder Overactivity in Cyclophosphamide Induced Rat Cystitis Model by Inhibiting Nuclear Factor-κB Phosphorylation

Cyclophosphamide (Sigma) is associated with urological complications, including irritative voiding symptoms and hemorrhagic cystitis. Evidence suggests that tumor necrosis factor-alpha (R&D Systems), interleukin-1beta and cyclooxygenase-2 are directly involved in the pathogenesis of cyclophosphamide induced cystitis and these molecules depend on transcription factor NF-kappaB for maximal secretion. Additionally, sesquiterpene lactone parthenolide has been shown to be a potent nuclear factor-kappaB inhibitor. We hypothesized that enhanced nuclear factor-kappaB activity contributes to cyclophosphamide induced cystitis and, therefore, it may be an attractive target for preventing cyclophosphamide cystitis. We determined whether parthenolide could be used as a preventive agent for hemorrhagic cystitis and bladder overactivity. Moreover, we determined the molecular mechanisms of parthenolide on the inhibitory action of nuclear factor-kappaB in inflammatory human benign urothelial cells. Rats were pretreated with parthenolide or vehicle solution and administered cyclophosphamide. Histological analysis and cystometry were performed 24 hours after cyclophosphamide administration. Human urothelial cells were pretreated with parthenolide and stimulated with tumor necrosis factor-alpha. Western blotting and immunofluorescence were performed to determine activation of the cyclooxygenase-2 and nuclear factor-kappaB pathway. Parthenolide pretreatment inhibited bladder inflammation as well as bladder overactivity and it was also associated with nuclear factor-kappaB activation in the bladder. Parthenolide dose dependently suppressed tumor necrosis factor-alpha induced cyclooxygenase-2 expression and prevented nuclear factor-kappaB phosphorylation as well as nuclear factor-kappaB nuclear translocation and IkappaBalpha phosphorylation/degradation. Nuclear factor-kappaB may have a crucial role in the pathogenesis of cyclophosphamide induced cystitis models. Parthenolide ameliorates bladder inflammation and bladder overactivity, and it might be a promising agent for preventing cyclophosphamide induced complications.

ANTAGONISM OF MACROPHAGE MIGRATION INHIBITORY FACTOR PREVENTS CYCLOPHOSPHAMIDE CYSTITIS IN MICE

You have accessJournal of Urology1 Apr 2009ANTAGONISM OF MACROPHAGE MIGRATION INHIBITORY FACTOR PREVENTS CYCLOPHOSPHAMIDE CYSTITIS IN MICE Pedro L Vera, Kenneth A Iczkowski, Daniel J Howard, Lixian Jiang, and Katherine L Meyer-Siegler Pedro L VeraPedro L Vera More articles by this author , Kenneth A IczkowskiKenneth A Iczkowski More articles by this author , Daniel J HowardDaniel J Howard More articles by this author , Lixian JiangLixian Jiang More articles by this author , and Katherine L Meyer-SieglerKatherine L Meyer-Siegler More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(09)60673-XAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "ANTAGONISM OF MACROPHAGE MIGRATION INHIBITORY FACTOR PREVENTS CYCLOPHOSPHAMIDE CYSTITIS IN MICE." The Journal of Urology, 181(4S), pp. 236–237 © 2009 by American Urological AssociationFiguresReferencesRelatedDetails Volume 181Issue 4SApril 2009Page: 236-237 Advertisement Copyright & Permissions© 2009 by American Urological AssociationMetricsAuthor Information Pedro L Vera More articles by this author Kenneth A Iczkowski More articles by this author Daniel J Howard More articles by this author Lixian Jiang More articles by this author Katherine L Meyer-Siegler More articles by this author Expand All Advertisement PDF downloadLoading ...

Cyclophosphamide necrosis of bladder causing calcification, contracture and reflux; treated by colocystoplasty.

SUMMARY Necrosis of the bladder in a patient with cyclophosphamide cystitis extended to urethra and vulva, caused calcification in the bladder wall, and resulted in vesico-ureteric reflux from a grossly contracted bladder. Colocystoplasty was performed. The patient's progress is described and the literature reviewed. We wish to thank Professor J. Ledingham for his help and criticism.

Upregulation of Macrophage Migration Inhibitory Factor (MIF) and CD74, Receptor for MIF, in Rat Bladder During Persistent Cyclophosphamide-Induced Inflammation

The objective of this study was to determine if macrophage migration inhibitory factor (MIF) is upregulated in the bladder during persistent cystitis. MIF is a pro-inflammatory cytokine found pre-formed in the urothelium. Previous findings showed that acute bladder inflammation increased MIF release into the bladder lumen while upregulating MIF and CD74 (MIF receptor) in the bladder. Because the effects of persistent cystitis on MIF and CD74 are not known, MIF and CD74 changes in the bladder were examined after short-term (1-day) or persistent (8-day) cyclophosphamide (CYP)-induced bladder inflammation. Anesthetized male Sprague-Dawley rats received either a single CYP treatment (150 mg/kg, ip; saline, control) and examined 1 day after treatment (short-term), or repeated CYP doses (20-75 mg/ kg, ip; saline, control; every third day for 8 days) and examined after 8 days of treatment (persistent). MIF protein levels in urine and bladder were determined. In addition, Mif, CD74, and cox-2 expression in the bladder was determined. Histology verified cystitis and MIF and CD74 immunoreactivity in the bladder. Repeated CYP doses were decreased to avoid toxicity. Short-term or repeated low CYP doses (40 mg/kg; 8 days) increased urinary MIF and decreased bladder MIF amounts while upregulating bladder Mif and CD74 mRNA expression. Persistent CYP-induced bladder inflammation (even at 40 mg/kg; 8-day treatment) also upregulated other inflammatory cytokines (CCL5, IL-11, iNOS) in the bladder. Short-term and persistent (low dose) CYP cystitis are associated with markedly increased MIF release into the urine and upregulation of Mif and CD74 in bladder. This supports the hypothesis that MIF and CD74 play a significant role in both acute and persistent stages of bladder inflammation.

Morphine and ABT-594 (a Nicotinic Acetylcholine Agonist) Exert Centrally Mediated Antinociception in the Rat Cyclophosphamide Cystitis Model of Visceral Pain

A visceral pain model incorporating use of cyclophosphamide (CP) to induce bladder inflammation has been described. CP treatment in rats produces changes in behavior (abnormal postures and eye closure) and respiration rate indicative of visceral pain. We characterized the dose-dependency and progression of CP-induced cystitis pain after intraperitoneal (i.p.) CP. The behavioral and respiration rate changes were ameliorated by systemic morphine and ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine], a neuronal nicotinic acetylcholine receptor agonist, in a manner reversible by naloxone and mecamylamine, respectively. Sites of antinociceptive actions of morphine and ABT-594 were investigated using systemic, intrathecal (i.t.), or intracerebroventricular (i.c.v.) administration of blood-brain barrier impenetrant antagonists. Naloxone methiodide produced a complete antagonism of morphine antinociception after i.c.v. but not i.p. or i.t. administration. Chlorisondamine blocked ABT-594 antinociception after i.c.v. but not i.p. administration. Further pharmacological characterization of behavioral and respiration changes in CP-cystitis was performed using standard analgesics. The alpha(2)-adrenoceptor agonist clonidine produced a weak attenuation of CP-pain behavior. NSAIDs (ibuprofen, acetaminophen, and celecoxib) and anticonvulsants (gabapentin and lamotrigine) were without effect. These results demonstrate that morphine and ABT-594 produce antinociception in CP-cystitis by a predominantly supraspinal site of action, and that mechanisms producing robust centrally-mediated antinociception could be beneficial in cystitis pain. In this article, potential antinociceptive effects of a variety of pharmacological agents were evaluated in a rat cystitis pain model. Morphine and a nicotinic acetylcholine receptor agonist ABT-594 were found to exert potent antinociception in this model. Findings presented here aid identification of agents to treat cystitis pain in the clinic.

Bacterial Cystitis is Accompanied by Increased Peripheral Thermal Sensitivity in Mice

Visceral inflammation and pain associated with chemical cystitis produce increased sensitivity to noxious stimuli in the sacral dermatomes. We determined whether a similar sensitization occurs in response to bacterial cystitis. Bacterial cystitis was induced by intravesical instillation of Escherichia coli 1677 in female C57BL/6N and C3H/OuJ mice (Jackson Laboratories, Bar Harbor, Maine). C3H/HeJ mice (Jackson Laboratories) served as a control because C3H/HeJ mice lack functional toll-like receptor 4, which is an essential component of cellular recognition of bacterial lipopolysaccharide. Hind paw sensitivity to thermal stimulus was quantitatively determined 1, 2, 7 and 14 days after infection. Intravesical instillation of E. coli produced infection in all strains of mice. Infection persisted in all C3H/OuJ and C3H/HeJ mice but it spontaneously cleared in some C57BL/6N mice. Increased sensitivity to thermal stimuli was observed in C57BL/6N and C3H/OuJ mice starting 1 to 2 days after E. coli instillation and it was still present 14 days after instillation. Increased sensitivity to thermal stimuli did not occur in C3H/HeJ mice. E. coli induced cystitis produced increased sensitivity to peripheral thermal stimuli in mice with competent toll-like receptor 4.

Endogenous Opioids Inhibit Early-Stage Pancreatic Pain in a Mouse Model of Pancreatic Cancer

The endogenous opioid system is involved in modulating the experience of pain, the response to stress, and the action of analgesic therapies. Recent human imaging studies have shown a significant tonic modulation of visceral pain, raising the question of whether endogenous opioids tonically modulate the pain of visceral cancer. Transgenic mice expressing the first 127 amino acids of simian virus 40 large T antigen, under the control of the rat elastase-1 promoter, that spontaneously develop pancreatic cancer were used to investigate the role of endogenous opioids in the modulation of pancreatic cancer pain. Visceral pain behaviors were assessed as degree of hunching and vocalization. Although mice with late-stage pancreatic cancer displayed spontaneous, morphine-reversible, visceral pain-related behaviors such as hunching and vocalization, these behaviors were absent in mice with early-stage pancreatic cancer. After systemic administration of the central nervous system (CNS)-penetrant opioid receptor antagonists naloxone or naltrexone, mice with early-stage pancreatic cancer displayed significant visceral pain-related behaviors, whereas systemic administration of the CNS-nonpenetrant opioid antagonist naloxone-methiodide did not induce an increase in visceral pain behaviors. Our findings suggest that a CNS opioid-dependent mechanism tonically modulates early and late-stage pancreatic cancer pain. Understanding the mechanisms that mask this pain in early stage disease and drive this pain in late-stage disease may allow improved diagnosis, treatment, and care of patients with pancreatic cancer.

Characterization of Cyclophosphamide Cystitis, a Model of Visceral and Referred Pain, in the Mouse: Species and Strain Differences

Existing animal models of visceral pain in the mouse are of limited practical usefulness since they are labor intensive or not visceral specific. Recently a rat model of cyclophosphamide (CP) cystitis was developed that requires only intraperitoneal injection and features inflammation confined to the bladder. We adapted this model for use in multiple mouse strains to investigate the genetic basis of variability in visceral nociception. Outbred CD-1 mice and 12 inbred mouse strains were tested for behavioral changes induced by CP (0 to 300 mg/kg intraperitoneally). We noted that despite the absence of postural changes or abdominal crises in CD-1 mice, CP produced dose dependent decreases in voluntary locomotor activity unaccompanied by ataxia measured on the rotarod test; referred hyperalgesia of the tail base region but not of the hind paw, which was inhibited in dose dependent fashion by morphine (0 to 20 mg/kg); and bladder inflammation corresponding to these behavioral indices. Furthermore, the extent of hypolocomotion was genotype dependent across 12 inbred strains. The simple and automatable nature of CP cystitis using hypolocomotion as a dependent measure renders it an attractive model in which to investigate the genetic and physiological bases of visceral pain. Comparison of strain sensitivity to CP induced hypolocomotion with other nociceptive assays suggests that genes specific to visceral nociception may exist.

Subsensitivity of P2X but not vanilloid 1 receptors in dorsal root ganglia of rats caused by cyclophosphamide cystitis

The application of cyclophosphamide to rats was used to induce interstitial cystitis. Behavioural studies indicated a strong pain reaction that developed within 2 h and levelled off thereafter causing a constant pain during the following 18 h. Neurons prepared from L6/S1 dorsal root ganglia innervating the urinary bladder responded to the application of capsaicin or α,β-methylene ATP (α,β-meATP) with an increase of intracellular Ca 2+ ([Ca 2+] i ). The [Ca 2+] i responses to capsaicin were identical in the dorsal root ganglion cells of cyclophosphamide- and saline-treated rats, whereas α,β-meATP induced less increase in [Ca 2+] i in the cyclophosphamide-treated animals than in their saline-treated counterparts. Hence, α,β-meATP-sensitive P2X 3 and/or P2X 2/3 receptors of L6/S1 dorsal root ganglion neurons were functionally downregulated during subacute pain caused by experimental cystitis. In contrast, capsaicin-sensitive vanilloid 1 receptors did not react to the same procedure. Thoracal dorsal root ganglia, not innervating the urinary bladder, were also unaltered in their responsiveness to α,β-meATP by cyclophosphamide treatment.

Deficits in Visceral Pain and Referred Hyperalgesia in Nav1.8 (SNS/PN3)-Null Mice

The tetrodotoxin-resistant sodium channel alpha subunit Nav1.8 is expressed exclusively in primary sensory neurons and is proposed to play an important role in sensitization of nociceptors. Here we compared visceral pain and referred hyperalgesia in Nav1.8-null mice and their wild-type littermates in five tests that differ in the degree to which behavior depends on spontaneous, ongoing firing in sensitized nociceptors. Nav1.8-null mice showed normal nociceptive behavior provoked by acute noxious stimulation of abdominal viscera (intracolonic saline or intraperitoneal acetylcholine). However, Nav1.8-null mutants showed weak pain and no referred hyperalgesia to intracolonic capsaicin, a model in which behavior is sustained by ongoing activity in nociceptors sensitized by the initial application. Nav1.8-null mice also showed blunted pain and hyperalgesia to intracolonic mustard oil, which sensitizes nociceptors but also provokes tissue damage. To distinguish between a possible role for Nav1.8 in ongoing activity per se and ongoing activity after sensitization in the absence of additional stimuli, we tried a visceral model of tonic noxious chemical stimulation, cyclophosphamide cystitis. Cyclophosphamide produces cystitis by gradual accumulation of toxic metabolites in the bladder. In this model, Nav1.8-null mice showed normal responses. There were no differences between null mutants and their normal littermates in tissue damage and inflammation evoked by any of the stimuli tested, suggesting that the behavioral differences are not secondary to impairment of inflammatory responses. We conclude that there is an essential role for Nav1.8 in mediating spontaneous activity in sensitized nociceptors.

EFFECTS OF THE NUCLEAR FACTOR-κB INHIBITORS 2-HYDROXY-4-TRIFLUOROMETHYLBENZOIC ACID AND ASPIRIN ON MICTURITION IN RATS WITH NORMAL AND INFLAMED BLADDER

We examined the effects of intravenous administration of the 2 nuclear factor-kappaB inhibitors aspirin and 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) on bladder filling and voiding in anesthetized and conscious rats. Disappearance of isovolumic bladder contractions after intravenous administration of different doses of aspirin and HTB in anesthetized, transurethrally catheterized rats was evaluated. Cystometry was performed in conscious rats during bladder infusion with saline or diluted acetic acid as well as in those with cyclophosphamide induced cystitis. Changes in bladder capacity and voiding pressure were evaluated after intravenous administration of test compounds. Aspirin induced a dose dependent disappearance of isovolumic bladder contractions in anesthetized rats with an extrapolated dose of 2.1 mg./kg. inducing 10 minutes of bladder quiescence. HTB was practically inactive, inducing a dose independent block of 3 to 4 minutes after intravenous administration of 1 to 10 mg./kg. In conscious rats with a bladder infused with saline aspirin was poorly active on bladder capacity, inducing a 20% increase 60 minutes after intravenous administration of 30 and 100 mg./kg. In rats with a bladder infused with acetic acid aspirin was much more active when injected at the initiation of inflammation and after 1 hour of irritant infusion. In this latter situation aspirin increased bladder capacity up to 60% after intravenous administration of 30 and 100 mg./kg. Similar results were obtained in rats with cyclophosphamide induced cystitis in which the bladder was infused with saline. In these cystometrography models 30 mg./kg. HTB intravenously was completely inactive. The results show that HTB is devoid of significant effects on the micturition reflex in the absence or presence of bladder inflammation, suggesting that acute inhibition of nuclear factor-kappaB does not influence bladder urodynamics in rats. In contrast, aspirin, which is a cyclooxygenase and nuclear factor-kappaB inhibitor, was always effective, indicating the important role of cyclooxygenase enzymes.

INVOLVEMENT OF HYPOGASTRIC AND PELVIC NERVES FOR CONVEYING CYSTITIS INDUCED NOCICEPTION IN CONSCIOUS RATS

We determined the sites of the antinociceptive action of morphine in the experimental model of cyclophosphamide induced cystitis and investigated the afferent nerve fibers involved in nociception transmission originating from the bladder. Cyclophosphamide (200 mg./kg.) given intraperitoneally was used to induce cystitis in male rats and their behavior was observed and scored. The effect of 2 mg./kg. systemic morphine given intravenously on cyclophosphamide induced behavioral modifications was tested when administered alone and after 100 microg. naloxone per rat given intrathecally at the L1 to L2 or L6 to S1 level. The spinal antinociceptive effect of morphine was also tested when administered intrathecally alone at 10, 100 and 200 microg. per rat at L1 to L2, alone at 100 microg. per rat at L1 to L2 or L6 to S1, alone at 100 microg. per rat at L1 to L2 and L6 to S1 simultaneously, alone at 200 microg. per rat at L1 to L2 and after 100 microg. naloxone per rat given intrathecally at L6 to S1 at 100 microg. per rat at L1 to L2. Cyclophosphamide induced marked modifications in the behavior of the rats, including a decreased breathing rate, eye closing and specific postures. Morphine given intravenously reversed these behavioral disorders and this reversal was completely prevented by pretreatment with intrathecal naloxone. A dose of 100 microg. per rat given intrathecally also reversed these behavioral disorders by about 25% at the L1 to L2 and L6 to S1 levels. In addition, a dose of 100 microg. morphine per rat administered intrathecally and simultaneously at L1 to L2 and L6 to S1 produced an effect equal to the sum of those observed when administered separately, that is about 50%, whereas morphine at an intrathecal dose of 200 microg. at L1 to L2 produced the same effect as 100 microg. given intrathecally at the same level or at L6 to S1 (25%). Also, 100 microg. naloxone per rat administered intrathecally at L6 to S1 prevented the effect of 100 microg. morphine at L1 to L2. These results confirm the previously reported antinociceptive effect of systemic morphine in this model of cyclophosphamide cystitis, suggest that this antinociceptive action is completely located at the spinal site and most importantly demonstrate by the pharmacological approach and behavioral analysis that nociceptive sensations originating from the bladder are conveyed by hypogastric and pelvic nerves in this cyclophosphamide cystitis model in the conscious rat.

Deficits in visceral pain and hyperalgesia of mice with a disruption of the tachykinin NK1 receptor gene

Studies in mice lacking genes encoding for substance P or its receptor (NK1), or with NK1 antagonists, have shown that this system contributes to nociception, but the data are complex. Here, we have further examined the role of NK1 receptors in pain and hyperalgesia by comparing nociceptive responses to mechanical and chemical stimulation of viscera and the resulting hyperalgesia and inflammation in NK1 knockout (−/−) and wild-type (+/+) mice. We concentrated on visceral nociception because substance P is expressed by a much greater proportion of visceral than cutaneous afferents. NK1 −/− mice showed normal responses to visceral mechanical stimuli, measured as behavioural responses to intraperitoneal acetylcholine or hypertonic saline or reflex responses to colon distension in anaesthetized mice, although −/− mice failed to encode the intensity of noxious colon distensions. In contrast, NK1 −/− mice showed profound deficits in spontaneous behavioural reactions to an acute visceral chemical stimulus (intracolonic capsaicin) and failed to develop referred hyperalgesia or tissue oedema. However, in an identical procedure, intracolonic mustard oil evoked normal spontaneous behaviour, referred hyperalgesia and oedema in −/− mice. The inflammatory effects of capsaicin were abolished by denervation of the extrinsic innervation of the colon in rats, whereas those of mustard oil were unchanged, showing that intracolonic capsaicin evokes neurogenic inflammation, but mustard oil does not. Tests of other neurogenic inflammatory stimuli in NK1 −/− mice revealed impaired behavioural responses to cyclophosphamide cystitis and no acute reflex responses or primary hyperalgesia to intracolonic acetic acid. We conclude that NK1 receptors have an essential role mediating central nociceptive and peripheral inflammatory responses to noxious stimuli that evoke neurogenic inflammation, and modulating responses to noxious mechanical stimuli. We propose that two separate hyperalgesia pathways exist, one of which is NK1 receptor dependent, whereas the other does not require intact substance P/NK1 signalling.