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Feb 16, 2016 - Animals. Fourteen adult female rats (Sprague Dawley, 200–250g;. Charles River, l'Arbresle, France) were kept at a constant temperature (22 Ж ...

Neurourology and Urodynamics 36:308–315 (2017)

Bladder Telemetry: A New Approach to Evaluate Micturition Behavior Under Physiological and Inflammatory Conditions ,1 Nathalie Vergnolle,2 Bruno Le Grand,1 James Gillespie,3 Nicolas Monjotin,1* Martine Farrie and Didier Junquero1 1

Institut de Recherche Pierre Fabre, Castres, France 2 INSERM, U1043, Toulouse, France 3 Newcastle University, Newcastle upon Tyne, England Aims: To establish a new approach to cystometry using telemetry in conscious rats and to use this technique to determine the role of conscious decision making processes with respect to the initiation of voiding in physiological, inflammatory, and painful conditions. Methods: The pressure transducer of a telemetric transmitter was implanted in the dome of the urinary bladder. After a recovery period of at least 1 month, several investigations of urodynamic parameters were performed after diuresis activation by a pulse of furosemide. The model was characterized by tolterodine and mirabegron under physiological conditions and same animals were reused to evaluate the modification of the voiding pattern under bladder inflammation induced by cyclophosphamide. Results: The quality of traces and measurement of parameters recorded telemetrically were comparable to those with conventional cystometry. Furosemide induced a reproducible transient increase of urine production and a series of voids that persisted for 60 min. Tolterodine reduced the amplitude of micturition contractions although mirabegron was devoid of any effect. Seven hours after injection of CYP, voiding frequency increased significantly and the micturition amplitude contraction was not altered. However, the mean volume voided during individual micturitions and the total voided volume decreased. During a second exposure to furosemide 24H after CYP injection, the micturition pattern returned to control, however, the micturition volume was still lower than in control. Conclusion: This telemetric model appears to be as accurate as previously described in conscious conventional cystometry, and allows the repeated evaluation of compounds which may modulate the voiding patterns. Neurourol. Urodynam. 36:308–315, 2017. # 2016 The Authors. Neurourology and Urodynamics published by Wiley Periodicals, Inc. Key words: bladder; interstitial cystitis; telemetry; urodynamics; voiding function


Storage mechanism of urinary bladder can be disturbed in many pathological situations, among them interstitial cystitis and painful bladder syndrome, which are characterized by pelvic pain, inflammation, and detrusor dysfunction leaded to urinary frequency and urgency.1 It is argued that animal models can contribute to a better understanding of bladder inflammation and dysfunction. In rats, haemorrhagic cystitis can be triggered by the chemotherapeutic agent cyclophosphamide (CYP). CYP-induced cystitis is a well-established experimental model of bladder dysfunction due to the accumulation of its metabolite acrolein in the bladder.2 In rats, an acute intraperitoneal administration of CYP induces signs of pelvic pain,3 inflammation,4,5 and alterations to urodynamic parameters.6,7 So-called ‘‘conventional’’ cystometry consists in a continuous bladder infusion via a catheter inserted into the bladder, at defined flow rates, to stimulate micturition reflex.8 Bladder pressures and voided volumes can be recorded in both anaesthetized and conscious animals. In conscious animals, bladder infusion cystometry may be affected by some experimental biases: the reduced movements of the animal, an under-evaluation of the sensory element for triggering the micturition cycle,9 and the composition of the infusion fluid. Radio-telemetry allows access to continuous recording of physiological parameters in fully unrestrained conditions. The bladder is not filled with saline and consequently operates in more physiological conditions. Importantly, the timing of a void #

is determined by the animal, a ‘‘conscious decision’’ and not only by the volume of urine in the bladder. A similar approach has been reported in beagle dogs10 or in minipigs.11 If such telemetric techniques can be applied to small animal models in which pathophysiological conditions such as interstitial cystitis or chronic bladder pain symptoms could be induced, this would represent a unique tool for in vivo pharmacology studies, and for a better understanding of micturition behaviors. The aim of this study was: (i) to develop an integrated model of cystometry and analysis using telemetry in conscious rats; (ii) to demonstrate the validity of the model with clinically relevant compounds; and (iii) to explore the possibility of using this approach to determine whether conscious decision making processes play a role in the initiation of voiding in physiologiThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made. Dr. Hashim Hashim led the peer-review process as the Associate Editor responsible for the paper. Potential conflicts of interest: Dr. Farrie, Dr. Gillespie, Dr. Junquero, Dr. Le Grand, and Dr. Monjotin report personal fees from the Institut de Recherche Pierre Fabre during the conduct of the study and outside the submitted work.  Correspondence to: Nicolas Monjotin, Institut de recherche Pierre Fabre, Centre d’evaluation Preclinique de Campans, Service de Pharmacologie, Bel Air, 81100 Castres, France. E-mail: [email protected] Received 2 October 2015; Accepted 25 January 2016 Published online 16 February 2016 in Wiley Online Library ( DOI 10.1002/nau.22970

2016 The Authors. Neurourology and Urodynamics published by Wiley Periodicals, Inc.

Bladder Telemetry: A New Approach To Evaluate Micturition Behavior


cal, inflammatory, and painful conditions. MATERIALS AND METHODS Animals

Fourteen adult female rats (Sprague Dawley, 200–250 g; Charles River, l’Arbresle, France) were kept at a constant temperature (22  28C) and in a regular light/dark cycle, with food and water provided ad libitum. All experimental protocols were in accordance with internal Ethic committee (CEA-CEPC-110), with the European Directive 2010/63/EU and French legislation decree n8 2013-118. Data Analysis

Results are expressed as mean  SEM, with n values indicating the number of animals used for a particular set of experiments. Statistical analysis was carried out using SigmaStat 3.5 software. After checking the normality, paired Student’s t-test and Mann Whitney Rank Sum test were used for statistical analysis. For multiple comparisons, one-way ANOVA followed by Kruskall Wallis on Ranks was used. P < 0.05 ( ) values were considered to show significant differences between means. Study Design Telemetric devices. Transmitter used for these experiments were PhysioTel PA-C40 (Data Science International), allowing pressure (accuracy  3 mmHg) and activity measurement in small animals (7.8 g and 4.5 cc). Pressure of each transmitter was calibrated in three points by the manufacturer. Calibration values were checked in three points (0, 30, and 100 mmHg) by a sphygmomanometer just before implantation and after animal euthanasia to ensure validity of data obtained. Surgical procedure. Pre-operative analgesic (buprenorphine and ketofen) and antibiotic (enrofloxacin) treatments were given to all animals before anaesthesia (isoflurane 2–4%). In aseptic conditions, a small laparotomy was performed. The transmitter was placed on the right side of the abdominal cavity and sutured on the abdominal wall (silk 4/0) in order to avoid contact with the bladder. The pressure transducer was inserted in the dome of the urinary bladder and secured with a purse string (silk 6/0). After checking pressure values, the incisions were closed with resorbable sutures (Vicryl 3/0 for abdominal wall and Vicryl 4/0 for skin). Animals received postoperative care (analgesic and antibiotic) for 2 days following the surgery. All animals were giving a further recovery period of at least 1 month before using for experiments. Characterization of the model. Animal received an oral treatment of vehicle (Carboxymethylcellulose 0.5% in water), tolterodine (1–10 mg.kg1) or mirabegron (1–10 mg.kg1) in a final volume of 5 ml.kg1. After 1 hr, furosemide 1 (Lasilix 10 mg.kg1, 2 ml.kg1) was injected subcutaneously (to stimulate the diuresis) and the rats were placed immediately in customized metabolic cages. Bladder pressure and voided volumes were continuously recorded (IOX2, EMKA Technologies) for 60 min following the furosemide injection. Each animal was studied on several occasions (up to seven times) and received the vehicle at least once. Acute bladder inflammation. The same cohort of animals was also used to evaluate the effect of bladder inflammation on urodynamics and the voiding pattern. At T0, CYP (150 mg.kg1, 5 ml.kg1), or its vehicle was injected intraperitoneally. At T þ 2 Neurourology and Urodynamics DOI 10.1002/nau

Fig. 1. Schematic representation of re-use of animal during the experiments. Case of an animal which received one furosemide pulse (O) to control telemetric parameters, further furosemide pulses 1 hr after different oral treatment (characterization of the model) and two others furosemide pulse 7H and 24H after CYP injection (") (Bladder inflammation).

H30, animals were anaesthetized by isoflurane and the bladder was emptied. A catheter (PE-10) was inserted in the bladder through the urethra and an intravesical administration of saline (600 ml) was performed. After 60 min of exposure, the bladder was emptied and the animals woke up. The furosemide pulse was performed 7H and 24H after CYP injection and bladder pressure and voided volumes were continuously recorded for the following 60 min in the same conditions as previously described. After the second furosemide evaluation, animals were killed by an overdose of pentobarbital followed by a cervical dislocation. Schematic representation of re-use of telemetered animals for the different steps is presented in Figure 1. In separate experiments, the same procedure of intravesical administration was performed on different animals 2H30 after CYP or vehicle injection. To evaluate a possible interaction between furosemide and CYP, the stimulation of diuresis was performed by an oral gavage (5 ml of water) 7H and 24H after CYP (n ¼ 11) or vehicle (n ¼ 6) injection. Urine was collected in metabolic cages for 1 hr. RESULTS Cystometric Parameters Determined by Telemetry in Conscious Rats After Furosemide Injection

Typical traces (Fig. 2A) shown an example of a complete experiment in a control animal: the upper record showing bladder pressure and the lower voided volume. The injection of furosemide has produced an increase of diuresis resulting in the rapid accumulation of urine and the triggering of a series of voids. In this representative trace, the amplitude of the micturition was relatively stable throughout the 60min recording. The pattern of voiding was such that the ICI increased following the furosemide injection as its diuretic effects waned. Interestingly, individual voided volumes were constant (Fig. 2A) despite the progressive increase in ICI. Figure 2B shows, on an expanded time-scale, one micturition cycle displaying the quality of traces and quantitative parameters recorded telemetrically. Results obtained were comparable to those with conventional cystometry (data not shown). Using such records the ‘‘classical’’ cystometry parameters, Basal Pressure (BP), Micturition Pressure (MP), Inter Contraction Interval (ICI), and voided volume (V), were determined. Cystometry parameters were evaluated for 13 out of 14 telemetered rats included in the study after a single oral administration of CMC at 0.5% in water (vehicle). One animal did not respond to the furosemide pulse in the vehicle group and was excluded. One hour later, furosemide injection induced a mean of 7  1 voids in 60 min for a total voided volume of


Monjotin et al.

Fig. 2. Representative traces of telemetric recording after a pulse of furosemide (10 mg.kg1, 2 ml.kg1). (A) One hour recording following the furosemide pulse. (B) Focus on one micturition cycle with recorded parameters: BP, basal pressure (mmHg); MP, micturition pressure (mmHg); A, amplitude of micturition ¼ MPBP (mmHg); ICI, inter-contraction interval (sec); V, voided volume (ml).

9.0  0.6 ml and 1.4  0.1 ml per void; the mean amplitude of micturition was 38.0  3.5 mmHg (Fig. 3). Pharmacological Characterization of the Effects of Tolterodine and Mirabegron in Physiological Conditions

Under physiological conditions, single doses of tolterodine, mirabegron (1, 3, or 10 mg.kg1 for both) or vehicle were administered orally after appropriate washout periods. Timematched vehicle-treated rats were evaluated also (seven animals). Tolterodine reduced the amplitude of micturition with a significant effect at 10 mg.kg1, when compared to vehicle (40.8  3.1 mmHg, n ¼ 19 for vehicle vs. 23.5  3.4 mmHg, n ¼ 12 for tolterodine 10 mg.kg1). Mirabegron, at all concentrations used did not affect the micturition amplitude (n ¼ 13) (Fig. 4). Regarding the other cystometry parameters, no modification of the number of micturitions induced by furosemide was observed with both tolterodine and mirabegron, as compared to vehicle-treated rats (Fig. 4, Table I). Mean voided volume per micturition (1.4  0.1 ml) was not modified by tolterodine or mirabegron, and no significant modification of total voided volume, within 60 min after the pulse of furosemide, was observed with both compounds compared to vehicle-treated rats (Table I).

The effect of tolterodine on voiding amplitude was highly related to the animal. A detailed analysis of an individual animal is shown in Figure 5, with typical traces illustrating a control experiment (vehicle) or with tolterodine (10 mg.kg1) (Fig. 5A and B). It can be seen that, for this animal, the voided volume is the same in the control situation compared to values after tolterodine (1.4  0.1 ml for tolterodine 10 mg.kg1 vs. 1.3  0.1 ml for vehicle). Note the significant fall in the amplitude of the voiding (14.5  0.4 mmHg for tolterodine vs. 55.3  1.7 mmHg for vehicle) throughout the recording. The total voided volume over the 60 min experiment duration was comparable to that of vehicle treatment (Fig. 5C). There was also no significant difference in the number of micturitions recorded in control compared to tolterodine (Table I). In Figure 6, the percentage change in pressure amplitude and voided volume obtained with tolterodine 10 mg.kg1 and vehicle was calculated for each animal (each animal served as its own control being treated with both vehicle and tolterodine). Each point is the average of all voids obtained during the 1 hr of recording period after furosemide injection. The expressed parameters corresponded to (ATolte–Aveh)/Aveh in percentage. The same calculation was applied for voided volume parameter. Hence, this representation took into account the inter-individual variability of cystometric measurement profile between rats. Under these experimental conditions, tolterodine 10 mg.kg1 decreased the amplitude of bladder contraction during micturition by 42%, without a notable modification of the voided volume (14%), suggesting that whatever the individual urodynamic and voiding patterns are, there was no relationship between micturition pressure and quantity of urine voided. Effect of Bladder Inflammation on Furosemide Induced Micturitions

Fig. 3. Effect of furosemide 10 mg.kg1 subcutaneous injection on urodynamics (amplitude of micturition in mmHg and number of micturitions) and associated voided volumes (total and per void volume). Each dot corresponds to one animal (n ¼ 12).

Neurourology and Urodynamics DOI 10.1002/nau

Telemetric evaluation after 7H. When compared to vehicle, CYP evoked a significant increase of the number of micturitions (18  5 vs. 5  1 for vehicle) associated with a decrease of ICI (Figs. 7 and 8A.) without modification of amplitude of bladder contraction (39.9  9.3 mmHg for CYP-treated group vs. 44.9  8.6 mmHg for vehicle) (Figs. 7 and 8B). CYP decreased the mean volume of individual micturitions and total voided volume within the 60 min recording (0.7  0.2 ml for CYPtreated group vs. 2.1  0.5 ml for vehicle) (Figs. 7, 8C and D). Telemetric evaluation after 24H. After a second injection of furosemide 24 hr after the CYP injection, the increase of the number of micturitions (18  5) returned to the level of vehicle

Bladder Telemetry: A New Approach To Evaluate Micturition Behavior


Fig. 4. Effect of tolterodine (Tolte) at 1, 3, and 10 mg.kg1 and mirabegron (Mira) at 1, 3, and 10 mg.kg1 on amplitude of micturition (MP-BP, A and B), on number of micturitions (C and D) and on voided volume per micturition (E and F). Each bar chart represents the average of parameters obtained in 60 min following the furosemide pulse (  P < 0.01 vs vehicle).

TABLE I. Effect of Tolterodine at 1, 3, and 10 mg.kg1 and Mirabegron at 1, 3, and 10 mg.kg1 on Urodynamic Parameters Parameter (mean  SEM)

Groups Vehicle Tolterodine 1 mg.kg1 Tolterodine 3 mg.kg1 Tolterodine 10 mg.kg1 Mirabegron 1 mg.kg1 Mirabegron 3 mg.kg1 Mirabegron 10 mg.kg1 

Number of animals

Amplitude of micturition (mmHg)

Number of micturitions

18 12 11 12 12 13 13

40.8  3.1 35.4  4 30.2  4.9 23.5  3.4 40  4.4 38.6  3.1 36.3  3.0

81 71 71 91 91 81 81

P < 0.05 versus vehicle.

Neurourology and Urodynamics DOI 10.1002/nau

ICI (sec)

Mean voided volume per micturition (ml)

Total voided volume in 60 min (ml)

415  28 471  37 477  31 354  27 380  44 412  47 397  39

1.4  0.1 1.8  0.2 1.6  0.2 1.2  0.1 1.3  0.1 1.4  0.2 1.3  0.2

9.9  0.5 10.4  0.3 10.0  0.7 10.5  0.4 10.5  0.6 10.1  0.6 9.3  0.5


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Fig. 7. Representative traces of effect of furosemide 10 mg.kg1 on naive (A) and 7H after cyclophosphamide 150 mg.kg1 injection i.p. (B), on bladder pressure and associated voided volumes.

Fig. 5. Effect of furosemide pulse on Bladder pressure and voided volumes obtained with the same animal 1H after vehicle (A) or tolterodine 10 mg.kg1 (B) administration. Effect of vehicle and tolterodine 10 mg.kg1 on amplitude of micturition against individual voided volume (C) for a high responsive rat.

group (4  1 micturitions in both groups). However, the mean volume of each micturition remained lower in CYP-treated group and comparable to that after the first pulse of furosemide (0.8  0.1 ml in CYP conditions vs. 1.9  0.1 ml in vehicle group). The reason for this reduction in voided volume despite a reduced voiding frequency was a significant fall in the rate of urine production. Consequently, another important difference was the total voided volume in 60 min, which was highly decreased 24H after CYP injection (2.9  0.4 ml in CYP conditions vs. 7.8  0.7 ml in vehicle group) (Table II). In the separate experiments of stimulation of diuresis by the oral gavage, a significant reduction (P < 0.05) of total voided volume between vehicle or CYP group was observed 24H after injection (2.3  0.4 ml in vehicle group, vs. 1.1  0.2 in CYP group) (Table II). These results are in accordance with those obtained after furosemide pulse and the decrease of the total voided volume combined with a lower voided volume per micturition observed in furosemide experiments suggest an impact of CYP on kidney function 24H after injection. The amplitude of micturition was comparable for both injections of furosemide and remained unchanged in CYP or vehicle conditions. DISCUSSION

Fig. 6. Integrative representation of the effect of tolterodine 10 mg.kg1 on amplitude of micturition against mean voided volume. Results are expressed as percentage of variation of tolterodine 10 mg.kg1 effect versus vehicle for the two parameters.

Neurourology and Urodynamics DOI 10.1002/nau

A novel approach to cystometry using radiotelemetric recording has been presented in conscious freely moving rats. We used well characterized drugs, namely tolterodine (a muscarinic antagonist), and mirabegron (a b3 adrenergic agonist) to explore the technique, and results obtained were in accordance with those reported under physiological conditions.12,13 Under pathological conditions such as inflammation, the key parameters of the micturition cycle and voiding could be assessed at the most critical time periods7 without any forced bladder infusion but rather upon endogenous stimulation of diuresis. Commonly used models of cystomanometry are based on the infusion of the bladder

Bladder Telemetry: A New Approach To Evaluate Micturition Behavior


Fig. 8. Effect of cyclophosphamide on number of micturitions (A and E), amplitude of bladder contraction (B and F) and mean (C and G) and total (D and H) voided volume 7H (A–D) or 24H (E–H) after injection.  P < 0.05 vs Vehicle/Vehicle.

with a determined flow rate of saline.8,14 The model presented herein is based on a different approach with a bladder filling by increased diuresis triggering micturitions by a more physiological way. Neurourology and Urodynamics DOI 10.1002/nau

It has been clearly demonstrated that anaesthesia modifies urodynamics in rats suggesting a reduction of bladder sensation.15–17 The anaesthetized rat only voids when the bladder volume of urine exceeds the micturition threshold.18 Every


Monjotin et al.

TABLE II. Effect of CYP (150 mg.kg1) on Bladder Pressure and Associated Voided Volumes 7H and 24H After Injection Parameter (mean  SEM)

Time after CYP

Amplitude of micturition (mmHg)

Number of micturitions

Mean voided volume per micturition (ml)

Total voided volume in 60 min after furosemide pulse (ml)

Total voided volume in 60 min after water load (ml)


7H 24H

44.9  8.6 42.1  6.9

51 51

2.1  0.5 1.9  0.4

9.6  0.6 7.8  0.7

2.8  0.3 2.3  0.4


7H 24H

45.6  9.1 46.0  15.7

18  5 41

0.7  0.2 0.8  0.1

7.6  0.4 2.9  0.4

3.0  0.3 1.1  0.2


P < 0.05 versus vehicle/vehicle.

anaesthetic decreased the bladder capacity and non-voiding contractions, even if urethane was described to have the lowest impacts on classical urodynamic parameters.15 In conscious cystometry, the catheter implantation into the dome of the bladder and exteriorisation at scapular level is commonly used,8 but it is described as inducing severe histological changes (oedema, haemorrhage, and granulation) and modification of the bladder function during early cystometry after surgery.10,19 Moreover, animals can mostly be used once. This telemetric model allowed an evaluation of urodynamics for several months in the same conscious animal without any modification of the voiding pattern. This approach allows several tests on the same animal to ensure quantitative and qualitative reproducibility of the results, to see control and drug effects in the same animal and to reduce the total number of animals. Hence, this novel unrestrained urodynamic approach, allowed conditioning of the animal, behavior and welfare without technical impairment of bladder function. Furosemide, a loop diuretic with a short half-life,20 was used to stimulate an intrinsic diuresis and increased the number of micturitions about sevenfold. The selected dose (10 mg.kg1) was chosen to ensure good reproducibility of the voiding pattern for 1 hr. The pharmacokinetic profile of furosemide enabled several injections in the same animal on different days producing the same regular increase in the number of voids during the 60 min of recording period. In this series of experiments, furosemide induced no modification of the voiding pattern even after many pulses in contrast to previous reports in dogs10 and in rats.21 These contradictory results regarding voided volumes per void may be explain by the difference of species, dose, and route of administration of furosemide (2 mg.kg1 in dogs, i.m.) and duration of the recording. Regarding bladder pressure levels in rats recorded by telemetry, they were comparable to conventional cystometry obtained in our laboratory (data not shown) and results reported previously.22,23 The differences of bladder pressure levels between conventional cystometry and telemetry reported by Moody et al.21 could be explain by the implantation techniques in the bladder dome. The latter described significant reductions in simultaneously measured basal and micturition pressures by telemetry when compared to conventional technique in rats. A substantial inter-individual variability was observed in this study, although comparable to that of conscious conventional cystometry, and it was due to the unrestrained conditions of moving rats and catheter positioning.24 In order to explore the use of the telemetric model the effects of mirabegron and tolterodine was evaluated on urodynamic parameters and voided volumes.24–26 It was observed that mirabegron induced no modification to either micturition pressure or frequency. The effect on bladder pressure was in Neurourology and Urodynamics DOI 10.1002/nau

accordance with data reported by Sadananda et al. but in this study, mirabegron increased the ICI.12 These differing results could be explained by the approach of the two methods: one was based on bladder infusion at constant rate which stimulated a forced bladder reflex and the other by the stimulation of the diuresis. Moreover, the relevance the b3 adrenergic impact of mirabegron on urodynamic parameters was demonstrated mostly in pathological conditions such as bladder over-activity.26 In keeping with previous studies using conventional cystometry tolterodine decreased the micturition amplitude but did not modify the micturition interval.13 However, an interesting observation was that the voided volumes were not reduced despite a lower micturition pressure suggesting that magnitude of contraction during micturition is not involved in the control of voiding. Determination of voiding could be probably more related to sensation as described by Saitoh et al. in a model of vesical pain, and this bladder discomfort correlated with a decreased bladder capacity or urgency.27 In conclusion, our results demonstrate, that the main effect of tolterodine was on bladder pressure not on voided volume. Inter-individual differences were probably not related to the pharmacological efficacy of tolterodine but rather to the specific urodynamic profile of individual rats. In order to evaluate a modification of the voiding pattern in a pathological condition a rat model of interstitial cystitis was induced by injection of CYP. The effect of furosemide was evaluated 7H after CYP injection, during the acute phase of bladder inflammation and associated pain28 with urothelium necrosis and cell apoptosis,29 as well as 24H later during the chronic phase of inflammation but when urothelial reconstruction had begun.29 Hughes et al. have recently demonstrated that acrolein-induced urothelium damages began 2 hr after CYP injection.30 The intravesical administration of saline for 1 hr, 2H30 after CYP allow a standardisation of the acrolein exposure for all the animals and ensure a better reproducibility of the model. Moreover, this technique was also developed for further pharmacological evaluation of drug candidates for IC management. The number of micturitions 7H after CYP injection was increased significantly compared to the respective physiological control experiment in the same animal. Moreover, the voiding pattern after CYP treatment was dramatically modified with a low voided volume per void. A correlation between the increase in number of voids and bladder pain/sensation was reported but without evaluation of urodynamics.31 With our model, the bladder reflex was not stimulated by an artificial mechanical filling but by a more physiological one via the kidneys. This suggests that when an animal decided to void, it was not only based on the volume but mostly on bladder

Bladder Telemetry: A New Approach To Evaluate Micturition Behavior 9

sensation confirming a modification of the bladder sensation related to function in rat interstitial cystitis elicited by CYP. 24H after CYP injection, the number of voids following the furosemide injection was reduced markedly. Comparable results were obtained after a water loading in the same inflammatory conditions demonstrating that this phenomenon was neither due to a possible interaction between furosemide and CYP or its metabolites, nor to a loss of the pharmacological efficacy of furosemide during the second pulse. These results rather suggested that CYP induced a major modification/reduction of the diuresis32 probably by a stringent water retention at the level of the proximal nephron.32 Such a model can demonstrate this effect with an urodynamic evaluation because in infusion models, only the bladder function is assessed and not all the lower urinary tract. Moreover, voided volume per void were similar at 7H and 24H after CYP injection, and much lower than in the physiological situation confirming that voiding pattern was still affected 24H after CYP injection as described in other studies.7,33 This set of experiments demonstrates that a telemetric approach allows the reliable evaluation of urodynamic parameters. When associated with measurements of voided volumes and linked to a furosemide induced diuresis it is possible to have reproducible patterns of voiding without modification of measured urodynamic ‘‘classical parameters.’’ Moreover, this work demonstrates that voiding pattern is certainly mostly triggered by the sensation and not by the volume, especially under inflammatory conditions. This telemetric model is likely to record more closely physiological and pathophysiological functions of the bladder, rather than previously described conscious conventional cystometry based on forced bladder infusion. Thus, this telemetric models in small animals (rats), will now allow the pharmacological evaluation of compounds, which could modulate the voiding pattern either by targeting detrusor contractility or afferent outflow.

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The authors are grateful to Mr. Albert Volant for expert animal care and surgery assistance and to Mr. Jean Michel Talmant for providing technical devices.




1. Hanno PM, Erickson D, Moldwin R, et al. Diagnosis and treatment of interstitial cystitis/bladder pain syndrome: AUA guideline amendment. J Urol 2015;193:1545–53. 2. Cox PJ. Cyclophosphamide cystitis-identification of acrolein as the causative agent. Biochem Pharmacol 1979;28:2045–9. 3. Saitoh C, Yokoyama H, Chancellor MB, et al. Comparison of voiding function and nociceptive behavior in two rat models of cystitis induced by cyclophosphamide or acetone. Neurourol Urodyn 2010;29:501–5. 4. Auge C, Chene G, Dubourdeau M, et al. Relevance of the cyclophosphamideinduced cystitis model for pharmacological studies targeting inflammation and pain of the bladder. Eur J Pharmacol 2013;707:32–40. 5. Lecci A, Birder LA, Meini S, et al. Pharmacological evaluation of the role of cyclooxygenase isoenzymes on the micturition reflex following experimental cystitis in rats. Br J Pharmacol 2000;130:331–8. 6. Hu VY, Zvara P, Dattilio A, et al. Decrease in bladder overactivity with REN1820 in rats with cyclophosphamide induced cystitis. J Urol 2005; 173: 1016–21. 7. Dornelles FN, Andrade EL, Campos MM, et al. Role of CXCR2 and TRPV1 in functional, inflammatory and behavioural changes in the rat model of

Neurourology and Urodynamics DOI 10.1002/nau





31. 32.



cyclophosphamide-induced haemorrhagic cystitis. Br J Pharmacol 2014;171: 452–67. Andersson KE, Soler R, Fullhase C. Rodent models for urodynamic investigation. Neurourol Urodyn 2011;30:636–46. Eastham JE, Gillespie JI. The concept of peripheral modulation of bladder sensation. Organogenesis 2013;9:224–33. McCafferty GP, Coatney RW, Laping NJ, et al. Urodynamic measurements by radiotelemetry in conscious, freely moving beagle dogs. J Urol 2009;181: 1444–51. Huppertz ND, Kirschner-Hermanns R, Tolba RH, et al. Telemetric monitoring of bladder function in female Gottingen minipigs. BJU Int 2015;116:823–32. Sadananda P, Drake MJ, Paton JF, et al. A functional analysis of the influence of beta3-adrenoceptors on the rat micturition cycle. J Pharmacol Exp Ther 2013;347:506–15. Yamazaki T, Muraki Y, Anraku T. In vivo bladder selectivity of imidafenacin, a novel antimuscarinic agent, assessed by using an effectiveness index for bladder capacity in rats. Naunyn Schmiedebergs Arch Pharmacol 2011;384: 319–29. Buyuknacar HS, Kumcu EK, Gocmen C, et al. Effect of phosphodiesterase type 4 inhibitor rolipram on cyclophosphamide-induced cystitis in rats. Eur J Pharmacol 2008;586:293–9. Cannon TW, Damaser MS. Effects of anesthesia on cystometry and leak point pressure of the female rat. Life Sci 2001;69:1193–202. Craft RM, McNiel DM. Agonist/antagonist properties of nalbuphine, butorphanol and ()-pentazocine in male vs. female rats. Pharmacol Biochem Behav 2003;75:235–45. Chang HY, Havton LA. Differential effects of urethane and isoflurane on external urethral sphincter electromyography and cystometry in rats. Am J Physiol Renal Physiol 2008;295:F1248–53. De Groat WC DJ, Levin RM, Long Lin AT, et al. Basic neurophysiology and neuropharmacology. In: Abrams PKS, Wein A, editors. Incontinence. Plymouth, UK: Plymbridge Distributors Ltd; 1999. 105–54. Morikawa K, Ichihashi M, Kakiuchi M, et al. Effects of various drugs on bladder function in conscious rats. Jpn J Pharmacol 1989;50:369–76. Agence Nationale de Securite du medicament (ANSM). Resume des caracteristiques produit (RCP) Furosemide 20 mg/2 ml, solution injectable. Update ANSM November, 2010. Moody BJ, Liberman C, Zvara P, et al. Acute lower urinary tract dysfunction (LUTD) following traumatic brain injury (TBI) in rats. Neurourol Urodyn 2014;33:1159–64. Breyer BN, Wang G, Lin G, et al. The effect of long-term hormonal treatment on voiding patterns during filling cystometry and on urethral histology in a postpartum, ovariectomized female rat. BJU Int 2010;106:1775–81. Castiglione F, Bergamini A, Bettiga A, et al. Perioperative betamethasone treatment reduces signs of bladder dysfunction in a rat model for neurapraxia in female urogenital surgery. Eur Urol 2012;62:1076–85. Hatanaka T, Ukai M, Watanabe M, et al. Effect of mirabegron, a novel beta3adrenoceptor agonist, on bladder function during storage phase in rats. Naunyn Schmiedebergs Arch Pharmacol 2013;386:71–8. Wefer J, Truss MC, Jonas U. Tolterodine: An overview. World J Urol 2001;19: 312–8. Gillespie JI, Palea S, Guilloteau V, et al. Modulation of non-voiding activity by the muscarinergic antagonist tolterodine and the beta(3)-adrenoceptor agonist mirabegron in conscious rats with partial outflow obstruction. BJU Int 2012;110:E132–42. Saitoh C, Chancellor MB, de Groat WC, et al. Effects of intravesical instillation of resiniferatoxin on bladder function and nociceptive behavior in freely moving, conscious rats. J Urol 2008;179:359–64. Lanteri-Minet M, Bon K, de Pommery J, et al. Cyclophosphamide cystitis as a model of visceral pain in rats: Model elaboration and spinal structures involved as revealed by the expression of c-Fos and Krox-24 proteins. Exp Brain Res 1995;105:220–32. Jezernik K, Romih R, Mannherz HG, et al. Immunohistochemical detection of apoptosis, proliferation and inducible nitric oxide synthase in rat urothelium damaged by cyclophosphamide treatment. Cell Biol Int 2003;27:863–9. Hughes FM, Jr., Corn AG, Nimmich AR, et al. Cyclophosphamide induces an early wave of acrolein-independent apoptosis in the urothelium. Adv Biosci Biotechnol 2013;4. Pessina F, Capasso R, Borrelli F, et al. Protective effect of palmitoylethanolamide in a rat model of cystitis. J Urol 2015;193:1401–8. Kim S, Jo CH, Park JS, et al. The role of proximal nephron in cyclophosphamideinduced water retention: Preliminary data. Electrolyte Blood Press 2011; 9:7–15. Chopra B, Barrick SR, Meyers S, et al. Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium. J Physiol 2005;562:859–71.

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