Research Article The Effects of Tempol on Cyclophosphamide ...downloads.hindawi.com/journals/tswj/2015/545048.pdf · following each micturition event in control and CYP-treated groups

Research ArticleThe Effects of Tempol on Cyclophosphamide-Induced OxidativeStress in Rat Micturition Reflexes

Eric J Gonzalez Abbey Peterson Susan Malley Mitchel Daniel Daniel LambertMichael Kosofsky and Margaret A Vizzard

Department of Neurological Sciences University of Vermont College of Medicine Burlington VT 05405 USA

Correspondence should be addressed to Margaret A Vizzard margaretvizzarduvmedu

Received 13 January 2015 Revised 3 March 2015 Accepted 5 March 2015

Academic Editor Ana Charrua

Copyright copy 2015 Eric J Gonzalez et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We hypothesized that cyclophosphamide- (CYP-) induced cystitis results in oxidative stress and contributes to urinary bladderdysfunction We determined (1) the expression of oxidative stress markers 3-nitrotyrosine (3-NT) reactive oxygen species(ROS)reactive nitrogen species (RNS) inflammatory modulators neuropeptides calcitonin gene-related peptide (CGRP)substance P (Sub P) and adenosine triphosphate (ATP) that contribute to the inflammatory process in the urinary tract and (2) thefunctional role of oxidative stress in urinary bladder dysfunction with an antioxidant Tempol (1mM in drinking water) combinedwith conscious cystometry In CYP-treated (4 hr or 48 hr 150mgkg ip) rats ROSRNS and 3-NT significantly (119875 le 001)increased in urinary bladder CYP treatment increased ATP Sub P and CGRP expression in the urinary bladder and cystometricfluid In CYP-treated rats Tempol significantly (119875 le 001) increased bladder capacity and reduced voiding frequency comparedto CYP-treated rats without Tempol Tempol significantly (119875 le 001) reduced ATP expression 3-NT and ROSRNS expression inthe urinary tract of CYP-treated rats These studies demonstrate that reducing oxidative stress in CYP-induced cystitis improvesurinary bladder function and reduces markers of oxidative stress and inflammation

1 Introduction

Bladder pain syndrome (BPS)interstitial cystitis (IC) is achronic syndrome characterized by pressure discomfort andpain thought to arise from the urinary bladder with at leastone urinary symptom [1 2] While the underlying etiology ofBPSIC is not known the majority of biopsies from BPSICpatients reveal inflammation [3] Mediators of inflamma-tion including cytokines chemokines growth factors andneuropeptides have been shown to contribute to urinarybladder dysfunction and somatic sensitivity in animalmodelsof cystitis and in the clinical syndrome of BPSIC [3 4] Inaddition reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) generated by inflammation result in oxidativestress and may contribute to urinary bladder dysfunction[5 6] The role(s) that oxidants may have in inducing inflam-mation has been extensively studied in diverse experimentalmodels [7] Although it is widely accepted that ROSRNSare fundamentally involved antioxidant therapy as a validmeans of arresting inflammation remains largely unresolvedespecially in the context of urinary bladder inflammation

Accompanying oxidative stress and inflammatorymedia-tor upregulation the urotheliummay also respond to cystitisby increasing the secretion of neuroactive factors such asATP which signal to the underlying nerve plexus [8] Alteredrelease of these neuromodulatory compounds has beensuggested to contribute to increased sensory transductionand result in urinary bladder dysfunction [4 8] In BPSICfor example distention-evoked release of ATP was increasedin tissue biopsies that may have resulted in the elevatedurinary ATP observed in patients with BPSIC [9] Similarlynumerous animal models of cystitis have demonstratedincreased distention-evoked release of ATP from the urothe-lium [10 11] Further purinergic receptor activation has beenassociated with increased cellular production and releaseof multiple inflammatory mediators including superoxideanion nitric oxide and other ROS [12] Purinergic receptoractivation induces ROS generation in numerous cell typesresulting in a variety of downstream effects including tran-scription factor activation [13] proinflammatory cytokinerelease [14 15] and cell death [16]

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 545048 13 pageshttpdxdoiorg1011552015545048

2 The Scientific World Journal

Using a rat model of urinary bladder inflammationinduced by cyclophosphamide (CYP) [3 4 17 18] wedetermined (1) the expression of oxidative stress markers(3-nitrotyrosine (3-NT) ROSRNS) and other modulatorsof inflammation the neuropeptides calcitonin gene-relatedpeptide (CGRP) and substance P (Sub P) in the urinary tract(2) the contribution of ROSRNS to ATP expression in theurinary bladder and urine with an antioxidant and superox-ide dismutase mimetic Tempol (1mM in drinking water)and (3) the role of ROSRNS in urinary bladder functionwith Tempol combined with open outlet and conscious andcontinuous intravesical infusion [19] Although the etiologyof BPSIC is unknown previous studies [3 4 17 18] havedemonstrated that the rat CYP model of urinary bladderinflammation is a reliable and reproducible model withface validity (eg increased voiding frequency and referredsomatic sensitivity) to BPSIC Previous studies have demon-strated roles for neuropeptides including CGRP and Sub Pin CYP-induced bladder dysfunction [20 21] and the currentstudies have continued to focus on these two neuropeptidesabundantly expressed in bladder sensory pathways

2 Materials and Methods

21 Animals Adult female Wistar rats (200ndash225 g CharlesRiver St Constant Canada) were used for this study Ratswere housed two per cage and maintained in standardlaboratory conditions with free access to food and water TheUniversity of Vermont Institutional Animal Care and UseCommittee approved all animal use procedures (protocol 08-085) Animal experimentation was carried out in accordancewith the National Institutes of Health Guide for the Careand Use of Laboratory Animals All efforts were made tominimize the potential for animal pain stress or distress

22 Induction of Cyclophosphamide- (CYP-) Induced CystitisRats were anesthetized under isoflurane (2) and acute cys-titis was induced with a single injection of CYP (150mgkgip) and used in studies at various time points (4 hours (hr) or48 hr) after treatment [22ndash24] Control rats received volume-matched injections of saline (09 ip) or no treatmentand no difference among the control groups was observedAll injections were performed under isoflurane (2) anes-thesia The CYP model of bladder inflammation producesan increase in voiding frequency with small micturitionvolumes and is associatedwith inflammatory cell infiltrates inthe urinary bladder including mast cells macrophages andneutrophils [25 26] Rats were euthanized using isoflurane(5) and a thoracotomy

For cystometry in conscious rats an unrestrained animalwas placed in a Plexiglas cage with a wire bottom Beforethe start of the recording the bladder was emptied and thecatheter was connected via a T-tube to a pressure transducer(Grass Model PT300 West Warwick RI) and microinjectionpump (Harvard Apparatus 22 South Natick MA) A SmallAnimal Cystometry Lab Station (MEDAssociates St AlbansVT) was used for urodynamic measurements [19 27] Salinesolution was infused at room temperature into the bladderat a rate of 10mLh to elicit repetitive bladder contractions

At least six reproducible micturition cycles were recordedafter the initial stabilization period of 25ndash30min [19 27]The following cystometric parameters were recorded in eachanimal baseline pressure (BP pressure at the beginning ofthe bladder filling) threshold pressure (TP bladder pressureimmediately prior to micturition) peak micturition pressure(MP) intercontraction interval (ICI time between micturi-tion events) bladder capacity (BC) void volume (VV) andpresence and amplitude of nonvoiding bladder contractions(NVCs) [19 27] NVCs were defined as rhythmic intravesicalpressure increases 7 cmH

2Oabove baseline during the filling

phase without the release of fluid from the urethra Bladdercapacity (BC) was measured as the volume of saline infusedinto the bladder at the time when micturition commenced[28] In these rats residual volume was less than 10 120583L there-fore VV and BC were similar After the initial stabilizationperiod cystometric fluid expelled during micturition eventswas collected and frozen on dry ice and stored at minus80∘C forfuture use in the assays described above At the conclusionof the experiment the rat was euthanized (5 isoflurane plusthoracotomy) the urinary bladder was harvested and used inthe assays described above

23 Split Bladder Preparation and Assessment of PotentialContamination of Bladder Layers The urothelium + sub-urothelium was dissected from the detrusor smooth muscleusing fine forceps under a dissecting microscope as previ-ously described [22 23] To confirm the specificity of oursplit bladder preparations urothelium + suburothelium anddetrusor samples were examined for the presence of 120572-smooth muscle actin (1 1000 Abcam Cambridge MA) anduroplakin II (1 25 American Research Products BelmontMA) by western blotting or reverse transcription PCR [2229] In urothelium + suburothelium layers only uroplakinII was present (data not shown) Conversely in detrusorsamples only 120572-smooth muscle actin was present (data notshown) All subsequent measurements of urinary bladderand cystometric fluid samples and conscious cystometry inrat groups were performed in a blind manner

24 Substance P (Sub P) Calcitonin Gene-Related Peptide(CGRP) and 3-Nitrotyrosine (3-NT) by Enzyme-Linked Im-munosorbent Assays (ELISAs) Tissue processing and ELISAswere performed as described previously [18 30] Briefly ratsfrom control (119899 = 6 each) and all experimental groups (119899 =6 each) were deeply anesthetized (4 isoflurane) and athoracotomy was performed Individual rat bladders weredissected weighed and placed in Tissue Protein ExtractionReagent (1 g tissue20mL Pierce Biotechnology WoburnMA) with complete protease inhibitor cocktail tablets (RocheApplied Science Mannheim Germany) and stored at minus80∘COn the day of assay individual bladders were disrupted witha Polytron homogenizer until being homogeneous and cen-trifuged (10000 rpm for 10min) [18 30] and the supernatantwas used for total protein estimation and CGRP (PhoenixPharmaceuticals Inc Burlingame CA) Sub P (PhoenixPharmaceuticals) and 3-NT (Millipore Corporation Beller-ica MA) quantification Total protein was determined by theCoomassie Plus Protein Assay Reagent Kit (Pierce) [18 30]

The Scientific World Journal 3

and CGRP Sub P and 3-NT were quantified using standard96-well ELISA plates (Phoenix Pharmaceuticals MilliporeCorporation) according to the manufacturerrsquos recommen-dations For determination of Sub P and CGRP contentin voided cystometric fluid void volumes were collectedfollowing each micturition event in control and CYP-treatedgroups with and without Tempol (only vehicle) administra-tion during conscious cystometry (see below) For Sub PCGRP and 3-NT measurements in voided cystometric fluid6ndash8 individual voidsanimal were collected and immediatelyfrozen on dry ice Data from multiple voids were averagedand the mean value was used for each animal

25 ELISAs for CRGP Sub P and 3-NT Expressions in Uri-nary Bladder and Voided Cystometric Fluid The microtiterplates (Phoenix Pharmaceuticals Millipore Corporation)were coatedwithmouse anti-rat CGRP anti-rat Sub P or anti-rabbit-NT antibody Sample and standard solutions were runin duplicate Horseradish peroxidase- (HRP-) streptavidin orHRP-conjugated goat anti-rabbit IgG and LumiGLO wereused to detect the antibody complex Tetramethylbenzidineor LumiGLO was the substrate and the enzyme activity orluminescence was measured The CRGP standard providedwith this protocol generated a standard curve from 0 to100 ngmL (1198772 = 0998 119875 le 00001) for bladder samples [30]The Sub P standard provided with this protocol generateda standard curve from 0 to 25 ngmL (1198772 = 0998 119875 le00001) for bladder samples [30] The nitrated-BSA standardprovided with this protocol generated a standard curve from15 to 100 120583gmL (1198772 = 0997 119875 le 00001) for bladdersamples The absorbance values of standards and sampleswere corrected by subtraction of the background value(absorbance due to nonspecific binding) [18 30] No sampleswere diluted and all samples had absorbance values that fellonto the linear portion of the standard curve Curve fitting ofstandards and evaluation of protein content of samples wereperformed using a least-squares fit

26 Measurement of Cellular Oxidative Stress The levels ofcellular oxidative stress were determined by a spectroflu-orimetric method using the dichlorofluorescein DiOxyQ(DCFH-DiOxyQ) assay OxiSelect in vitro ROSRNS AssayKit (Green Fluorescence Cell Biolabs Inc San Diego CAUSA) The urinary bladder was removed from control (119899 = 6each) and all experimental groups (119899 = 6 each) and rapidlyhomogenized in 50mM TrisndashCl pH 74 The homogenatewas centrifuged at 2400 g for 15min at 4∘C and a low-speedsupernatant fraction was used for assays To determine levelsof cellular oxidative stress the supernatant from the urinarybladder homogenate was diluted (1 10) in 50mM TrisndashHCl (pH 74) and incubated with 10 120583L of 2101584071015840-DCHF-DA(1mM) at 37∘C for 30min The DCHF-DA is enzymaticallyhydrolyzed by intracellular esterases to form nonfluorescentDCFH which is then rapidly oxidized to form highly flu-orescent 2101584071015840-dichlorofluorescein (DCF) in the presence ofcellular oxidative stress species DCF fluorescence intensity isproportional to the amount of cellular oxidative stress speciesthat is formed The DCF fluorescence intensity emission was

recorded at 520 nm (with 480 nm excitation) 30 min after theaddition of DCHF-DA to the sample The cellular oxidativespecies levels were expressed as fluorescence arbitrary units(FAU)

27 Collection and Measurement of Urinary ATP

271 Sample Collections For determination of ATP in uri-nary bladder urinary bladder harvest and subsequent pro-cessing were performed as described above for ELISAs Fordetermination of ATP content in voided cystometric fluidvoid volumes were collected following eachmicturition eventin animal groups (control CYP-treated groups with andwithout Tempol) during conscious cystometry For ATPmeasurements in voided cystometric fluid 6ndash8 individualvoidsanimal were collected and immediately frozen on dryice and stored at minus80∘C until use Undiluted cystometric fluidsamples were defrosted till 25∘C and centrifuged at 3000 g atroom temperature for 20 seconds to remove cellular debrisand the supernatant was separated Data frommultiple voidswere averaged and the mean value was used for each animal

272 ATP Determination A mixture of luciferin-luciferasewas added according to the manufacturer instructionsusing the ATP Bioluminescence Assay Kit HS II (adeno-sine triphosphate (ATP) Bioluminescent Assay Kit Sigma-Aldrich St Louis MO USA) as previously described [31]ATP detection was evaluated using a multimode microplatereader (Synergy HT BioTek Instruments Inc VermontUSA) controlled with Gen5 Data Analysis Software (BioTek)Sample bioluminescence was compared to that of standardamounts of ATP used in the same concentration rangestandard ATP samples were prepared daily All samples wererun in duplicate Cystometric void data were obtained fromat least three voidsanimal from control (119899 = 6 each) and allexperimental groups (119899 = 6 each) and urinary bladder datawere obtained from control (119899 = 6 each) and all experimentalgroups (119899= 6 each) Vehicle for Tempol did not affect the ATPdeterminationsTheATP in cystometric fluid or ATP contentin urinary bladder was calculated relative to the standardcurve and expressed as nmol per total protein or nmol permL of urine

28 Conscious Cystometry and Effects of Tempol a Super-oxide Dismutase (SOD) Mimetic Rats were anesthetizedwith isoflurane (3-4) a lower midline abdominal incisionwas made and polyethylene tubing (PE-50 Clay AdamsParsippany New Jersey) was inserted into the bladder domeand secured with nylon purse-string sutures (6-zero) [19 27]The end of the PE tubing was heat flared but the catheterdid not extend into the bladder body or neck and it was notassociated with inflammation or altered cystometric function[19 27] The distal end of the tubing was sealed tunneledsubcutaneously and externalized at the back of the neck outof reach of the animal [19 27] Abdominal and neck incisionswere closed with nylon sutures (4-zero) Postoperative anal-gesics were given and animals were maintained for 72 to 96hours after survival surgery to ensure recovery


Tempol (4-hydroxy-2266-tetramethylpiperidine-N-oxyl) a superoxide dismutase (SOD) mimetic is a stablenitroxyl antioxidant Previous studies have suggested thatTempol is protective in disorders involving ROS [32ndash35]Theeffects of Tempol on urinary bladder function in CYP-treated(4 hr and 48 hr 119899 = 6 each) rats and control rats (119899 = 6each) were assessed using conscious open outlet cystometrywith continuous instillation of intravesical saline [19 27]Tempol was prepared daily with distilled water purified witha Millipore Milli-Q system and administered in the drinkingwater (1mM) that was provided to rats ad libitum untreatedrats received water ad libitum Covered bottles were used tominimize degradation by light The addition of Tempol tothe drinking water did not affect water consumption (datanot shown) For CYP-treated rat groups (4 hr 48 hr) Tempolwas provided for approximately two weeks (13ndash15 days) whilethe intravesical tube was implanted on day 10 CYP or vehiclewas injected on day 13 and rats were used in experimentson days 13ndash15 Due to the daily administration and routeof delivery (oral) of the Tempol these experiments wereperformed in different groups of control and CYP-treatedrats treated with vehicle or Tempol The concentration(1mM) of Tempol and duration of treatment used in thesestudies were based upon previous studies [36 37]

29 Exclusion Criteria Rats were removed from the studywhen adverse events occurred that included 20 reductionin body weight postsurgery a significant postoperative eventlethargy pain or distress not relieved by our IACUC-approved regimen of postoperative analgesics or hematuriain control rodents [19 27] In the present study no ratswere excluded from the study In addition behavioral move-ments such as grooming standing walking and defecationrendered bladder pressure recordings during these eventsunusable

210 Statistical Analyses All values represent mean plusmn SEMCystometry data were compared using repeated measuresANOVA where each animal served as its own controlAnimals processed and analyzed on the same day weretested as a block in the ANOVAWhen 119865-ratios exceeded thecritical value (119875 le 005) the Newman-Keuls or Dunnettrsquospost hoc tests were used to compare group means Dataobtained from the ATP assays violated the assumptionsof the ANOVA Thus these data were analyzed using anonparametric analysis the Mann-Whitney Rank Sum testWhen 119865 ratios exceeded the critical value (119875 le 005)the Newman-Keuls post hoc test was used to compare theexperimental means 119875 le 005 (two-tailed) values wereconsidered statistically significant

3 Results

31 ROSRNS Expression in Urinary Bladder with CYP-Induced Cystitis and the Effects of Tempol We determinedthe levels of reactive oxygen species (ROS) and reactivenitrogen species (RNS) in the urinary bladder following CYPtreatment (4 hr 48 hr) and in the presence of the antioxidantTempol 4 hr and 48 hr CYP-induced cystitis significantly

(119875 le 001) increased the total free radical presence inthe detrusor and urothelium that was significantly reduced(119875 le 001) by Tempol (1mM) delivered in the drinking water(Figure 1(a)) With both 4 hr and 48 hr CYP-induced cystitisthe increase in total free radical presence was significantly(119875 le 001) greater in the detrusor compared to the urotheli-um (Figure 1(a)) Basal expression of total free radical pres-ence in the detrusor was significantly (119875 le 001) greater inthe detrusor compared to the urothelium (Figure 1(a))

32 3-Nitrotyrosine (3-NT) Expression in Urinary Bladderwith CYP-Induced Cystitis and the Effects of Tempol 3-NTa product of tyrosine nitration mediated by RNS such asperoxynitrite anion and nitrogen dioxide is considered amarker of NO-dependent RNS-induced nitrative stress [38]4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001)increased 3-NT expression in the urinary bladder that wassignificantly reduced (119875 le 001) by Tempol (1mM) deliveredin the drinking water (Figure 1(b)) 3-NT expression in theurinary bladder was significantly (119875 le 001) greater following4 hr CYP-induced cystitis compared to 48 hr CYP-inducedcystitis (Figure 1(b))

33 Substance P (Sub P) and Calcitonin Gene-Related Peptide(CGRP) Expression in Urinary Bladder and Cystometric Fluidwith CYP-Induced Cystitis and the Effects of Tempol Theneuropeptides Sub P and CGRP are known modulators ofinflammation and may contribute to the pathogenesis ofmany diseases includingmigraine asthma and urinary blad-der inflammation [4 20 39 40] 4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001) increased Sub P andCGRP expression in the urinary bladder (Figures 2(a) and3(a)) and cystometric fluid (Figures 2(b) and 3(b)) that wassignificantly reduced (119875 le 001) by Tempol (1mM) deliveredin the drinking water (Figures 2(a) 2(b) 3(a) and 3(b)) Sub Pexpression in the urinary bladder was similar following 4 hrand 48 hr CYP-induced cystitis in the urinary bladder andcystometric fluid (Figures 2(a) and 2(b)) In contrast 48 hrCYP-induced cystitis resulted in significantly (119875 le 001)greater CGRP expression in the urinary bladder and cysto-metric fluid compared to the 4 hr time point (Figures 3(a)and 3(b))

34 Adenosine Triphosphate (ATP) Expression in UrinaryBladder and Cystometric Fluid with CYP-Induced Cystitis andthe Effects of Tempol Numerous studies have described arole(s) for ATP in urinary bladder dysfunction pain andaltered ATP release mechanisms in animal models and clini-cal studies of BPSIC [8] 4 hr and 48 hr CYP-induced cystitissignificantly (119875 le 001) increased ATP expression in theurinary bladder and cystometric fluid that was significantlyreduced (119875 le 001) by Tempol (1mM) delivered in thedrinking water (Figures 4(a) and 4(b)) ATP expression in theurinary bladder was similar following 4 hr and 48 hr CYP-induced cystitis (Figure 4(a)) In contrast 4 hr CYP-inducedcystitis resulted in significantly (119875 le 001) greater ATPexpression in the cystometric fluid compared to the 48 hrtime point (Figure 4(b))


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Figure 1 Cyclophosphamide- (CYP-) induced cystitis increases reactive oxygen species (ROS)reactive nitrogen species (RNS) and 3-nitrotyrosine (3-NT) in the urinary bladder and the antioxidant Tempol reduces expression (a) 4 hr and 48 hr CYP-induced cystitissignificantly (119875 le 001) increased ROSRNS expression in urothelium and detrusor that was significantly (119875 le 001) reduced with TempolBasal expression and CYP-induced ROSRNS expression were significantly (119875 le 001) greater in detrusor compared to urothelium Tempolwas without effect on ROSRNS expression in urothelium and detrusor from control (no CYP) rats (b) 4 hr and 48 hr CYP-induced cystitissignificantly (119875 le 001) increased ROSRNS expression in urinary bladder that was significantly (119875 le 001) reduced with Tempol Tempolwas without effect on 3-NT expression in urinary bladder from control (no CYP) rats lowast119875 le 001 119899 = 6 for control and treatment groups

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Figure 2 Cyclophosphamide- (CYP-) induced cystitis increases substance P (Sub P) in the urinary bladder and cystometric fluid and theantioxidant Tempol reduces expression (a) 4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001) increased Sub P expression inurinary bladder that was significantly reduced with Tempol Tempol was without effect on Sub P expression in urinary bladder from control(no CYP) rats (b) 4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001) increased Sub P expression in cystometric fluid that wassignificantly (119875 le 001) reduced with Tempol Tempol was without effect on Sub P expression in cystometric fluid from control (no CYP)rats lowast119875 le 001 119899 = 6 for control and treatment groups

35 Effect of an Antioxidant Tempol on Bladder FunctionConscious open outlet cystometry with continuous intrav-esical infusion of saline was performed in separate groups(119899 = 6 each) of control and CYP-treated (48 h) rats withor without Tempol (vehicle only) in the drinking water todetermine bladder function (Figures 5ndash8)

351 Control (NoCYPTreatment) Tempol (1mM) treatmenthad no effects on ICI BC or VV in control rats (no CYP

treatment) compared with control rats (no CYP treatment)treated with vehicle (Figure 5) There were no changes in BPTP or peak MP with Tempol treatment compared to controlrats (no CYP treatment) treated with vehicle (Figure 6)Residual volume in control rats with or without Tempol(vehicle) treatment was minimal (le10 120583L)

352 CYP Treatment As previously demonstrated [22ndash24] and confirmed here CYP treatment (4 hr and 48 hr)


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Figure 3 Cyclophosphamide- (CYP-) induced cystitis increases calcitonin gene-related peptide (CGRP) in the urinary bladder andcystometric fluid and the antioxidant Tempol reduces expression (a) 4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001) increasedCGRP expression in urinary bladder that was significantly (119875 le 001) reduced with Tempol Tempol was without effect on CGRP expressionin urinary bladder from control (no CYP) rats (b) 4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001) increased CGRP expressionin cystometric fluid that was significantly (119875 le 001) reduced with Tempol lowast119875 le 001 119899 = 6 for control and treatment groups

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Figure 4 Cyclophosphamide- (CYP-) induced cystitis increases adenosine triphosphate (ATP) in the urinary bladder and cystometric fluidand the antioxidant Tempol reduces expression (a) 4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001) increased ATP expressionin urinary bladder that was significantly (119875 le 001) reduced with Tempol Tempol was without effect on ATP expression in urinary bladderfrom control (no CYP) rats (b) 4 hr and 48 hr CYP-induced cystitis significantly (119875 le 001) increased ATP expression in cystometric fluidthat was significantly (119875 le 001) reduced with Tempol Tempol was without effect on ATP expression in cystometric fluid from control (noCYP) rats lowast119875 le 001 119899 = 6 for control and treatment groups

increased void frequency and decreased BC ICI and VVcompared with control rats (no CYP treatment) (Figures5ndash8) Additionally 48 hr CYP-induced cystitis significantly(119875 le 001) increased BP and TP (Figure 6) Tempol in thedrinking water (1mM) of CYP-treated rats (4 hr and 48 hr)significantly (119875 le 001) increased the ICI (ie decreasedvoiding frequency) (Figure 5(a) 20ndash29-fold) increased BC(Figure 5(c) 28ndash31-fold) and increased VV (Figure 5(b)29ndash54-fold) compared to rats treated with CYP (4 hr and48 hr) receiving vehicle (Figures 7 and 8) Tempol treatmentof CYP-treated rats (4 hr and 48 hr) increased BC to 70

of control rats (no CYP treatment) (Figures 5(c) 7 and 8)Effects of Tempol on bladder function in CYP-treated ratspersisted for at least 2 hr Residual volume in CYP-treated(4 hr and 48 hr) rats with or without Tempol treatment wasminimal and similar to that observed in control (no CYPtreatment) (le10 120583L) CYP-treated rats (4 hr and 48 hr) treatedwith or without Tempol (vehicle) exhibited no differences inBP TP or peak MP (Figures 6(a)ndash6(c)) Tempol also signifi-cantly (119875 le 001) reduced the number (24 plusmn 04micturitioncycle versus 06 plusmn 02micturition cycle) and amplitude(112 plusmn 13 cm H

2O versus 75 plusmn 05 cm H

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L)

(c)

Figure 5 Summary histograms of the effects of Tempol (1mM) on intercontraction interval (ICI s) bladder capacity (BC 120583L) and voidvolume (VV mL) in CYP-treated (4 hr 48 hr) rats Tempol in the drinking water significantly (119875 le 001) increased ICI (a) VV (b) and BC(c) in CYP-treated (4 hr and 48 hr) rats Tempol was without effect in control (no CYP treatment) rats 119875 le 001 compared to control +vehicle (between-group difference) lowast119875 le 001 compared to 4 hr or 48 hr CYP + vehicle (within-group difference) Sample sizes are 119899 = 6 incontrol and treatment groups

(increases in bladder pressure during the filling phasewithoutthe release of fluid) in 4 hr CYP-treated rats (Figure 7)The effects of Tempol on NVCs in the 48 hr CYP-treatedgroup were not determined due to the dramatically increasedvoiding frequency that made the presence of NVCs difficultto determine

4 Discussion

The present studies demonstrate several novel findings withrespect to the induction and reduction of oxidative stresswith the antioxidant Tempol following cyclophosphamide-(CYP-) induced cystitis CYP-induced cystitis (4 hr and48 hr) increased ROSRNS and 3-NT expression in theurinary bladder In addition CYP-induced cystitis increasedexpression of neuropeptides CGRP and Sub P in the uri-nary bladder as well as cystometric fluid collected duringconscious cystometry CYP-induced cystitis also increasedATP in the urinary bladder and cystometric fluid Providingrats with the antioxidant Tempol in the drinking waterprior to and during the induction of CYP-induced cystitis

significantly reduced the expression of ROSRNS CGRPSub P and ATP in urinary bladder and cystometric fluidFurther Tempol decreased voiding frequency and increasedthe intercontraction interval and bladder capacity withouteffects on urinary bladder pressures (baseline threshold andpeak) in rats with CYP-induced cystitis Tempol did not alterbladder function in control (no CYP treatment) rats Thesestudies demonstrate that CYP-induced cystitis is associatedwith oxidative stress in the urinary tract and that use of theantioxidant Tempol reduces oxidative stress and improvesurinary bladder function

Molecular oxygen reduction results in the productionof several reactive intermediates that must be actively scav-enged [41] Under conditions of inflammation there maybe insufficient scavenging of reactive intermediates that canlead to oxidative stress and damage to cellular structure andfunction Recent studies suggest that the generation of thesereactive intermediates contributes to the pathogenesis of uri-nary bladder dysfunction with CYP administration [42ndash44]The contribution of oxidative stress in bladder inflammationfollowing CYP is further supported by the attenuation of


VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70

Control 4hr CYP 48hr CYP

MP

(cm

H2O

)

(c)

Figure 6 Tempol in the drinking water was without effect on baseline pressure ((a) BP) threshold pressure ((b) TP) or peak micturitionpressure ((c) MP cm H

2O) in control or CYP-treated (4 hr 48 hr) rats Sample sizes are 119899 = 6 in control and treatment groups 119875 le 001

compared to control + vehicle (between-group difference)

tissue damage following the administration of agents withantioxidant properties like taurine [42] flavonoid [43] beta-carotene and others [44] Our studies using the CYP modelof urinary bladder inflammation at two time points (4 hrand 48 hr) confirm the expression of reactive intermediatesincludingROSRNS and 3-NT in the urinary bladder Expres-sion of ROSRNS following CYP-induced cystitis was signifi-cantly greater in the detrusor smoothmuscle compared to theurothelium but both tissues exhibited increased ROSRNScompared to control (no CYP treatment) urinary bladder 3-NT expression has been previously demonstrated in lowerurinary tract tissues following injury or inflammation Partialoutlet obstruction in rabbit is associated with increased NTin mucosa [45] that is correlated with progressive decreasein contractility of detrusor smooth muscle [46] Previousstudies demonstrated iNOS and NT immunoreactivity inthe urothelium and inflammatory infiltrates in the laminapropria of individuals with BPSIC with a Hunnerrsquos lesion[47] Consistent with this clinical study the present studydemonstrates increased 3-NT in the urinary bladder follow-ing CYP-induced cystitis (4 hr and 48 hr) in a rat model Theamino acid tyrosine is particularly susceptible to nitrationand the formation of 3-NT may represent a biomarker for

the generation of reactive nitrogen intermediates in vivo[47] In addition to being a biomarker 3-NT could alsohave a detrimental impact on cell function and viability byinhibiting protein phosphorylation by tyrosine kinases andinterfering with the signal transduction mechanism [48]In addition in vitro nitration of a specific tyrosine residueinactivates manganese superoxide dismutase [49] that maylead to a greater concentration of ROSRNS perpetuatingtissue damage and altered function

As amajor sensory component of the urinary bladder theurothelium is able to respond to various extracellular stimuliby releasing neuroactive factors like ATP acetylcholine andnitric oxide [8]The sustained release of these factors such aswhat may occur with inflammation may underlie the devel-opment of urinary bladder dysfunction and lower urinarytract symptoms The role of oxidative stress with ATP releasein epithelial cells however is not well defined In endothelialcells oxidative stress has been demonstrated to mediate thedirect release of ATP and inhibit the catabolism of extra-cellular ATP [50 51] Further purinergic receptor activationhas been associated with increased cellular production andrelease of multiple inflammatory mediators including super-oxide anion nitric oxide and other ROS Purinergic receptor


75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)

Figure 7 Representative cystometrogram recordings using continuous intravesical infusion of saline in conscious rats with an open outletfrom a CYP-treated (4 hr) rat with vehicle ((a1)ndash(a3)) and a CYP-treated (4 hr) rat with Tempol (1mM (b1)ndash(b3)) ((a) (b)) Bladder functionin a CYP-treated (4 hr) rat without Tempol (vehicle only (a1)ndash(a3)) and in a CYP-treated (4 hr) rat with Tempol (1mM in the drinking water(b1)ndash(b3)) during continuous intravesical infusion of saline Bladder function recordings in (a) and (b) are from different rats Infused volume(IF 120583L (a1) (b1)) bladder pressure (BP cm H

2O (a2) (b2)) and void volume (VV mL (a3) (b3)) with vehicle ((a1)ndash(a3)) and with Tempol

treatment ((b1)ndash(b3)) are shown Arrows ((a2) (b2)) indicate examples of nonvoiding contractions that were significantly (119875 le 001) reducedin number and amplitude in CYP-treated (4 hr) rats with Tempol

activation induces ROS generation in numerous cell typesresulting in a variety of downstream effects including tran-scription factor activation [13] proinflammatory cytokinerelease [14 15] and cell death [16] Our studies determinedthe contribution of ROSRNS to extracellular ATP expres-sion with CYP-induced cystitis and Tempol CYP-inducedcystitis increased ATP expression in the urinary bladder andcystometric fluid that was significantly reduced by Tempoladministration It is not known from the present studieswhether antioxidant treatment directly inhibits the releaseof ATP through purinergic receptor blockade Additionalstudies in CYP-treated rats involving assessment of oxidativestress in the presence of purinergic receptor (P2) blockadeas well as the effects of P2 activation on oxidative stress incontrols should be considered

Tempol is a membrane-permeable redox-cycling agentthat scavenges superoxide anions and decreases the forma-tion of hydroxyl radicals [35]Theprotective effects of Tempolto tissues with inflammation and oxidative damage have beenwidely established For example Tempol has been shown to

decreaseNF-kappaB activationwith acute inflammation [52]decrease neutrophil infiltration and PARP activation withperiodontitis [53] and decrease cytokine release stimulatedby an inflammatory soup [54] In animal studies puriner-gic neuromuscular transmission and propulsive motilitywere significantly restored in the inflamed colon treatedwith the free radical scavenger Tempol [55] Furthermoreintrathecal Tempol administration has been demonstratedto decrease thermal and mechanical hypersensitivity withneuropathic pain [56] Due to the central and peripheral anti-inflammatory properties of Tempol our studies determinedthe role of oxidative stress on bladder function with CYP-induced cystitis and Tempol administration Pretreatmentwith Tempol and continued treatment with Tempol dur-ing the progression of CYP-induced cystitis significantlyimproved urinary bladder function In CYP-treated (4 hrand 48 hr) rats Tempol in the drinking water increased theintercontraction interval and bladder capacity and reducedurinary frequency The presence and amplitude of NVCsduring the filling phase of the urinary bladder were also


Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)

Figure 8 Representative cystometrogram recordings using continuous intravesical infusion of saline in conscious rats with an open outletfromaCYP-treated (48 hr) ratwith vehicle ((a1)ndash(a3)) and aCYP-treated (48 hr) ratwithTempol (1mM (b1)ndash(b3)) ((a) (b)) Bladder functionin a CYP-treated (48 hr) rat without Tempol (vehicle only (a1)ndash(a3)) and in a CYP-treated (48 hr) rat with Tempol (1mM in the drinkingwater (b1)ndash(b3)) during continuous intravesical infusion of saline Bladder function recordings in (a) and (b) are from different rats Infusedvolume (IF 120583L (a1) (b1)) bladder pressure (BP cm H

2O (a2) (b2)) and void volume (VV mL (a3) (b3)) with vehicle ((a1)ndash(a3)) and with

Tempol treatment ((b1)ndash(b3)) are shown

reduced in 4 hr CYP-treated rats given Tempol Given thedemonstration of Tempol decreased mechanical hypersen-sitivity from neuropathic pain [56] future studies shouldexamine the effects of Tempol administration on somatic (iepelvic and hindpaw) sensitivity in CYP-treated rats

Previous studies from our laboratory have demon-strated roles for neuropeptides including pituitary adenylatecyclase-activating polypeptide (PACAP) CGRP and SubP in CYP-induced bladder dysfunction [20 21] Increasedexpression of PACAP CGRP and Sub P was demonstrated inthe urinary bladder lumbosacral spinal cord and dorsal rootganglia of CYP-treated rats and pharmacological blockade ofthe PACAP specific PAC1 receptor improved urinary bladderfunction inCYP-treated rats [20 21]The present studies con-firm increased expression of Sub P and CGRP in the urinarybladder and cystometric fluidwithCYP-induced cystitis (4 hrand 48 hr) Tempol treatment significantly reduced Sub Pand CGRP expression in the urinary bladder and cystometricfluid Previous studies demonstrated that Sub P via neu-rokinin (NK) receptor facilitates bladder afferent signaling

and ROS formation in bladder in association with neuro-genic inflammation [57] Increased Sub P release increasedROS in the bladder via increased mast cell degranulationintercellular adhesion molecule expression and leukocyteadhesion Future studies can determine the involvement ofSub PNK receptor signaling with these sources of ROS inthe inflamed bladder following CYP-induced cystitis Theneuropeptides PACAP27 PACAP38 and VIP evoked ATPrelease from rat urothelial cell cultures that was significantlyblocked by the PAC1 receptor selective antagonist M65 [31]The present studies suggest two possibilities by which CYP-induced cystitis increases ATP expression in the urinarybladder and cystometric fluid (1) neuropeptide (Sub P andCGRP) evoked release of ATP and (2) ROS evoked ATPrelease Current research is consistent with the suggestionthat neuropeptide and ROS signaling are regulators of blad-der physiology at the level of the urinary bladder and possiblythe urothelium [31]The present studies demonstrate that useof the antioxidant Tempol not only reduces the presence ofoxidative stress markers in the urinary tract but also reduces


modulatorsmediators of inflammation including neuropep-tides (CGRP Sub P) also known to contribute to micturitionreflex plasticity and dysfunction with CYP-induced cystitisIn the context of CYP-induced cystitis Tempol treatmentmay be more beneficial given its broader impact on oxidativestress markers and other modulators of urinary bladderinflammationwhile also improving urinary bladder function

The present studies demonstrate oxidative stress in theurinary tract following CYP-induced cystitis and improve-ment in urinary bladder function and markers of oxidativestress with antioxidant treatment however there are addi-tional issues to be considered Future studies may include (1)determining the effects of Tempol after the induction of CYP-induced cystitis rather than as a pretreatment on urinarybladder function (2) determining if Tempol treatment canreduce somatic sensitivity in the CYP model of urinary blad-der inflammation with referred somatic hypersensitivity (3)determining the effects of Tempol in a more chronic modelof CYP-induced urinary bladder inflammation that we haveused extensively [25 26] The present studies demonstratethat CYP-induced cystitis results in oxidative stress in the uri-nary tract and that the antioxidant Tempol ameliorates CYP-induced bladder dysfunctionThese studies suggest that phar-macological interventions directed at oxidative stress media-torsmarkers may be a promising strategy to address inflam-mation of the urinary tract and target organ dysfunction

Conflict of Interests

The authors declare that there is no conflict of interests in thisstudy

Acknowledgments

This work was funded by National Institutes of Health(NIH) Grants DK051369 (MAV) DK060481 (MAV) andDK065989 (MAV) This publication was also supported byGrants from the National Center for Research Resources (5P30 RR 032135) and the National Institute of GeneralMedicalSciences (8 P30 GM 103498) from the NIH Mr MitchelDaniel was supported by the National Science Foundation(NSF) Research Experience for Undergraduates (REU no1262786) through the Summer Undergraduate NeuroscienceFellowship Program at UVM

References

[1] G K Kartha H Kerr and D A Shoskes ldquoClinical phenotypingof urologic pain patientsrdquo Current Opinion in Urology vol 23no 6 pp 560ndash564 2013

[2] P Hanno A Lin J Nordling et al ldquoBladder pain syndromeinternational consultation on incontinencerdquo Neurourology andUrodynamics vol 29 no 1 pp 191ndash198 2010

[3] E J Gonzalez L Arms and M A Vizzard ldquoThe role(s) ofcytokineschemokines in urinary bladder inflammation anddysfunctionrdquo BioMed Research International vol 2014 ArticleID 120525 17 pages 2014

[4] E J Gonzalez L Merrill and M A Vizzard ldquoBladder sen-sory physiology neuroactive compounds and receptors sen-sory transducers and target-derived growth factors as targetsto improve functionrdquo The American Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 306 no12 pp R869ndashR878 2014

[5] MWWitthaus F Nipa J-H Yang Y Li L B Lerner andKMAzadzoi ldquoBladder oxidative stress in sleep apnea contributes todetrusor instability and nocturiardquoThe Journal of Urology 2014

[6] MNomiya K EAndersson andOYamaguchi ldquoChronic blad-der ischemia and oxidative stress new pharmacotherapeutictargets for lower urinary tract symptomsrdquo International Journalof Urology vol 22 no 1 pp 40ndash46 2015

[7] A A Geronikaki and A M Gavalas ldquoAntioxidants andinflammatory disease synthetic and natural antioxidants withanti-inflammatory activityrdquo Combinatorial Chemistry and HighThroughput Screening vol 9 no 6 pp 425ndash442 2006

[8] L Birder and K-E Andersson ldquoUrothelial signalingrdquo Physio-logical Reviews vol 93 no 2 pp 653ndash680 2013

[9] Y Sun S Keay P G de Deyne and T C Chai ldquoAugmentedstretch activated adenosine triphosphate release from bladderuroepithelial cells in patients with interstitial cystitisrdquo Journalof Urology vol 166 no 5 pp 1951ndash1956 2001

[10] L A Birder S R Barrick J R Roppolo et al ldquoFeline intersti-tial cystitis results in mechanical hypersensitivity and alteredATP release from bladder urotheliumrdquo American Journal ofPhysiologymdashRenal Physiology vol 285 no 3 pp F423ndashF4292003

[11] C P Smith V M Vemulakonda S Kiss T B Boone and G TSomogyi ldquoEnhanced ATP release from rat bladder urotheliumduring chronic bladder inflammation effect of botulinum toxinArdquo Neurochemistry International vol 47 no 4 pp 291ndash2972005

[12] A N Guerra M L Gavala H S Chung and P J BerticsldquoNucleotide receptor signalling and the generation of reactiveoxygen speciesrdquo Purinergic Signalling vol 3 no 1-2 pp 39ndash512007

[13] S Wesselborg M K A Bauer M Vogt M L Schmitz and KSchulze-Osthoff ldquoActivation of transcription factor NF-120581B andp38 mitogen-activated protein kinase is mediated by distinctand separate stress effector pathwaysrdquoThe Journal of BiologicalChemistry vol 272 no 19 pp 12422ndash12429 1997

[14] C M Cruz A Rinna H J Forman A L M Ventura P MPersechini and D M Ojcius ldquoATP activates a reactive oxygenspecies-dependent oxidative stress response and secretion ofproinflammatory cytokines in macrophagesrdquo The Journal ofBiological Chemistry vol 282 no 5 pp 2871ndash2879 2007

[15] J Hewinson S F Moore C Glover A G Watts and A BMacKenzie ldquoA key role for redox signaling in rapid P2X7receptor-induced IL-1120573 processing in human monocytesrdquo TheJournal of Immunology vol 180 no 12 pp 8410ndash8420 2008

[16] M Furuhata E Takada TNoguchi H Ichijo and JMizuguchildquoApoptosis signal-regulating kinase (ASK)-1 mediates apoptosisthrough activation of JNK1 following engagement ofmembraneimmunoglobulinrdquo Experimental Cell Research vol 315 no 20pp 3467ndash3476 2009

[17] M A Vizzard ldquoUp-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways afterchronic cystitisrdquoThe Journal of ComparativeNeurology vol 420no 3 p 335 2000

[18] M A Vizzard ldquoChanges in urinary bladder neurotrophicfactor mRNA and NGF protein following urinary bladder


dysfunctionrdquo Experimental Neurology vol 161 no 1 pp 273ndash284 2000

[19] B Schnegelsberg T T Sun G Cain et al ldquoOverexpression ofNGF in mouse urothelium leads to neuronal hyperinnervationpelvic sensitivity and changes in urinary bladder functionrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 298 no 3 pp R534ndashR547 2010

[20] L Merrill B Girard L Arms P Guertin and M A VizzardldquoNeuropeptidereceptor expression and plasticity in micturi-tion pathwaysrdquo Current Pharmaceutical Design vol 19 no 24pp 4411ndash4422 2013

[21] K M Braas V May P Zvara et al ldquoRole for pituitary adenylatecyclase activating polypeptide in cystitis-induced plasticity ofmicturition reflexesrdquo The American Journal of PhysiologymdashRegulatory Integrative and Comparative Physiology vol 290 no4 pp R951ndashR962 2006

[22] B P Cheppudira B M Girard S E Malley K C Schutz VMay and M A Vizzard ldquoUpregulation of vascular endothelialgrowth factor isoform VEGF-164 and receptors (VEGFR-2Npn-1 and Npn-2) in rats with cyclophosphamide-inducedcystitisrdquoTheAmerican Journal of PhysiologymdashRenal Physiologyvol 295 no 3 pp F826ndashF836 2008

[23] M B Klinger B Girard and M A Vizzard ldquop75NTR expres-sion in rat urinary bladder sensory neurons and spinal cordwithcyclophosphamide-induced cystitisrdquo Journal of ComparativeNeurology vol 507 no 3 pp 1379ndash1392 2008

[24] M B Klinger andM A Vizzard ldquoRole of p75NTR in female raturinary bladder with cyclophosphamide-induced cystitisrdquo TheAmerican Journal of PhysiologymdashRenal Physiology vol 295 no6 pp F1778ndashF1789 2008

[25] V Y Hu S Malley A Dattilio J B Folsom P Zvara and MA Vizzard ldquoCOX-2 and prostanoid expression in micturitionpathways after cyclophosphamide-induced cystitis in the ratrdquoTheAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 284 no 2 pp R574ndashR585 2003

[26] MAVizzard ldquoAlterations in spinal cord Fos protein expressioninduced by bladder stimulation following cystitisrdquo AmericanJournal of PhysiologymdashRegulatory Integrative and ComparativePhysiology vol 278 no 4 pp R1027ndashR1039 2000

[27] E J Gonzalez BMGirard andMAVizzard ldquoExpression andfunction of transforming growth factor-120573 isoforms and cognatereceptors in the rat urinary bladder following cyclophospham-ide-induced cystitisrdquo The American Journal of PhysiologymdashRenal Physiology vol 305 no 9 pp F1265ndashF1276 2013

[28] G M Herrera M J Pozo P Zvara et al ldquoUrinary bladderinstability induced by selective suppression of the murine smallconductance calcium-activated potassium (SK3) channelrdquo Jour-nal of Physiology vol 551 no 3 pp 893ndash903 2003

[29] K A Corrow and M A Vizzard ldquoPhosphorylation of extra-cellular signal-regulated kinases in urinary bladder in ratswith cyclophosphamide-induced cystitisrdquo American Journal ofPhysiologymdashRegulatory Integrative and Comparative Physiologyvol 293 no 1 pp R125ndashR134 2007

[30] C Dugan S Malley L Arms V May and M A Vizzard ldquoRoleof c-Jun N-terminal kinase (JNK) activation in micturitionreflexes in cyclophosphamide (CYP)-induced cystitis in femaleratsrdquo Journal of Molecular Neuroscience vol 54 no 3 pp 360ndash369 2014

[31] B M Girard A Wolf-Johnston K M Braas L A BirderV May and M A Vizzard ldquoPACAP-mediated ATP releasefrom rat urothelium and regulation of PACAPVIP and recep-tor mRNA in micturition pathways after cyclophosphamide

(CYP)-induced cystitisrdquo Journal of Molecular Neuroscience vol36 no 1ndash3 pp 310ndash320 2008

[32] T Miyauchi E P Wei and J T Povlishock ldquoEvidence forthe therapeutic efficacy of either mild hypothermia or oxygenradical scavengers after repetitive mild traumatic brain injuryrdquoJournal of Neurotrauma vol 31 no 8 pp 773ndash781 2014

[33] Q Liang A D Smith S Pan et al ldquoNeuroprotective effects ofTEMPOL in central and peripheral nervous system models ofParkinsonrsquos diseaserdquo Biochemical Pharmacology vol 70 no 9pp 1371ndash1381 2005

[34] S Cuzzocrea M C McDonald E Mazzon et al ldquoTempol amembrane-permeable radical scavenger reduces dinitroben-zene sulfonic acid-induced colitisrdquo European Journal of Phar-macology vol 406 no 1 pp 127ndash137 2000

[35] P K Chatterjee S Cuzzocrea P A J Brown et al ldquoTempola membrane-permeable radical scavenger reduces oxidantstress-mediated renal dysfunction and injury in the ratrdquoKidneyInternational vol 58 no 2 pp 658ndash673 2000

[36] H Nakamura S Matoba E Iwai-Kanai et al ldquoP53 promotescardiac dysfunction in diabetic mellitus caused by excessivemitochondrial respiration-mediated reactive oxygen speciesgeneration and lipid accumulationlipid accumulationrdquo Circula-tion Heart Failure vol 5 no 1 pp 106ndash115 2012

[37] L Gao Y Li H D Schultz et al ldquoDownregulated Kv43expression in the RVLM as a potential mechanism for sym-pathoexcitation in rats with chronic heart failurerdquo AmericanJournal of PhysiologymdashHeart and Circulatory Physiology vol298 no 3 pp H945ndashH955 2010

[38] M C Franco andA G Estevez ldquoTyrosine nitration asmediatorof cell deathrdquoCellular andMolecular Life Sciences vol 71 no 20pp 3939ndash3950 2014

[39] A C Raddant and A F Russo ldquoCalcitonin gene-related peptidein migraine intersection of peripheral inflammation and cen-tral modulationrdquo Expert Reviews in Molecular Medicine vol 13article e36 18 pages 2011

[40] S Frydas G Varvara G Murmura et al ldquoImpact of capsaicinonmast cell inflammationrdquo International Journal of Immunopa-thology and Pharmacology vol 26 no 3 pp 597ndash600 2013

[41] I Fridovich ldquoThe biology of oxygen radicalsrdquo Science vol 201no 4359 pp 875ndash880 1978

[42] A RAAbd-AllahAMGadoAAAl-MajedAAAl-Yahyaand O A Al-Shabanah ldquoProtective effect of taurine againstcyclophosphamide-induced urinary bladder toxicity in ratsrdquoClinical and Experimental Pharmacology and Physiology vol 32no 3 pp 167ndash172 2005

[43] A Ozcan A Korkmaz S Oter and O Coskun ldquoContributionof flavonoid antioxidants to the preventive effect of mesna incyclophosphamide-induced cystitis in ratsrdquo Archives of Toxicol-ogy vol 79 no 8 pp 461ndash465 2005

[44] S Sadir S Deveci A Korkmaz and S Oter ldquoAlpha-tocop-herol beta-carotene and melatonin administration protectscyclophosphamide-induced oxidative damage to bladder tissuein ratsrdquo Cell Biochemistry and Function vol 25 no 5 pp 521ndash526 2007

[45] Y S JuanW Y Lin C Kalorin B A Kogan RM Levin andAMannikarottu ldquoThe effect of partial bladder outlet obstructionon carbonyl and nitrotyrosine distribution in rabbit bladderrdquoUrology vol 70 no 6 pp 1249ndash1253 2007

[46] A Mannikarottu A D-Y Lin C Whitebeck R LeggettB Kogan and R Levin ldquoEffect of partial bladder outletobstruction on nitrotyrosine levels and their correlation with


contractile functionrdquo Neurourology and Urodynamics vol 25no 4 pp 397ndash401 2006

[47] B Balint S A Kharitonov T Hanazawa et al ldquoIncreasednitrotyrosine in exhaled breath condensate in cystic fibrosisrdquoEuropean Respiratory Journal vol 17 no 6 pp 1201ndash1207 2001

[48] A van der Vliet J P Eiserich M K Shigenaga and C ECross ldquoReactive nitrogen species and tyrosine nitration in therespiratory tract epiphenomena or a pathobiologic mechanismof diseaserdquo American Journal of Respiratory and Critical CareMedicine vol 160 no 1 pp 1ndash9 1999

[49] F Yamakura H Taka T Fujimura and K Murayama ldquoInac-tivation of human manganese-superoxide dismutase by perox-ynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosinerdquoThe Journal of Biological Chemistry vol 273 no23 pp 14085ndash14089 1998

[50] T K Aalto and K O Raivio ldquoMetabolism of extracellular ade-nine nucleotides by human endothelial cells exposed to reactiveoxygen metabolitesrdquo The American Journal of PhysiologymdashCellPhysiology vol 264 no 2 pp C282ndashC286 1993

[51] S Ahmad A Ahmad M Ghosh C C Leslie and C WWhite ldquoExtracellular ATP-mediated Signaling for Survivalin Hyperoxia-induced Oxidative Stressrdquo Journal of BiologicalChemistry vol 279 no 16 pp 16317ndash16325 2004

[52] S Cuzzocrea B Pisano L Dugo et al ldquoTempol reduces theactivation of nuclear factor-kappaB in acute inflammationrdquo FreeRadical Research vol 38 no 8 pp 813ndash819 2004

[53] R Di Paola E Mazzon D Zito et al ldquoEffects of Tempola membrane-permeable radical scavenger in a rodent modelperiodontitisrdquo Journal of Clinical Periodontology vol 32 no 10pp 1062ndash1068 2005

[54] T Volk M Hensel H Schuster and W J Kox ldquoSecretion ofMCP-1 and IL-6 by cytokine stimulated production of reactiveoxygen species in endothelial cellsrdquo Molecular and CellularBiochemistry vol 206 no 1-2 pp 105ndash112 2000

[55] J A Roberts L Durnin K A Sharkey V N Mutafova-Yambolieva and G M Mawe ldquoOxidative stress disruptspurinergic neuromuscular transmission in the inflamed colonrdquoThe Journal of Physiology vol 591 no 15 pp 3725ndash3737 2013

[56] M Tanabe Y Nagatani K Saitoh K Takasu and H OnoldquoPharmacological assessments of nitric oxide synthase isoformsand downstream diversity of NO signaling in the maintenanceof thermal and mechanical hypersensitivity after peripheralnerve injury in micerdquo Neuropharmacology vol 56 no 3 pp702ndash708 2009

[57] C-T Chien H-J Yu T-B Lin M-K Lai and S-M HsuldquoSubstance P via NK1 receptor facilitates hyperactive bladderafferent signaling via action of ROSrdquo The American Journal ofPhysiologymdashRenal Physiology vol 284 no 4 pp F840ndashF8512003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Using a rat model of urinary bladder inflammationinduced by cyclophosphamide (CYP) [3 4 17 18] wedetermined (1) the expression of oxidative stress markers(3-nitrotyrosine (3-NT) ROSRNS) and other modulatorsof inflammation the neuropeptides calcitonin gene-relatedpeptide (CGRP) and substance P (Sub P) in the urinary tract(2) the contribution of ROSRNS to ATP expression in theurinary bladder and urine with an antioxidant and superox-ide dismutase mimetic Tempol (1mM in drinking water)and (3) the role of ROSRNS in urinary bladder functionwith Tempol combined with open outlet and conscious andcontinuous intravesical infusion [19] Although the etiologyof BPSIC is unknown previous studies [3 4 17 18] havedemonstrated that the rat CYP model of urinary bladderinflammation is a reliable and reproducible model withface validity (eg increased voiding frequency and referredsomatic sensitivity) to BPSIC Previous studies have demon-strated roles for neuropeptides including CGRP and Sub Pin CYP-induced bladder dysfunction [20 21] and the currentstudies have continued to focus on these two neuropeptidesabundantly expressed in bladder sensory pathways

2 Materials and Methods

21 Animals Adult female Wistar rats (200ndash225 g CharlesRiver St Constant Canada) were used for this study Ratswere housed two per cage and maintained in standardlaboratory conditions with free access to food and water TheUniversity of Vermont Institutional Animal Care and UseCommittee approved all animal use procedures (protocol 08-085) Animal experimentation was carried out in accordancewith the National Institutes of Health Guide for the Careand Use of Laboratory Animals All efforts were made tominimize the potential for animal pain stress or distress

22 Induction of Cyclophosphamide- (CYP-) Induced CystitisRats were anesthetized under isoflurane (2) and acute cys-titis was induced with a single injection of CYP (150mgkgip) and used in studies at various time points (4 hours (hr) or48 hr) after treatment [22ndash24] Control rats received volume-matched injections of saline (09 ip) or no treatmentand no difference among the control groups was observedAll injections were performed under isoflurane (2) anes-thesia The CYP model of bladder inflammation producesan increase in voiding frequency with small micturitionvolumes and is associatedwith inflammatory cell infiltrates inthe urinary bladder including mast cells macrophages andneutrophils [25 26] Rats were euthanized using isoflurane(5) and a thoracotomy

For cystometry in conscious rats an unrestrained animalwas placed in a Plexiglas cage with a wire bottom Beforethe start of the recording the bladder was emptied and thecatheter was connected via a T-tube to a pressure transducer(Grass Model PT300 West Warwick RI) and microinjectionpump (Harvard Apparatus 22 South Natick MA) A SmallAnimal Cystometry Lab Station (MEDAssociates St AlbansVT) was used for urodynamic measurements [19 27] Salinesolution was infused at room temperature into the bladderat a rate of 10mLh to elicit repetitive bladder contractions

At least six reproducible micturition cycles were recordedafter the initial stabilization period of 25ndash30min [19 27]The following cystometric parameters were recorded in eachanimal baseline pressure (BP pressure at the beginning ofthe bladder filling) threshold pressure (TP bladder pressureimmediately prior to micturition) peak micturition pressure(MP) intercontraction interval (ICI time between micturi-tion events) bladder capacity (BC) void volume (VV) andpresence and amplitude of nonvoiding bladder contractions(NVCs) [19 27] NVCs were defined as rhythmic intravesicalpressure increases 7 cmH

2Oabove baseline during the filling

phase without the release of fluid from the urethra Bladdercapacity (BC) was measured as the volume of saline infusedinto the bladder at the time when micturition commenced[28] In these rats residual volume was less than 10 120583L there-fore VV and BC were similar After the initial stabilizationperiod cystometric fluid expelled during micturition eventswas collected and frozen on dry ice and stored at minus80∘C forfuture use in the assays described above At the conclusionof the experiment the rat was euthanized (5 isoflurane plusthoracotomy) the urinary bladder was harvested and used inthe assays described above

23 Split Bladder Preparation and Assessment of PotentialContamination of Bladder Layers The urothelium + sub-urothelium was dissected from the detrusor smooth muscleusing fine forceps under a dissecting microscope as previ-ously described [22 23] To confirm the specificity of oursplit bladder preparations urothelium + suburothelium anddetrusor samples were examined for the presence of 120572-smooth muscle actin (1 1000 Abcam Cambridge MA) anduroplakin II (1 25 American Research Products BelmontMA) by western blotting or reverse transcription PCR [2229] In urothelium + suburothelium layers only uroplakinII was present (data not shown) Conversely in detrusorsamples only 120572-smooth muscle actin was present (data notshown) All subsequent measurements of urinary bladderand cystometric fluid samples and conscious cystometry inrat groups were performed in a blind manner

24 Substance P (Sub P) Calcitonin Gene-Related Peptide(CGRP) and 3-Nitrotyrosine (3-NT) by Enzyme-Linked Im-munosorbent Assays (ELISAs) Tissue processing and ELISAswere performed as described previously [18 30] Briefly ratsfrom control (119899 = 6 each) and all experimental groups (119899 =6 each) were deeply anesthetized (4 isoflurane) and athoracotomy was performed Individual rat bladders weredissected weighed and placed in Tissue Protein ExtractionReagent (1 g tissue20mL Pierce Biotechnology WoburnMA) with complete protease inhibitor cocktail tablets (RocheApplied Science Mannheim Germany) and stored at minus80∘COn the day of assay individual bladders were disrupted witha Polytron homogenizer until being homogeneous and cen-trifuged (10000 rpm for 10min) [18 30] and the supernatantwas used for total protein estimation and CGRP (PhoenixPharmaceuticals Inc Burlingame CA) Sub P (PhoenixPharmaceuticals) and 3-NT (Millipore Corporation Beller-ica MA) quantification Total protein was determined by theCoomassie Plus Protein Assay Reagent Kit (Pierce) [18 30]














3 Results







0200400600800

10001200140016001800

DetrusorUrothelium

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowast

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

DCF

relat

ive fl

uore

scen

ce

(a)

02040608

1121416

lowast

lowast

lowast lowast

lowast

0

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

ng3

-NT120583

g to

tal p

rote

in

(b)


0

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

pg S

ub P

120583g

tota

l pro

tein

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

0

ng S

ub P

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)







0

05

1

15

2

25

3

lowast

lowast

lowastlowast

lowastpg

CG

RP120583

g to

tal p

rote

in

Con

trol+

vehi

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Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

10

20

30

40

50

60

lowast

lowast

lowast

lowast

lowast

ng C

GRP

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)


0

1

2

3

4

5

6

7

8lowast

lowast

lowast

lowast

pM A

TP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

002

004

006

008

01lowast

lowast

lowast lowast

lowast

nM A

TPm

L ur

ine

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)





2O) of NVCs


050

100150200250300350400450

Control

VehicleTempol

lowast

lowast

ICI (

s)

4hr CYP 48hr CYP

(a)

VehicleTempol

0

02

04

06

08

1

12

14

Control

lowast

lowast

4hr CYP 48hr CYP

VV

(mL)

(b)

VehicleTempol

0

200

400

600

800

1200

Control

lowast

lowast

1000

4hr CYP 48hr CYP

BC (120583

L)

(c)



4 Discussion





VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70


MP

(cm

H2O

)

(c)








75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results







0200400600800

10001200140016001800

DetrusorUrothelium

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowast

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

DCF

relat

ive fl

uore

scen

ce

(a)

02040608

1121416

lowast

lowast

lowast lowast

lowast

0

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

ng3

-NT120583

g to

tal p

rote

in

(b)


0

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

pg S

ub P

120583g

tota

l pro

tein

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

0

ng S

ub P

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)







0

05

1

15

2

25

3

lowast

lowast

lowastlowast

lowastpg

CG

RP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

10

20

30

40

50

60

lowast

lowast

lowast

lowast

lowast

ng C

GRP

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)


0

1

2

3

4

5

6

7

8lowast

lowast

lowast

lowast

pM A

TP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

002

004

006

008

01lowast

lowast

lowast lowast

lowast

nM A

TPm

L ur

ine

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)





2O) of NVCs


050

100150200250300350400450

Control

VehicleTempol

lowast

lowast

ICI (

s)

4hr CYP 48hr CYP

(a)

VehicleTempol

0

02

04

06

08

1

12

14

Control

lowast

lowast

4hr CYP 48hr CYP

VV

(mL)

(b)

VehicleTempol

0

200

400

600

800

1200

Control

lowast

lowast

1000

4hr CYP 48hr CYP

BC (120583

L)

(c)



4 Discussion





VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70


MP

(cm

H2O

)

(c)








75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results







0200400600800

10001200140016001800

DetrusorUrothelium

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowast

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

DCF

relat

ive fl

uore

scen

ce

(a)

02040608

1121416

lowast

lowast

lowast lowast

lowast

0

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

ng3

-NT120583

g to

tal p

rote

in

(b)


0

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

pg S

ub P

120583g

tota

l pro

tein

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

0

ng S

ub P

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

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Tem

pol

(b)







0

05

1

15

2

25

3

lowast

lowast

lowastlowast

lowastpg

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RP120583

g to

tal p

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in

Con

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vehi

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Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

10

20

30

40

50

60

lowast

lowast

lowast

lowast

lowast

ng C

GRP

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)


0

1

2

3

4

5

6

7

8lowast

lowast

lowast

lowast

pM A

TP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

002

004

006

008

01lowast

lowast

lowast lowast

lowast

nM A

TPm

L ur

ine

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)





2O) of NVCs


050

100150200250300350400450

Control

VehicleTempol

lowast

lowast

ICI (

s)

4hr CYP 48hr CYP

(a)

VehicleTempol

0

02

04

06

08

1

12

14

Control

lowast

lowast

4hr CYP 48hr CYP

VV

(mL)

(b)

VehicleTempol

0

200

400

600

800

1200

Control

lowast

lowast

1000

4hr CYP 48hr CYP

BC (120583

L)

(c)



4 Discussion





VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70


MP

(cm

H2O

)

(c)








75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0200400600800

10001200140016001800

DetrusorUrothelium

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast lowast

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

DCF

relat

ive fl

uore

scen

ce

(a)

02040608

1121416

lowast

lowast

lowast lowast

lowast

0

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

ng3

-NT120583

g to

tal p

rote

in

(b)


0

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

pg S

ub P

120583g

tota

l pro

tein

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

5

10

15

20

25

30

35lowast

lowast

lowast

lowast

0

ng S

ub P

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)







0

05

1

15

2

25

3

lowast

lowast

lowastlowast

lowastpg

CG

RP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

10

20

30

40

50

60

lowast

lowast

lowast

lowast

lowast

ng C

GRP

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)


0

1

2

3

4

5

6

7

8lowast

lowast

lowast

lowast

pM A

TP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

002

004

006

008

01lowast

lowast

lowast lowast

lowast

nM A

TPm

L ur

ine

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)





2O) of NVCs


050

100150200250300350400450

Control

VehicleTempol

lowast

lowast

ICI (

s)

4hr CYP 48hr CYP

(a)

VehicleTempol

0

02

04

06

08

1

12

14

Control

lowast

lowast

4hr CYP 48hr CYP

VV

(mL)

(b)

VehicleTempol

0

200

400

600

800

1200

Control

lowast

lowast

1000

4hr CYP 48hr CYP

BC (120583

L)

(c)



4 Discussion





VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70


MP

(cm

H2O

)

(c)








75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25

3

lowast

lowast

lowastlowast

lowastpg

CG

RP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

10

20

30

40

50

60

lowast

lowast

lowast

lowast

lowast

ng C

GRP

mL

fluid

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)


0

1

2

3

4

5

6

7

8lowast

lowast

lowast

lowast

pM A

TP120583

g to

tal p

rote

in

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(a)

0

002

004

006

008

01lowast

lowast

lowast lowast

lowast

nM A

TPm

L ur

ine

Con

trol+

vehi

cle

Con

trol+

Tem

pol

4hr

CYP

+

vehi

cle

4hr

CYP

+

Tem

pol

48

hr C

YP+

vehi

cle

48

hr C

YP+

Tem

pol

(b)





2O) of NVCs


050

100150200250300350400450

Control

VehicleTempol

lowast

lowast

ICI (

s)

4hr CYP 48hr CYP

(a)

VehicleTempol

0

02

04

06

08

1

12

14

Control

lowast

lowast

4hr CYP 48hr CYP

VV

(mL)

(b)

VehicleTempol

0

200

400

600

800

1200

Control

lowast

lowast

1000

4hr CYP 48hr CYP

BC (120583

L)

(c)



4 Discussion





VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70


MP

(cm

H2O

)

(c)








75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















050

100150200250300350400450

Control

VehicleTempol

lowast

lowast

ICI (

s)

4hr CYP 48hr CYP

(a)

VehicleTempol

0

02

04

06

08

1

12

14

Control

lowast

lowast

4hr CYP 48hr CYP

VV

(mL)

(b)

VehicleTempol

0

200

400

600

800

1200

Control

lowast

lowast

1000

4hr CYP 48hr CYP

BC (120583

L)

(c)



4 Discussion





VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70


MP

(cm

H2O

)

(c)








75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

BP (c

m H

2O

)

(a)

VehicleTempol

0

5

10

15

20

25

Control

4hr CYP 48hr CYP

TP (c

m H

2O

)

(b)

VehicleTempol

0

10

20

30

40

50

60

70


MP

(cm

H2O

)

(c)








75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















75

50

25

0

400

200

600

0

04

02

06

Time (s)

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + vehicle

(a1)

(a2)

(a3)

(a)

Time (s)

75

50

25

0

400

200

600

0

04

02

06

0

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

4hr CYP + Tempol

(b1)

(b2)

(b3)

(b)








Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Time (s)

0

75

50

25

0

600

400

200

0

06

04

02

(a1)

(a2)

(a3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + vehicle

(a1)

(a2)

(a)

0

75

50

25

0

600

400

200

Time (s)

0

06

04

02

(b1)

(b2)

(b3)

100 s

BP (c

m H

2O

)IV

(120583L)

VV

(mL)

48hr CYP + Tempol

(b1)

(b2)

(b)












Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article The Effects of Tempol on Cyclophosphamide ...downloads.hindawi.com/journals/tswj/2015/545048.pdf · following each micturition event in control and CYP-treated groups