URODYNAMIC EFFECTS OF THE BLADDER C-FIBER AFFERENT ACTIVITY MODULATION IN CHRONIC MODEL OF OVERACTIVE BLADDER IN RATS

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1 JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 29, 6, 4, K. JUSZCZAK 1,2, A. ZIOMBER 1, M. WYCZOLKOWSKI 2, P.J. THOR 1 URODYNAMIC EFFECTS OF THE BLADDER C-FIBER AFFERENT ACTIVITY MODULATION IN CHRONIC MODEL OF OVERACTIVE BLADDER IN RATS 1 Department of Pathophysiology, Jagiellonian University, Medical College, Cracow, Poland; 2 Department of Urology, Rydygier Memorial Hospital, Cracow, Poland Previous studies have suggested that, different types of unmyelinated bladder afferent C-fibres, such as capsaicin-sensitive and capsaicin-resistant mediate the voiding reflex in overactive bladder (OAB). Considering its polymodal features, we explored the urodynamic effect of primary afferent neurons modulation on detrusor activity in normal and OAB rats. Experiments were performed on 48 female rats. OAB was induced by intraperitoneal administration of cyclophosphamide. All the surgical procedures and urodynamic studies were performed under urethane anaesthesia. Cystometry was done after a 1 h recovery period from the surgical procedure. All animals were randomly divided into six groups: control, chronic OAB, chronic OAB after capsaicin or lidocaine instillation, control capsaicin or lidocaine instillation. The measurements represent the average of five bladder micturition cycles. We analyzed: basal, threshold, micturition voiding pressure; intercontraction interval; compliance; functional bladder capacity; motility index; detrusor overactivity index. We used chronic cyclophosphamide OAB model for further investigations. In healthy rats, intravesical instillation of capsaicin caused complete inhibition of detrusor contractility preventing from proper voiding function of the bladder. Contrary, lidocaine has no influence on micturition cycles in intact animals. Also, intravesical instillation of capsaicin and lidocaine reduced the severity of detrusor overactivity of OAB rats leading to improvement of cystometric parameters. Keywords: afferent C-fibres, overactive bladder, cystometry, capsaicin, lidocaine, rats INTRODUCTION The most important afferents for the micturition are myelinated Aδ-fibres and unmyelinated C-fibres, which are believed to be insensitive in naive conditions (1). Afferent C-fibres are responding to mechanical, thermal and chemical stimuli (2, 3). Its stimulation leads to micturition activation. This alternative C- fibres mediated spinal reflex, play a key role in pathogenesis of bladder overactivity development, increment of OAB symptoms severity, as well as in patients after spinal cord injuries (4). The pivotal backgrounds for OAB development are as follow: the C- fibres sensitisation (increment of sensitivity to various stimuli acting on urotelium) and local effector function of afferent C- fibres endings leading to neurogenic inflammation. Previous studies have suggested that different types of unmyelinated afferent C-fibres, such as capsaicin-sensitive and capsaicinresistant mediate the voiding reflex in overactive bladder (5). Considering the polymodal features of afferent C-fibre we explored the urodynamic effect of primary afferent neurons modulation on detrusor activity in normal and overactive bladder model rats. MATERIAL AND METHODS Animals Experiments were performed on forty-eight adult female Wistar rats (weight: g). Rats were housed individually per cage. The animal room was maintained at a constant temperature of 23 C, humidity and a 12:12 h alternating light-dark cycle. They were fed with animal s food (Labofeed; Kcynia, Poland) with any restraint to water. The study has been approved by the Animals Ethical Committee of Jagiellonian University (Cracow, Poland). Animal model of overactive bladder Chronic chemical cystitis leading to bladder overactivity was induced by cyclophosphamide (CYP). CYP (Endoxan, BaxterOncology, Germany) was administrated intraperitonealy in four doses of 75 mg/kg, every 3 rd day for 7 days of experiment (6). Anaesthesia All the surgical procedures and urodynamic studies were performed under anaesthesia with intraperitonealy injection of 1.2 g/kg urethane (Sigma-Aldrich, St. Louis, USA) (6) and in case of capsaicin instillation with.4 g/kg. Administration of drugs The following drugs were used: capsaicin (Sigma-Aldrich, Germany) and 2% lidocaine (Polfa, Warsaw, Poland). Capsaicin (final concentration 1 mm) was dissolved in special solution composed of.9% saline (8%), absolute ethanol (%) and Tween 8 (Sigma-Aldrich, Germany) (%) (the volume

2 86 participation of individual components in solvent expressed in the per cent, is provided in square brackets). Under urethane anaesthesia, the bladder was catheterized through the urethra and emptied. Volume of.3 ml of 1 mm capsaicin or 2% lidocaine were injected through the catheter (group V, VI) or.2 ml (group III, IV) at a rate of.15 ml/min. Capsaicin and lidocaine were left contact with the mucosa for 15 and 3 minutes, respectively. The bladder was emptied again and flushed out using.5 ml.9% saline at a rate of.15 ml/min. (7-9). Surgical procedure Bladder catheter implantation: under urethane anaesthesia, the abdomen was opened through a midline incision and the bladder end of the polyethylene catheter (o.d..97 mm/i.d..58 mm; BALT, Poland) was passed through a 1 mm incision at the apex of the bladder dome and secured in place by silk ligature 4-, as previously described (6). Urodynamic studies Cystometry was performed under urethane anaesthesia after a 1 h recovery period from the surgical procedure. Room temperature saline solution was infused at a rate of.46 ml/min continuously into the bladder. The free end of the implanted catheter was connected via T-stopcock to a pressure transducer (UFI, MorroBay, CA, USA) and injection pump (Unipan34A, Poland). Cystometry was recorded using ML1-BridgeAmp (ADInstruments, Australia) hardware and PowerLab/8SP (ADInstruments, Castle Hill, Australia) software, as previously described (). Study protocol All animals were randomly divided into six groups: group I control group (n=12), group II rats with chronic OAB (n=12), group III rats with chronic OAB after capsaicin instillation (n=6), group IV rats with chronic OAB after lidocaine instillation (n=6), group V healthy rats after capsaicin instillation (n=6), group VI - healthy rats after lidocaine instillation (n=6). Cystometry was performed 1 h after surgical procedure in all groups. The surgical procedure was performed after 24 h of the 4 th CYP dose administration in group II, after 24 h of the 4 th CYP dose administration and capsaicin instillation in group III, after 24 h of the 4 th CYP dose administration within 3 min after lidocaine instillation in group IV, after 24 h of the capsaicin instillation in group V, after 3 min of the lidocaine instillation in group VI, as previously described with slightly modification (6, 11). The measurements in each animal represent the average of five bladder micturition cycles, after obtaining repetitive voiding. The following cystometrogram s (CMGs) parameters were recorded: BP - basal pressure (cmh 2 ), PT threshold pressure (cmh 2 ), MVP micturition voiding pressure (cmh 2 ), ICI intercontraction interval (min), Compliance (ml/cmh 2 ), fbc functional bladder capacity (ml). Moreover we calculated MI motility index (cmh 2 O x s/min) in -minutes intervals. MI is defined as the enclosed area between the sampled data and their minimum on the selected interval. In addiction we analysed DI detrusor index (cmh 2 /ml) in group I and DOI - detrusor overactivity index (cmh 2 /ml) in other groups (12), depicted as quotient of the sum of amplitudes of all detrusor contractions during filling phase and functional bladder capacity (13). Statistical analysis The results are expressed as mean and standard deviation (±SD). Kruskal-Wallis test was used to compare between groups and post hoc multiple comparison tests for statistically significant results. Statistical significance was set at p.5 for all tests. RESULTS Chronic cyclophosphamide-induced cystitis The chronic CYP-treated rats exhibited macroscopical signs of bladder inflammation, i.e. redness, oedema and also wall thickening, mucosal erosions, ulcerations, petechial hemorrhages on the serosal surface. In some animals the urine contained blood. As shown in Figs. 1, 2 and Table 1, CMGs performed in chronic bladder cystitis were different from in control group. Chronic CYP administration leads to bladder overactivity. After chronic CYP administration we observed Zapis CMG - badanie kontrolne :31: : 36:4 38:2 4: 41:4 43:2 45: 46:4 48:2 5: 51:4 Fig. 1. Cystometrogram trace in control group. The (- 5) - 4 cmh 2 O range.

3 87 Zapis CMG - model przewlekły OAB :6: :4 23:2 25: 26:4 28:2 3: 31:4 33:2 35: 36:4 38:2 Fig. 2. Cystometrogram trace in chronic OAB. The (-5) 4 cmh 2 O range. Table 1. Cystometrogram s parameters in normal and after chronic CYP administration rats. Group I: CONTROL Group II: chronic OAB P Value BP [cmh 2 O] 1,4 ±,6 3,31 ± 1,85 p<,1* PT [cmh 2 O] 5,68 ± 1,22 6,88 ± 1,93 NS MVP [cmh 2 O] 27,41 ± 4,86 21,67 ± 1,94 p<,1* ICI [min.] 5,278 ± 1,549 2,149 ±,35 p<,1* Compliance [ml/cmh 2 O],59 ±,19,3 ±,7 p=,5* fbc [ml],243 ±,71,99 ±,16 p<,1* DI / DOI [cmh 2 O/ml] 121,92 ± 32,98 384,3 ± 181,68 p<,1* MI [cmh 2 O x s/min.] 185,64 ± 45,95 261, ± 33,92 p=,4* *statistically significant differences between group I and group II (p,5). significant decrease of micturition voiding pressure (21.5%), intercontraction intervals (59%), functional bladder capacity (59.2%) and compliance (49.1%). Also increase of basal pressure (136.4%), detrusor activity (215%) and motility index (4.6%) were observed. The threshold pressure was not significantly changed. Effect of intravesical instillation of capsaicin and lidocaine on bladder activity in normal rats Intravesical instillation of 1 mm capsaicin (CAP) produced complete inhibition of detrusor contractility preventing from proper voiding function of the bladder. On the basis of performed CMGs we observed complete disorganization of micturition cycles (Fig. 3). In storage phase of micturition cycles we observed increased spontaneous detrusor overactivity, evaluated as phasic detrusor contractions of low amplitude with accompanying increased intravesical pressure. In voiding phase these was no proper detrusor contractility (generation of maximal voiding pressure MVP). As a consequence of the lack of periodically generated MVP and incomplete bladder empting, constantly lasting urine retention occured. In case of critical bladder fulfill achievement (maximal cystometric capacity) we recorded constant, dripping flow of urine through the urethra. Because of, no proper micturition cycles, bladder activity was estimated using only motility index (Table 2). Contrary, intravesical instillation of 2% lidocaine (LDK) has no influence on micturition cycles (Fig. 4). Compared to control group, we observed significant increase of intercontraction interval (47.2%), compliance (79.7%), functional bladder capacity (47%) and detrusor index (49.8%). The basal, threshold and maximal voiding pressure was not significantly changed. As well, there were no significant changes of motility index after CAP or LDK intravesical instillation, compared to control group (Table 2). Effect of intravesical instillation of capsaicin and lidocaine on bladder activity in rats with chronic OAB. Intravesical administration of 1 mm capsaicin, as well 2% lidocaine reduced the severity of detrusor overactivity, leading to the improvement of cystometric parameters (Figs. 5, 6, Table 3). Compared to rats with chronic OAB we observed, statistical significant increase of intercontraction interval (172.3% and 126%, respectively), functional bladder capacity (173% and 125.2%, respectively), and compliance (8% and 12%, respectively). Also statistical significant decrease of detrusor

4 88 * Zapis CMG - 1mM KAPSAICYNA :57:.93 3 Constant, dripping urine leakage through urethra 2 : 11:4 13:2 15: 16:4 18:2 2: 21:4 23:2 25: 26:4 3 Fig. 3. Cystometrogram trace in healthy rats after capsaicin instillation. The (-5) 4 cmh 2 O range. Zapis CMG - 2% LIDOKAINA :4: :3: 1:31:4 1:33:2 1:35: 1:36:4 1:38:2 1:4: 1:41:4 1:43:2 1:45: 1:46:4 1:48:2 1:5: Fig. 4. Cystometrogram trace in healthy rats after lidocaine instillation. The (-5) 4 cmh 2 O range. Table 2. Cystometrogram s parameters in normal and after intravesical instillation of capsaicin or lidocaine. Group I: CONTROL Group V: CONTROL + CAPSAICIN Group VI: CONTROL + LIDOCAINE P Value BP [cmh 2 O] 1,4 ±, ,7 ±,58 NS PT [cmh 2 O] 5,68 ± 1, ,33 ±,88 NS MVP [cmh 2 O] 27,41 ± 4, ,33 ±,7 NS ICI [min.] 5,278 ± 1, ,77 ± 1,38 p=,3* Compliance [ml/cmh 2 O],59 ±,19 ---,6 ±,29 p<,1* fbc [ml],243 ±,71 ---,357 ±,6 p=,4* DI / DOI [cmh 2 O/ml] 121,92 ± 32, ,62 ± 38,75 p=,3* MI [cmh 2 O x s/min.] 185,64 ± 45,95 23,75 ± 54, 29,98 ± 42,86 * statistically significant differences between group I and group VI (p,5). Test K-W p=,543

5 89 Zapis CMG - model przewlekły OAB + 1mM KAPSAICYNA :25: :6:4 1:8:2 1:: 1:11:4 1:13:2 1:15: 1:16:4 1:18:2 1:2: 1:21:4 Fig. 5. Cystometrogram trace in chronic OAB after capsaicin instillation. The (-5) 4 cmh 2 O range. Zapis CMG - model przewlekły OAB + 2% LIDOKAINA :59: :2 1:55: 1:56:4 1:58:2 2:: 2:1:4 2:3:2 2:5: 2:6:4 2:8:2 2:: 2:11:4 Fig. 6. Cystometrogram trace in chronic OAB after lidocaine instillation. The (-5) 4 cmh 2 O range. Table 3. Cystometrogram s parameters in rats with chronic OAB and after intravesical instillation of capsaicin or lidocaine. Grupa II: chronic OAB Grupa III: chronic OAB + CAPSAICIN Grupa IV: chronic OAB + LIDOCAINE P Value BP [cmh 2 O] 3,31 ± 1,85 3,23 ±,31 2,2 ±,28 Test K-W p=,163 PT [cmh 2 O] 6,88 ± 1,93 8,28 ±,77 5,42 ±,42 p=,* MVP [cmh 2 O] 21,67 ± 1,94 23,43 ± 1,33 28,93 ± 2,68 p<,1** ICI [min.] 2,149 ±,35 5,852 ±,886 4,853 ±,792 p<,1*** Compliance [ml/cmh 2 O],3 ±,7,54 ±,,66 ±, p<,1*** fbc [ml],99 ±,16,27 ±,4,223 ±,36 p<,1*** DI / DOI [cmh 2 O/ml] 384,3 ± 181,68 91,48 ± 15,13 137,93 ± 27, p<,1**** MI [cmh 2 O x s/min.] 261, ± 33,92 211,3 ± 15,57 25,8 ± 36,8 p=,2**** * statistically significant differences between group III and group IV (p,5). ** statistically significant differences between group II and group IV, as well as between group III and IV (p,5). *** statistically significant differences between all groups (p,5). **** statistically significant differences between group II and group III, IV (p,5).

6 9 overactivity index (319.8% and 178.4%, respectively) and motility index (23.5% and 27.3%, respectively), were observed. Surprisingly, the amplitude of maximal voiding pressure (MVP) was significant higher after lidocaine instillation, compared to control animals with chronic OAB (28.93±2.68 vs ±1.94 cm H 2 O, p<.1). Contrary, capsaicin has no such effect, as lidocaine on MVP amplitude. The differences of basal and threshold pressure were not statistically significant. DISCUSSION Cyclophosphamide treatment causes mucosal inflammatory response as indicated by macroscopic and microscopic changes in bladder histology and the presence of inflammatory cell infiltrates. Also it has been stated, that chemical CYP-cystitis initiates urinary bladder hyperactivity by sensitizing mechanosensitive afferents and/or recruitment of silent afferents which are unresponsive to mechanical stimuli in healthy conditions such as bladder distension (14). Similarly, mild gastric mucosa irritation sensitizes the abdominal vagal afferents, which conduct the sensory information in the C-fiber range, and alters the gastric pacemaker activity which seems to contribute to the dyspeptic sensations (15). Additionally sensitization of abdominal afferents can be induced by mucosal lesions secondary to bacterial infection leading to clinical symptoms. This fact was reinforced by Zycinska et al. (16) study which revealed that H. pylori infection is associated with gastroduodenal mucosal lesions in patients with Wegener s granulomatosis who undergo a complex therapy with glucocorticosteroids, cyclophosphamide, and non-steroidal antiinflammatory drugs. After chronic CYP treatment we observed significant decrease of micturition voiding pressure, intercontraction interval, functional bladder capacity, compliance and also an increase of non-voiding contraction (NVCs) frequency. These results are close to that reported previously by several authors in spite of slight modification of experimental protocol (, 17). Surprisingly, we observed an increase of basal pressure and no significant changes of threshold pressure. Contrary Giuliano et al. (18) observed an increase of basal and micturition voiding pressure for voiding and non-voiding contractions in CYP-treated rats under isoflurane anaesthesia. Moreover, Hu et al. (19) observed bladder hyperactivity with increase of basal, threshold and micturition voiding pressure in acute and intermediate CYPtreated rats under no anaesthesia. It seems that basal pressure changes are caused by the disturbances of detrusor muscle cells ability to relax, which could be concerned with alterations in gene expression and synthesis of neurotransmitters within bladder and afferent nerves. Moreover, Vizzard et al. (11) observed that neurotransmitters release is linked with bladder inflammation, which can alter the properties of sensory pathways leading to a reduction in a pain threshold (allodynia) and an amplification of painful sensations (hyperalgesia). On the other hand, micturition voiding pressure changes are probably resulting in influence of anaesthetics (e.g. urethane, isoflurane). Urethane could disturb the activity of supraspinal micturition regulation organs and consequently alter the effector response to stimuli leading to weakness detrusor muscle cells contractions. Silent afferent capsaicin-sensitive neurons have positive expression to polymodal TRPV1 receptors (transient receptor potential ion channel of the vanilloid type 1) (2). So far, it was believed that TRPV1 expressing unmyelinated bladder afferent nerves have no impact on micturition regulation in physiological condition, because of no response to mechanical stimuli (4). Surprisingly, Birder et al. observed significant difference in voiding behaviour and cystometrogram s traces of TRPV1 knockout mice compared to healthy mice. His finding indicates that TRPV1 bladder afferents participates in normal bladder function (21). Also other human and animal studies involving capsaicin-evoked desensitization have shown that capsaicinsensitive neurons are important in bladder physiology, particularly in the development of bladder overactivity (22, 23). Our experiments reveal that the modulation of bladder C- fibers activity by neurotoxin capsaicin leads to disorganization of micturition cycles in healthy rats, as well as to improvement of bladder function (storage and voiding phase of micturition) in rats with chronic model of overactive bladder. In rats with overactive bladder, C-fibres desensitization by capsaicin caused a significant increase of intercontraction interval, functional bladder capacity and compliance, as well as a decrease of detrusor overactivity index, motility index and non-voiding contraction (NVCs) frequency. These observations are close to that reported by Komiyama et al. (7) who obtained the inhibition of rhythmic bladder contractions after capsaicin and resiniferotoxin instillation in normal and chronic spinal cord injury rats. Exemplified studies revealed that stimulation of TRPV1 by vanilloids or other agents (bradykinin, protons), vanilloid-sensitive nerve terminals may release neuropeptides like substance P (SP), calcitonin gene-related peptide (CGRP) and interleukins, generating responses in blood vessels, mast cells and lymphocytes causing neurogenic inflammation and in consequence leading to overactivity of bladder (4, 24). Capsaicin, as agonist of TRPV1 receptors, initially stimulates and then subsequently desensitized, temporarily, the afferent C-fibres causing incomplete suppression of bladder overactivity, which confirms the presence of capsaicinresistant C-fibre afferents (25). Intravesical instillation of capsaicin in patients with OAB causes pain and discomfort. The background of decreased OAB severity caused by intravesical capsaicin in chronic OAB rats may be the result of the suppression of C-fibres and it weakens the inflammatory neurogenic response of C-fibres to stimuli. Furthermore, local intravesical anaesthesia of the bladder with lidocaine, before capsaicin administration, has been used to reduce pungent effects of capsaicin (26, 27). Contrary to intravesical instillation of capsaicin, lidocaine has no impact on disorganization of micturition cycles in healthy rats. Similarly to capsaicin modulation, the intravesical administration of lidocaine attenuates the severity of detrusor overactivity, leading to the improvement of urodynamic parameters in rats with chronic overactive bladder. In overactive bladder rats, lidocaine caused a significant increase of intercontraction interval, compliance, functional bladder capacity and detrusor index, compared to control group. The higher value of detrusor overactivity index is the result of the intense detrusor contractile activity (ability to generate detrusor contractions of higher amplitude at the end part of storage phase). Additionally, an increase of micturition voiding pressure after lidocaine instillation in rats with overactive bladder was recorded. It seems that there are two underlying mechanisms responsible for the increase of micturition voiding pressure in response to lidocaine, as compared to capsaicin. Primarily, there is no over-stimulation of detrusor muscle in response to neuropeptides release by C-fibres as on stimulation of capsaicin, causing partial exhaustion of detrusor. Secondarily, after lidocaine the desensitization phase of C-fibres does not occur which does not lead to impairment of modulating activity of non-adrenergic, non-cholinergic autonomic nervous system (NANC) on activity of bladder efferent nerves. Local anaesthetic, such as lidocaine decreases the likelihood of neural depolarization by binding to voltage-gated sodium membrane channels and leading to the blockage the admission of sodium ions into the neurons. However, recently discovered TRPA1 receptors (transient receptor potential ion channel of the ankyrin type A1) on bladder afferent C-fibres, probably, also contribute to micturition. Du et al. (28) evaluated that agonist of

7 91 TRPA1 leads to bladder overactivity in rats. TRPA1 is coexpressed with TRPV1 in sensory neurons within lower urinary tract (24). Leffler et al. study showed that lidocaine activates, sensitizes TRPV1 and TRPA1, as well as causes strong, acute desensitization of TRPV1 in rodent sensory neurons (29). This term might suggest that local anaesthetics, such as lidocaine can also act directly on bladder afferent C-fibres and modulate a micturition cycles. CONCLUSION Our results obtained, that chronic chemical CYP-induced cystitis leads to the overactivity of urinary bladder in rats. Additionally, the modulation of C-fibres activity by capsaicin and lidocaine reduces the severity of detrusor overactivity in rats with chronic overactive bladder, and improve its urodynamic estimation. This observations confirm the hypothesis, that in pathophysiology of overactive bladder the pivotal role play two types of unmyelinated bladder afferent C-fibres, both capsaicinsensitive and capsaicin-resistant. Conflict of interests statement: None declared. REFERENCES 1. Chancellor MB. New frontiers in the treatment of overactive bladder and incontinence. Rev Urol 22; 4(4): S5-S Habler HJ, Janig W, Koltzenburg M. Activation of unmyelinated afferent fibres by mechanical stimuli and inflammation of the urinary bladder in the cats. J Physiol 199; 425: Fall M, Lindstrom S, Mazieres L. A bladder-to-bladder cooling reflex in the cats. J Physiol 199; 427: de Ridder D, Baert L. Vanilloids and the overactive bladder. BJU Int 2; 86: Magii CA, Conte B. Effect of urethane anesthesia on the micturition reflex in capsaicin treated rats. J Auton Nerv Syst 199; 3(3): Dinis P, Churrua A, Avelino A, Cruz F. 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The Vanilloid receptor TRPV1 is activated and sensitized by local anesthetics in rodent sensory neurons. J Clin Incest 28; 2: R e c e i v e d: November 7, 28 Accepted: November 6, 29 Author s address: Kajetan Juszczak M.D., Departament of Pathophysiology, Jagiellonian University, Medical College, Czysta Street 18, Cracow, Poland; Phone: , Fax: ; kajus13@poczta.onet.pl