INTRODUCTIONCyclophosphamide (CYP) treatment causes mucosal

inflammatory response as indicated by macroscopic andmicroscopic changes in the urinary bladder and the presence ofinflammatory cell infiltrations. Such acute and chronic CYPtreatment induces cystitis leading to the urinary bladderoveractivity (OAB) in rats (1, 2). Bladder hyperactivity seems tobe initiated by sensitizing mechanosensitive afferents and/orrecruitment of silent afferents, which are unresponsive tomechanical stimuli in healthy conditions (3). Neurogenicinflammation responses have recently been linked to overactivebladder and painful bladder syndrome development. Itencompasses a series of vascular and non-vascular inflammatoryresponses, triggered by the activation of primary sensoryneurons and the subsequent release of inflammatoryneuropeptides (4). In addition, mastocytes play a pivotal role inneurogenic inflammation reflex. Some studies revealed closerelationship between these cells and peptidergic nerve fibres,including primary sensory afferents (5, 6).

The aim of this study was to estimate the effect of acute andchronic CYP treatment on rat urinary bladder function,histological structure and mastocytes numbers.

MATERIAL AND METHODSAnimals

Experiments were performed on forty-two adult femaleWistar rats (weight: 195-275 g). Rats were housed individuallyper cage. The animal room was maintained at a constanttemperature of 23°C, humidity and a 12:12-hours alternatinglight-dark cycle. They were fed with animal food (Labofeed;Kcynia, Poland) without any restraint to water. The study hasbeen approved by the Animals Ethical Committee ofJagiellonian University (Cracow, Poland).Animal models of overactive bladder (OAB)

Acute and chronic chemical cystitis leading to OAB wasinduced by CYP. CYP (Endoxan, BaxterOncology, Germany)was administrated intraperitonealy in a single dose of 200 mg/kgand four doses of 75 mg/kg (in 1st, 3rd, 5th, 7th day of experiment),respectively (1, 7).Bladder catheter implantation

Under urethane anaesthesia, the abdomen was openedthrough a midline incision and the bladder end of the

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2010, 61, 4, 477-482www.jpp.krakow.pl

K. JUSZCZAK1,2, K. GIL1, M. WYCZOLKOWSKI2, P.J. THOR1

FUNCTIONAL, HISTOLOGICAL STRUCTURE AND MASTOCYTES ALTERATIONS IN RAT URINARY BLADDERS FOLLOWING ACUTE AND CHRONIC

CYCLOPHOSPHAMIDE TREATMENT

1Department of Pathophysiology, Jagiellonian University Medical College, Cracow, Poland; 2Department of Urology, Rydygier Memorial Hospital, Cracow, Poland

Neurogenic inflammation is linked to urinary bladder overactivity development. Cyclophosphamide (CYP) damages allmucosal defence lines of urinary bladder and induces cystitis with overactivity. The aim of this study was to estimate theeffect of CYP on rat urinary bladder function, histological structure and mastocytes numbers following acute and chronicCYP treatment. Fourty two female rats were divided into four groups: I (control), II (acute cystitis), III (chronic cystitis),IV (sham group). Acute and chronic cystitis were induced by CYP in single dose and four doses (1st, 3rd, 5th, 7th day),respectively. In group I–III the cystometric evaluation was performed. Sections of the bladder were stained with HE andtoluidine blue for the detection of mastocytes. The severity of inflammation was examined according to mucosal abrasion,haemorrhage, leukocyte infiltration and oedema. Acute and chronic CYP treatment caused inflammatory macroscopic andmicroscopic changes (mucosal abrasion, haemorrhage, oedema) and increased infiltration of inflammatory cells in urinarybladder. Acute treatment induced the infiltration of mastocytes within bladder wall contrary to chronic one decrement.Acute treatment caused more severe mucosal abrasion, whereas chronic one revealed more developed haemorrhagechanges. Additionally, cystometric evaluation revealed urinary bladder overactivity development in both types of cystitis.Basal pressure and detrusor overactivity index after acute treatment increased considerably in comparison with the increaseobtained after chronic one. Our results proved that acute model of CYP-induced cystitis in rats is more credible for furtherevaluation of neurogenic inflammation response in pathogenesis of overactive bladder as compared to chronic one.

K e y w o r d s : overactive bladder, cyclophosphamide, mastocytes, cystitis, cystometry, afferent sensory fibers

polyethylene catheter (o.d. 0,97 mm/ i.d. 0,58 mm; BALT,Poland) was passed through a small incision at the apex of thebladder dome and secured in place by silk ligature 4-0, aspreviously described (2, 8). The implantation was performedafter 5 hours of CYP administration in group II and after 24hours of the 4thCYP dose administration in group III.

Study protocolAll animals were randomly divided into four groups: group

I – control (n=12), group II – acute cystitis (n=12), group III –chronic cystitis (n=12), group IV – sham group (n=6). In groupI – III cystometry was performed under urethane anaesthesia(dose: 1.2 g/kg urethane - Sigma-Aldrich, St. Louis, USA) aftera one-hour recovery period from the catheter implantation.Saline solution at room temperature was infused at a rate of0.046 ml/min continuously into the bladder. The free end of theimplanted catheter was connected via T-stopcock to a pressuretransducer (UFI, MorroBay, CA, USA) and injection pump(Unipan340A, Poland). Cystometry was recorded usingML110-BridgeAmp (ADInstruments, Australia) hardware andPowerLab/8SP (ADInstruments, Castle Hill, Australia)software, as previously described (1, 9). The measurements ineach animal represent the average of five bladder micturitioncycles, after obtaining repetitive voiding. The followingparameters were recorded: BP - basal pressure (cmH20), PT –threshold pressure (cmH20), MVP – micturition voidingpressure (cmH20), ICI – intercontraction interval (min),compliance (ml/cmH20), fBC – functional bladder capacity(ml), MI – motility index (cmH2O x s/min) DI – detrusor index(cmH20/ml) in group I and DOI - detrusor overactivity index(cmH20/ml) in group II and III (2). After cystometric evaluationthe rats were sacrificed by pentobarbital overdose (200 mg/kg)and urinary bladders were removed in group I-III. The bladdersfrom sham group (group IV) were also removed to estimate theeffect of bladder catheter implantation of hyperemia degreewithin bladder walls.

Histological evaluationThe bladder was removed through a lower midline

abdominal incision. After the ureters were ligated the bladderwas stored in 10% formalin solution. The specimen was splitlongitudinally and processed for histological examination.

Staining: Specimens were 10% formalin fixed andembedded in paraffin. Thin sections of the bladder were cut andstained with HE and toluidine blue for the detection ofmastocytes. In each fragment 10 consecutive cross section wereexamined.

Severity of inflammation: Previously described techniques(Cayan 2002, Bjorling 1999) were used to determine the severityof inflammation resulted from CYP administration. The severityof inflammation was examined using optical microscopeAxiophot (Zeiss, Germany) in each section according to 4criteria, including mucosal abrasion, haemorrhage, leukocyteinfiltration and oedema (10, 11).

Mucosal abrasion: Mucosal abrasion was defined as erosionof the mucosa. The presence or absence of mucosal abrasion perindividual field was determined at 100x magnification on a scaleof 0 - no abrasion and 1 - visible abrasion. The total score of allview fields was divided by the maximum possible score andmultiplied by 100.

Haemorrhage: The presence or absence of haemorrhage perindividual field was determined at 100x magnification on a scaleof 0 - no haemorrhage and 1 - visible haemorrhage. The totalscore of all view fields was divided by the maximum possiblescore and multiplied by 100.

Leukocyte infiltration: Leukocyte infiltrations (neutrophilsand mononuclear cells) were evaluated in each of the viewfields at 400x magnifications on a scale of 0 - no extravascularleukocytes, 1 - less than 20, 2 - 20 to 45 and 3 - greater than 45leukocytes per high power field. The total score of all viewfields was divided by the maximum possible score andmultiplied by 100.

Oedema: Oedema in each view fields was scored at 200xmagnification on a scale of 0 - no oedema, 1 - mild oedema (noalteration in the width of the submucosa), 2 – moderate oedema(an increase of less than twice the width of the submucosa) and3 - severe oedema (an increase of more than twice the width ofthe submucosa). The total score of all view fields was divided bythe maximal possible score and multiplied by 100.

Mastocytes: The total number of mastocytes was counted at200x magnification in 10 random sections of the bladder ofeach rat.

Statistical analysisThe results were expressed as mean and standard deviation

(±S.D.). Kruskal-Wallis test was used to compare betweengroups and “post hoc” multiple comparison tests for statisticallysignificant results. Statistical significance was set at p≤0.05 forall tests.

RESULTSAfter acute CYP treatment, rats display piloerection and the

characteristic immobile rounded back posture with the headaligned with the body axis, while none of control rats and ratsduring chronic CYP treatment displayed this position. Thesebehaviours were interpreted to be pain related condition (3).

The CYP-treated rats exhibited macroscopical signs ofurinary bladder inflammation, i.e. redness, oedema (in group II,III) and also wall thickening, mucosal erosions, ulcerations,petechial haemorrhages on the serosal surface (in group III). Insome animals of the group III the urine contained blood. Rats inthe groups I and IV had healthy bladders and normal urine.Microscopic evaluations of acute and chronic cystitis revealedincreased severity of inflammatory cells infiltration of bladderwall (neutrophils and mononuclear cells) – 32.65±18.14 vs.69.43±18.22 vs. 64.58±15.09 (p=0.017), respectively. Comparedto the control group, a single dose of CYP caused increasednumber of mastocytes from 6.73±2.09 to 11.97±6.02 (p=0.002).However, chronic administration of CYP decreased the numberof mastocytes within bladder wall (6.73±2.09 vs. 1.48±0.71,p=0.002). Furthermore, CYP-treated rats showed clear signs ofinflammation; however, the alteration of bladder structuredepends on the mode of CYP administration. Acute modelcaused more severe mucosal abrasion when compared to chronicone which revealed more developed haemorrhage changeswithin bladder wall. Additionally, in acute and chronic cystitiswe observed similar tissue oedema changes (Fig. 1AB-3AB).

Optional comparison of bladder architecture and hyperemiadegree between rats after (group I) and without (group IV)bladder catheter implantation showed no significant changes.

Compared to control group, acute and chronic CYPtreatment caused significant decrease of MVP (27.41±4.86,21.51±3.12, 21.67±1.94 cmH20, p<0.001), ICI (5.278±1.549,1.503±0.736, 2.149±0.350 min, p<0.001), fBC (0.243±0.071,0.069±0.034, 0.099±0.016 ml, p<0.001), compliance(0.059±0.019, 0.036±0.031, 0.030±0.007 ml/cmH20, p=0.005),and increase of BP (1.40±0.60, 4.57±1.20, 3.31±1.85 cmH20,p<0.001), DOI (121.92±32.98, 824.36±327.57, 384.03±181.68cmH20/ml, p<0.001), MI (185.64±45.95, 309.90±135.03,

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261.00±33.92 cmH2O x s/min, p=0.004). The PT was notsignificantly changed in acute treatment (5.68±1.22 and7.50±2.11 cmH20, p=0.050). Significant changes of BP and DOIbetween acute and chronic CYP treatment were observed. Thepercentage changes of cystometric parameters were presented inTable 2.

DISCUSSIONCYP treatment is associated with various urological

complications in rats including reduction of micturition voidingpressure, intercontraction intervals and functional bladdercapacity, as well as an increase of non-voiding contraction

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Fig. 1A. A histological appearance of urinary bladder wall incontrol rats (hematoxylin and eosin staining).Fig. 1B. A histological appearance of urinary bladder wall withmastocytes in control rats (Toluidine Blue staining).

A

BFig. 2A. A histological appearance of urinary bladder wall in ratswith acute cystitis (hematoxylin and eosin staining).Fig. 2B. A histological appearance of urinary bladder wall withmastocytes in rats with acute cystitis (Toluidine Blue staining).

A

B

GROUP

Histological parameters I

Control

II

Acute cystitis

III

Chronic cystitis

Kruskal-

Wallis

test

Mastocytes 6.73 ± 2.09 11.97 ± 6.02 1.48 ± 0.71

p=0.002

*

Mucosal abrasion 8.33 ± 6.45 52.08 ± 16.61 31.25 ± 18.96

p=0.003

**

Hemorrhage 16.67 ± 17.08 33.33 ± 24.58 83.33 ± 20.41

p=0.004

***

Edema 26.38 ± 11.07 75.70 ± 11.00 82.67 ± 7.18

p=0.002

****

Leukocyte infiltration 32.65 ± 18.14 69.43 ± 18.22 64.58 ± 15.09

p=0.017

****

* statistically significant differences between group I and group II, III, as well as between group II and III (p≤0.05); ** statisticallysignificant differences between group I and group II (p≤0.05); *** statistically significant differences between group I and group III,as well as between group II and III (p≤0.05); **** statistically significant differences between group I and group II, III (p≤0.05).

Table 1. Histological evaluations of urinary bladder wall and mastocytes in control group and with (acute & chronic) cystitis rats.

frequency (1). These signs are strongly correlated with bladdercystitis.

Our results showed that acute and chronic CYP treatmentcaused inflammatory response as indicated by macroscopic andmicroscopic changes in bladder (mucosal abrasion, oedema,haemorrhages) and increased infiltration of inflammatory cells(neutrophils, mononuclear cells) of bladder wall. These results

are close to that reported previously by several authors despite aslight modification of experimental protocol (10, 11). Also short-term CYP administration induced the infiltration of mastocyteswithin bladder wall (mucosa and muscular layer) contrary tochronic one which decreased the number of mastocytes.

Additionally, cystometric evaluation in acute and chroniccystitis revealed urinary bladder overactivity development.However, more severe overactivity occured in acute cystitis. Weobtained significant increase of basal pressure and detrusoroveractivity index after acute CYP treatment as compared tochronic one. We postulated that this fact might be the result oftwo mechanisms. Firstly, that more severe mucosal damage(abrasion) in case of acute cystitis led to extensive exposition ofsubmucosal bladder afferent C-fibres. Its activation by acrolein,as well as vide range of mediators excreted by inflammatorycells stimulated these nerves leading to generation of non-voiding bladder contractions (NVCs), which was reflected in thelarger increment of detrusor overactivity index. Secondarily,increased number of mastocytes, the cells which are pivotal inafferent C fibres stimulation, enabled their intensive stimulationand in consequence more frequent bladder NVCs generation.Conversely, in chronic cystitis the changes in basal pressure anddetrusor overactivity index are softer than in acute one.Nevertheless, the mastocytes number decreased the mucosaldamage (abrasion) occurred. Therefore, it could be stated thatextensive bladder afferent C-fibres exposition caused bymucosal damage (abrasion) seems to be crucial in bladder NVCsgeneration. On the other hand rhythmic detrusor activity isconsidered to be due to interstitial cells of Cajal (ICC) presentedin urinary bladder. In detrusor muscle bundles spontaneousaction potentials are initiated in non typical ICC-like cellssituated at the border of the bundles (12). Therefore theincreased frequency of NVCs could be a result ofoverstimulation of ICC by acrolein.

Neurogenic inflammation responses have recently beenlinked to overactive bladder in animals and humans. Keith et al.study revealed a close relationship between mastocytes andpeptidergic afferent nerve fibres of urinary bladder (5). Herevealed that stimulation of vanilloid (TRPV1) and ancyrin(TRPA1) transient potential receptors by vanilloids as well asother agents (bradykinin, protons) caused neuropeptides releaselike substance P (SP), calcitonin gene-related peptide (CGRP)and interleukins from nerve terminals. Those factors generateresponses in blood vessels diameter, mastocytes andlymphocytes activation causing neurogenic inflammation and inconsequence overactive bladder (3, 4). Previous studies suggestthat CGRP acts synergistically with SP in spinal cord, thereforeCGRP may facilitate the SP-evoked chemonociceptive reflex(13). Additionally, TRPV1 and TRPA1 receptors are alsotargeted by environmental irritants (e.g. acrolein) that areassociated with nociceptive and inflammatory effects of smog,cigarette smoke and the metabolic product of chemotherapeuticagents – CYP (14, 15). Ercan et al. (16) study showed that SPlocalized in capsaicin-sensitive sensory nerve endings (TRPV1positive nerves) is important in stress response. Inactivation ofsensory neurons with capsaicin prevents acute stress-inducedepithelial damage and mastocytes proliferation in the rat bladder.It was postulated by Saban et al. (17) that it is very likely thatduring stress, discharge of neuropeptides (particularly SP) fromprimary afferent fibres activates mastocytes in the bladder andinduces inflammation. Purinergic molecules such as ATP, ADPand adenosine play a role of a neurotransmitter in nonadrenergic,noncholinergic nerves supplying the urinary bladder (18).

Additionally, mastocytes seem to be equally important ininitiation and progression of inflammation in the bladder.Increased numbers of mastocytes were observed in bladderbiopsies obtained from patients with interstitial cystitis (19). Many

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Fig. 3A. A histological appearance of urinary bladder wall in ratswith chronic cystitis (hematoxylin and eosin staining).Fig. 3B. A histological appearance of urinary bladder wall withmastocytes in rats with chronic cystitis (Toluidine Bluestaining).

A

B

Cystometric

parameters

Acute

cystitis

Chronic

cystitis

BP ↑ 226.4% * ↑ 136.4% *

PT ↑ 32.0% NS

MVP ↓ 21.5% ↓ 21.0%

ICI ↓ 71.5% ↓ 59.0%

Compliance ↓ 39.0% ↓ 49.1%

fBC ↓ 71.6% ↓ 59.2%

DOI ↑ 576.1% * ↑ 215% *

MI ↑ 66.9% ↑ 40.6%

* significant changes between acute and chronic cystitis.

Table 2. The percentage changes of cystometric parametersfollowing acute and chronic cystitis in comparison with controlgroup.

experiments show that SP can induce mastocytes degranulation(20) as well as it mediates the inflammatory responses induced bymastocyte tryptase (21). The responses to SP and othertachykinins are regulated by G protein-coupled neurokininreceptors. Among these receptors neurokinin-1 receptor (NK1R)is the predominant receptor subtype activated by SP which isinvolved in inflammation and is expressed by endothelial cells,submucosal glands, and circulating leukocytes. NK1R seems alsoto modulate plasma extravasation in the urinary bladder inducedby SP (22). The fundamental role of NK1R in cystitis wasdemonstrated in NK1R knockout mice. These mice were resistantto bladder inflammation induced by antigen challenge (20).Stress- and SP-injected centrally, increased the number of bothgranulated and degranulated mast cells. Consequently, it leads tourothelial degeneration with vacuolization in the cytoplasm anddilated intercellular spaces. Both central and peripheral injectionof antagonist of SP receptor (NK1R) - CP 99994 prevented stress-induced urothelial degeneration as well as stress-induced mast celldegranulation (23). These findings imply a link between substanceP and mastocytes in the pathogenesis of bladder inflammation.

The decrease of mastocytes number in reaction to chronicCYP administration seems to be the result of three mechanisms.Firstly, the direct cytotoxic effect of CYP acting on mastocytesoccured. Secondarily, permanent irritating stimulation by CYPand its metabolites caused complete degranulation ofmastocytes, which became invisible in toluidine blue staining.Thirdly, chronic CYP administration led to depletion of bladderafferent C-fibres. Hence, the probable, coexistingpathomechanism of mastocytes restraint depended on dramaticdecrement of substance P released peripherally (within bladder)by C-fibres endings. Although, Vizzard et al. (24) studydemonstrated dramatic changes in the expression of CGRP andSP in micturition pathways following chronic CYP-inducedcystitis. However, both neuropeptides were present inlumbosacral (L6-S1) spinal cord segments and dorsal rootganglia involved in micturition reflexes in control animals,urinary bladder inflammation dramatically increased CGRP andSP immunoreactivity in these regions.

In summary, the acute and chronic CYP treatment causedinflammatory macroscopic and microscopic changes andincreased infiltration of inflammatory cells in urinary bladder.Acute CYP treatment induced the infiltration of mastocyteswithin bladder wall which was not observed in a chronic one.Additionally, cystometric evaluation revealed urinary bladderoveractivity development in both types of cystitis.

Our current functional and morphological results proved thatacute model of CYP-induced cystitis in rats is more credible forfurther evaluation of neurogenic inflammation response inoveractive bladder as compared to chronic one.

Conflict of interests: None declared.

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24. Vizzard MA. Alterations in neuropeptide expression inlumbosacral bladder pathways following chronic cystitis. JChem Neuroanat 2001; 21: 125-138.R e c e i v e d : January 3, 2010A c c e p t e d : July 15, 2010Author’s address: Dr. Kajetan Juszczak, Department of

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