Vardenafil Decreases Bladder Afferent Nerve Activity in Unanesthetized, Decerebrate, Spinal Cord Injured Rats

Transcription

1 EUROPEAN UROLOGY 59 (2011) available at journal homepage: Neuro-urology Vardenafil Decreases Bladder Afferent Nerve Activity in Unanesthetized, Decerebrate, Spinal Cord Injured Rats Delphine Behr-Roussel a,d, Stephanie Oger a,d, Stéphanie Caisey a,d, Peter Sandner b, Jacques Bernabé a,d, Laurent Alexandre a, Francois Giuliano c,d, * a Pelvipharm, Orsay, France b Bayer Schering Pharma, Global Drug Discovery, Wuppertal, Germany c AP-HP, Neuro-Uro-Andrology, Department of Physical Medicine and Rehabilitation, Raymond Poincaré Hospital, Garches, France d EA 4501 Université Versailles Saint Quentin en Yvelines, Garches, France Article info Article history: Accepted October 15, 2010 Published online ahead of print on October 26, 2010 Keywords: Bladder afferent signaling Phosphodiesterase type 5 inhibitor Neurogenic detrusor overactivity Storage symptoms Nonvoiding contractions Spinal cord injury Abstract Background: Phosphodiesterase type 5 inhibitors (PDE5-Is) improve storage symptoms in benign prostatic hyperplasia patients, despite a lack of effect on peak urinary flow rate. Moreover, vardenafil improves urodynamic parameters in spinal cord-injured (SCI) patients with neurogenic detrusor overactivity (NDO). SCI rats also display NDO characterized by nonvoiding contractions (NVCs) during bladder filling, resulting in an increased bladder afferent nerve firing (BANF). Objective: We postulated that vardenafil could improve urodynamic parameters by reducing BANF. The effect of vardenafil has been investigated on intravesical pressure by cystometry experiments while recording BANF in response to bladder filling. Design, setting, and participants: Complete T7 T8 spinalization was performed in 15 female adult Sprague-Dawley rats ( g). Measurements: At d postspinalization, fine filaments were dissected from the L6 dorsal roots and placed across a bipolar electrode. Bladder afferent nerve fibers were identified by electrical stimulation of the pelvic nerve and bladder distension. SCI rats were decerebrated before cystometry experiments. Bladders were filled to determine the maximal bladder filling volume (BFV) for each rat. Then, after bladder stabilization at 75% of maximal BFV, saline (n = 7) or vardenafil 1 mg/kg (n = 8) was delivered intravenously. NVCs and BANF were recorded for 45 min. Results and limitations: In all SCI rats, BANF was already present and regular at resting conditions ( spikes per second). During bladder filling, intravesical pressure (IVP) slowly increased with transient NVCs superimposed. Concomitantly, BANF progressively increased up to 2.4-fold at maximal BFV ( ml). After stabilization at submaximal BFV, BANF was increased by %. Vardenafil injection induced an immediate decrease in NVCs compared to saline ( p < 0.001) and BANF (52% decrease vs 28% in saline after 45 min; p < 0.001). Conclusions: Systemic vardenafil reduced both NVCs and BANF in unanesthetized, decerebrate, SCI rats. These findings provide new insights into the mechanism of action by which PDE5-Is improve storage symptoms in SCI patients. # 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. AP-HP, Neuro-Uro-Andrology, Department of Physical Medicine and Rehabilitation, Raymond Poincaré Hospital, 104 bd Raymond Poincaré, Garches, France. Tel ; Fax: address: giuliano@cyber-sante.org (F. Giuliano) /$ see back matter # 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 EUROPEAN UROLOGY 59 (2011) Introduction Phosphodiesterase type 5 inhibitors (PDE5-Is) improve voiding and storage lower urinary tract symptoms (LUTS) suggestive of benign prostate hyperplasia (BPH), with a more pronounced effect on the latter [1 4]. Moreover, a controlled, pilot clinical study in spinal cord-injured (SCI) patients with neurogenic detrusor overactivity (NDO) demonstrates that a single dose of vardenafil significantly decreases maximum detrusor pressure and detrusor overactivity while improving bladder capacity [5]. However, the exact mechanism and sites of action of PDE5-Is on male LUTS remain yet to be established. Recently it was shown that PDE5-Is could relax human detrusor, albeit to a limited extent [6]. Thus, the main effects observed clinically with this class of compound may reside elsewhere. In fact, the beneficial effects of PDE5-Is on storage LUTS may be exerted through an inhibitory effect on the sensory component of the micturition reflex. Several in vivo studies in rats have previously suggested that nitric oxide (NO) may modulate the micturition reflex by influencing the excitability of bladder afferents [7 13]. The SCI rat model mimics the voiding patterns of patients with NDO due to SCI by displaying nonvoiding contractions (NVCs) during the filling phase due to functional and morphologic plasticity of bladder afferent pathways. Furthermore, previous studies have shown that an increase in bladder afferent nerve firing (BANF) is involved in the initiation of NVCs during bladder filling in chronic SCI rats [14,15]. We postulated that vardenafil could improve urodynamic parameters by reducing BANF and subsequent NVCs in SCI rats. We investigated concomitantly the effect of vardenafil on intravesical pressure (IVP) by cystometry experiments while recording BANF in response to bladder filling in decerebrate, unanesthetized SCI rats. 2. Material and methods Experiments were performed in compliance with animal care regulations in force in France (Ministry of Agriculture Authorization Agreement No. A , May 2009) Animal preparation Female adult Sprague-Dawley rats (250 g; Charles River Laboratories International Inc, Les Oncins, France) were housed with free access to chow and water. Rats were anesthetized with isoflurane (1.2%; Centravet SA, Plancoët, France) and body temperature maintained at 37 8C. A dorsal midline incision was made to expose from the 6th to the 10th thoracic vertebrae, then a T7 T8 laminectomy was performed. The dura and spinal cord were cut with fine scissors and a sterile gelform sponge (Gelita-Spon, Gelita Medical BV, Amsterdam, The Netherlands) was inserted between the cut ends of the spinal cord. The overlying muscle and skin were sutured. Postoperatively, the animals were treated with antibiotics to prevent urinary infection [16]. Alternately, cefovecin (20 mg/kg; Pfizer Animal Health, Tadworth, Surrey, UK) and enrofloxacin (20 mg/kg per day; Bayer HealthCare AG, Leverkusen, Germany) were administered or sulfamethoxazole/trimethoprim (50/10 mg/kg per day; Roche Laboratories, Basel, Switzerland) were delivered every week. Rats were kept at 30 8C during the first 72 h after spinalization. During the first week postspinalization, the bladder was manually emptied by Credé maneuver three times daily, and then twice daily until an abnormal micturition reflex was totally established Cystometry and bladder afferent nerve firing recording experiments At d postspinalization, rats were anesthetized with isoflurane (1.2%) and a catheter was placed into the jugular vein. The bladder dome wasexposedviaamidlineabdominalincision, the ureters and urethra were ligated close to the bladder, the bladder was emptied, and a catheter inserted into the bladder through the bladder dome and secured in place. The free tip of the bladder catheter was connected to an Elcomatic EM 750 pressure transducer (Elcomatic, Glasgow, Scotland, UK) for pressure monitoring and to a KDS-200 syringe pump (Phymep, Paris, France) for perfusion. The left pelvic nerve was cleared from surrounding tissue proximal to the major pelvic ganglion, and a bipolar stimulation electrode was put into position and sealed with nonreactive Wacker gel (Wacker Chemie, Munich, Germany). To minimize somatic afferent activity, thesciaticnerveandallnervesat the base of the tail were cut. Since anesthesia may confound the evaluation of nerve-firing activity, precollicular decerebration was performed by ligating both carotid arteries followed by removal of the forebrain.cottonimmersedin1%thrombinsolutionwasplacedinthe intracranial cavity and covered with agar [17,18]. Isoflurane anesthesia was then gradually discontinued. Experiments started at least 2 h after decerebration. Finally, the lumbosacral spinal cord was exposed by laminectomy (L1 S1) and the rat was suspended in a stereotaxic frame. The dura membrane was carefully removed and the spinal cord covered with warm mineral oil. The L6 dorsal root was decentralized close to its entry to the spinal cord and distally cut. Fine filaments were teased out and placed on a bipolar recording electrode. Spiking activity was recorded continuously, amplified , and was filtered using a khz bandpass filter (model 1700 AC amplifier; A-M Systems, Sequim, WA, USA) Design of experiments First, a bladder afferent fiber was identified that was both reactive to manual filling of the bladder and displayed spikes after pelvic nerve electrical stimulation (1 ms squarewave pulse at 1 8 ma). Although initially bladder afferent fibers were classified according to their conduction velocity into Ad and C fibers, with the afferent limb of the micturition reflex thought to be carried out by unmyelinated C fibers in SCI rats versus myelinated Ad fibers in normal rats, more recent reports have challenged this concept in normal rats [15,19]. Moreover, a very recent electrophysiological study demonstrated that the properties of mechanoreceptors sensitive to bladder filling are not different between both types of afferents, even in SCI rats [14]. Therefore, such differentiation was not taken into account in this study. Complete filling of the bladder at a constant flow rate with saline (10 ml/h) was performed and analyzed using Elphy data-acquisition software (CNRS, Gif sur Yvette, France) to determine the volume necessary to reach fullness for each rat (maximal bladder filling volume [BFV]) and to verify that BANF is related to the degree of bladder filling. Then the bladder was emptied and the BANF allowed to rest. Second, another filling cycle was started until 75% of the maximal BFV of that rat was reached (filling period). Saline infusion was then stopped for the bladder to stabilize for 10 min (stabilization period). Vardenafil (1 mg/kg; n = 7) or saline (n = 8) was delivered intravenously (300 ml over 3 min), and the recording continued for another 45 min (treated period) until sacrifice with an overdose of urethane (1.4 g/kg).

3 274 EUROPEAN UROLOGY 59 (2011) Data analysis 3. Results NVCs were characterized in terms of amplitude (in millimeters of mercury) and frequency (in Hertz). BANF (spikes per second) was measured and averaged during the last minute before filling (resting BANF), during the last 5 min of the stabilization period (baseline BANF), and every 5 min during the treated period. All results were expressed as mean plus or minus standard error of the mean (SEM) and as a percentage of the stabilization period where appropriate and were compared using the student t-test or two-way analysis of variance (ANOVA) followed by Bonferroni s post hoc analysis. A p value < 0.05 was considered significant. At d after spinalization, body weights ( g in saline vs g in vardenafil; nonsignificant [ns], student t-test) and mean duration of Credé maneuver were not different between the two groups of rats ( d for saline vs d for vardenafil; ns, student t-test). Resting BANF was present and already quite regular in all rats ( spikes per second) with no significant differences between rats Drugs and chemicals Vardenafil was provided by Bayer Schering AG. Antibiotics were purchased from Centravet (Plancoët, France). All other chemicals were purchased from Sigma (Lyon, France). [()TD$FIG] 3.1. Characterization of the filling phase and concomitant bladder afferent nerve firing (first step) IVP changes during filling of the bladder consisted of a slowly increasing component with transient NVCs Fig. 1 Determination of mean maximal bladder filling volume in a decerebrate, unanesthetized, spinal cord-injured rat. (A) Representative cystometrogram tracing and (B) its concomitant recording of bladder afferent nerve firing (BANF). (C) Close-up view of a cystometrogram tracing and its concomitant recording of BANF before receiving saline or vardenafil injection. IVP = intravesical pressure.

4 [()TD$FIG] EUROPEAN UROLOGY 59 (2011) Fig. 2 (A) Relationship between bladder afferent nerve firing (BANF) and intravesical pressure (IVP) and infused volume during bladder filling with saline in decerebrate, unanesthetized, spinal cord injured rats before receiving saline or vardenafil injection. (B) Effect of bladder filling with saline on BANF. Afferent spike rate is plotted against the bladder volume, which was divided into 10 equal parts for each rat. Afferent spike rate was then averaged for each part and expressed as a percentage of its resting value. Data are mean plus or minus standard error of the mean. BFV = bladder filling volume. superimposed until fullness of the bladder was achieved (Fig. 1A). Mean maximal BFV was not different between those rats that later received saline or vardenafil ( ml vs ml, respectively; ns, student t-test). For each rat, the filling phase was divided into 10 identical parts corresponding to graded degrees of bladder filling and BANF was averaged for each part. The average unitary activity was then plotted as a function of the IVP for all rats (Fig. 2). A progressive and significant increase in BANF was noted, with filling of the bladder reaching a 2.4-fold increase at maximal BFV (Figs. 1B and 2). Interestingly, the increase in the frequency of BANF was repeatedly noticeable before the occurrence of an NVC on close-up views of simultaneous recording of IVP and BANF (Fig. 1C) Effect of intravenous vardenafil on intravesical pressure and bladder afferent nerve firing To evaluate the effects of vardenafil on a non-noxious state of bladder distension, we arbitrarily chose to fill the bladders to 75% of maximal BFV and let the IVP and BANF stabilize before injecting vardenafil. This level of filling corresponded to ml in vardenafil rats versus ml in the saline group (saline rats), yielding an increase in BANF of % of resting BANF similar in both groups of rats before treatment ( spikes per second in saline vs spikes per second in vardenafil; ns, student t- test). Likewise, amplitude and frequency of NVCs were comparable before treatment (data not shown). Compared to saline, there was an immediate and significant decrease in both amplitude and frequency of NVCs after vardenafil injection ( p < 0.001, two-way ANOVA; Figs. 3, 5A, and 5B), although post hoc analysis yielded statistically significant differences only in amplitudes and not in frequencies of NVCs in vardenafil-treated rats starting at 15 min postinjection. Concomitantly, the mean spike rate of BANF also decreased significantly and very rapidly after vardenafil injection ( p < 0.001, two-way ANOVA; Figs. 3 and 5c). This decrease was not statistically different after post hoc analysis at any time point. After 45 min of observation, BANF had decreased by 28% after

5 276 [()TD$FIG] EUROPEAN UROLOGY 59 (2011) Fig. 3 Representative cystometrogram tracing and its concomitant recording of bladder afferent nerve firing (BANF) in a decerebrate, unanesthetized, spinal cord injured rat after (A) vardenafil 1 mg/kg or (B) saline intravenous injection.

6 [()TD$FIG] EUROPEAN UROLOGY 59 (2011) Fig. 4 Effect of vardenafil (VAR) 1 mg/kg and saline intravenous injection on bladder compliance after submaximal filling of the bladder in unanesthetized, decerebrate, spinal cord-injured rats. Data are mean plus or minus standard error of the mean. saline injection compared to 52% after vardenafil injection. Importantly, the absence of confounding influence of a change in bladder compliance was verified after vardenafil injection (ns, two-way ANOVA; Fig. 4). 4. Discussion In the present study, acute intravenous (IV) vardenafil 1 mg/kg strongly reduced both NVCs and BANF elicited by submaximal filling of the bladder in unanesthetized, decerebrate, SCI rats. Sensation associated with urinary bladder filling is conveyed primarily by the pelvic and hypogastric nerves to the spinal cord. Most afferent fibers innervating the detrusor pass through the pelvic nerve, whereas most afferent endings in the bladder submucosa are derived from the hypogastric nerve [20]. These two sets of afferent fibers thus have different signaling roles: mechanical stimulation (eg, bladder distension) and chemical stimulation (eg, inflammation) [21,22]. In the present study, BANF was recorded from the L6 dorsal root since 84% of the pelvic afferent fibers from the lower urinary tract enter the spinal cord via the L6 dorsal root [23]. Mechanosensitive afferent fibers in the urinary bladder were first described in multiunit recordings in cats and dogs [24 26] as sensitive to bladder distension with periodic BANF bursts associated to detrusor contraction, thereby highlighting the close relationship between BANF and IVP. In the present study, it was repeatedly observed that an increase of BANF preceded an NVC on close-up views of simultaneous recording of IVP and BANF. This aspect requires further investigation. Nonetheless, a recent study has also documented the increase in sensitivity of the afferent fibers to mechanical stimulation in the anesthetized SCI rat in concert with NVCs [14]. This corroborates the present results where a close relationship between the degree of filling of the bladder and BANF was evidenced in decerebrate, unanesthetized, SCI rats. In the present study, BANF in unanesthetized, decerebrate, SCI rats was recorded. Interestingly, in all SCI rats tested, BANF was present even at resting conditions. Unfortunately, the comparison with control rats in the same experimental conditions (eg, unanesthetized and decerebrate) was not possible since such data are not available. Nonetheless, it has been reported that urethaneanesthetized control rats display a resting BANF of one to five spikes per second [27]. Although not in the scope of the present study, it would have been of utmost interest to demonstrate an increased resting BANF in decerebrate, unanesthetized, SCI rats compared to normal rats to confirm the Iijima et al study in anesthetized SCI rats [14]. Indeed, the volume of BANF has been described very recently as afferent noise [19]. If, as suspected, BANF indeed is increased in SCI rats compared to control rats, it would corroborate this concept of increased bladder afferent noise. Such an increase could also be suspected in patients with SCI and could represent by itself a valid therapeutic target. In fact, the transduction from mechanical bladder stretch into afferent potentials is very complex and might involve different steps and mediators, including NO. These mediators exert their effect directly on afferent endings or indirectly by acting on the suburothelial myofibroblasts, thus modulating bladder afferent input through their communications with the urothelium, the afferent nerve endings, and the detrusor smooth muscle. The response to stretch involving NO has been shown to exert inhibitory effects on the micturition reflex in normal rats and in chronic models of bladder inflammation [7 13], suggesting that NO may modulate the micturition reflex by reducing the excitability of bladder afferents. However, this is the first report evidencing a direct inhibiting effect of a PDE5-I, vardenafil, on both BANF and occurrence of NVCs in SCI rats. Interestingly, our findings are in direct line with the results from Gacci et al reporting urodynamic benefits of acute vardenafil in SCI patients with NDO [5]. Moreover, PDE5-Is were shown to improve storage symptoms in men with LUTS/BPH [1 3]. It would be very tempting to verify whether modulation of BANF by vardenafil can also occur in different physiopathology models, such as rats with bladder outlet obstruction (BOO). Indeed, efficacy of vardenafil in reducing the frequency of NVCs in this model was recently reported [28,29]. This indicates that vardenafil might also be able to exert such an inhibitory effect on BANF in the BOO model. Such results could also help understand the differences in efficacy of PDE5-Is noted in clinical studies depending on the patient population (patients with BPH, overactive bladder, or NDO). Since the emergence of NVCs in SCI rats is associated to bladder afferent fiber hyperexcitability [14,15], we propose that vardenafil decreases the SCI-induced NDO by exerting an inhibitory effect on the hyperexcitability of bladder afferent fibers, besides its proposed relaxing effect on the detrusor [6]. Indeed, although the effect on NVCs appears to be more drastic and occurs earlier, it may well be that only a small reducing effect on the frequency of BANF is sufficient to go below the threshold level necessary to induce NVCs. Hence, although the extent of the effect on BANF seems to be more progressive than on NVCs, these results may

7 278 [()TD$FIG] EUROPEAN UROLOGY 59 (2011) support the notion that vardenafil exerts its effect on BANF, with a resulting drastic effect seen on the NVCs. However, it is to be noted that vardenafil 1 mg/kg IV also leads to a significant drop in blood pressure, which may also directly influence the micturition cycle, as previously indicated [30]. This hypothesis has recently been confirmed in spontaneously hypertensive rats [31]. It would be worthwhile exploring whether a sudden drop in blood pressure due to vardenafil also yields an immediate drop in bladder blood flow and subsequent inhibition of NVCs in SCI rats as well. 5. Conclusions We show for the first time that vardenafil reduces both NVCs and BANF elicited by submaximal bladder filling in unanesthetized, decerebrate, SCI rats. These findings provide new insights into the mechanism of action by which PDE5-Is improve storage symptoms in SCI patients. Conflicts of interest Author contributions: François Giuliano had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Behr-Roussel, Oger. Acquisition of data: Caisey, Bernabé. Analysis and interpretation of data: Behr-Roussel, Bernabé. Drafting of the manuscript: Behr-Roussel. Critical revision of the manuscript for important intellectual content: Giuliano, Sandner. Statistical analysis: Behr-Roussel. Obtaining funding: Giuliano, Sandner, Alexandre. Administrative, technical, or material support: Bernabé. Supervision: Bernabé, Giuliano. Other (specify): None. Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: P. Sandner is an employee of Bayer Schering Pharma. Funding/Support and role of the sponsor: Bayer Schering Pharma supported this study with a restricted grant and approved the manuscript. Acknowledgment statement: The authors gratefully acknowledge the technical advice of Dr. Mitsuharu Yoshiyama and Dr. Naoki Aizawa. Fig. 5 Effect of vardenafil 1 mg/kg and saline intravenous injection on (A)amplitudeand(B)frequencyofnonvoiding contractions and (C) bladder afferent nerve firing after submaximal filling of the bladder in unanesthetized, decerebrate, spinal cord-injured rats. Data are mean plus or minus standard error of the mean. ***p < 0.001, two-way analysis of variance. # p < ## p < ### p < 0.001, Bonferroni s post hoc analysis. References [1] McVary KT, Monnig W, Camps Jr JL, et al. Sildenafil citrate improves erectile function and urinary symptoms in men with erectile dysfunction and lower urinary tract symptoms associated with benign prostatic hyperplasia: a randomized, double-blind trial. J Urol 2007; 177: [2] Roehrborn CG, McVary KT, Elion-Mboussa A, Viktrup L. Tadalafil administered once daily for lower urinary tract symptoms second-

8 EUROPEAN UROLOGY 59 (2011) ary to benign prostatic hyperplasia: a dose finding study. J Urol 2008;180: [3] Stief CG, Porst H, Neuser D, Beneke M, Ulbrich E. A randomised, placebo-controlled study to assess the efficacy of twice-daily vardenafil in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia. Eur Urol 2008;53: [4] Tamimi NA, Mincik I, Haughie S, et al. A placebo-controlled study investigating the efficacy and safety of the phosphodiesterase type 5 inhibitor UK-369,003 for the treatment of men with lower urinary tract symptoms associated with clinical benign prostatic hyperplasia. BJU Int 2010;106: [5] Gacci M, Del Popolo G, Macchiarella A, et al. Vardenafil improves urodynamic parameters in men with spinal cord injury: results from a single dose, pilot study. J Urol 2007;178: [6] Oger S, Behr-Roussel D, Gorny D, et al. Signalling pathways involved in sildenafil-induced relaxation of human bladder dome smooth muscle. Br J Pharmacol 2010;160: [7] Burnett AL, Calvin DC, Chamness SL, et al. Urinary bladder-urethral sphincter dysfunction in mice with targeted disruption of neuronal nitric oxide synthase models idiopathic voiding disorders in humans. Nat Med 1997;3: [8] Masuda H, Kim JH, Kihara K, et al. Inhibitory roles of peripheral nitrergic mechanisms in capsaicin-induced detrusor overactivity in the rat. BJU Int 2007;100: [9] Ozawa H, Chancellor MB, Jung SY, et al. Effect of intravesical nitric oxide therapy on cyclophosphamide-induced cystitis. J Urol 1999; 162: [10] Persson K, Igawa Y, Mattiasson A, Andersson KE. Inhibition of the arginine/nitric oxide pathway causes bladder hyperactivity in the rat. Acta Physiol Scand 1992;144: [11] Sasatomi K, Hiragata S, Miyazato M, et al. Nitric oxide-mediated suppression of detrusor overactivity by arginase inhibitor in rats with chronic spinal cord injury. Urology 2008;72: [12] Caremel R, Oger-Roussel S, Behr-Roussel D, Grise P, Giuliano FA. Nitric oxide/cyclic guanosine monophosphate signalling mediates an inhibitory action on sensory pathways of the micturition reflex in the rat. Eur Urol 2010;58: [13] Yoshimura N, Seki S, de Groat WC. Nitric oxide modulates Ca(2+) channels in dorsal root ganglion neurons innervating rat urinary bladder. J Neurophysiol 2001;86: [14] Iijima K, Igawa Y, Wyndaele JJ, De Wachter S. Mechanosensitive primary bladder afferent activity in rats with and without spinal cord transection. J Urol 2009;182: [15] De Groat WC, Yoshimura N. Changes in afferent activity after spinal cord injury. Neurourol Urodyn 2010;29: [16] Salomon J, Denys P, Merle C, et al. Prevention of urinary tract infection in spinal cord-injured patients: safety and efficacy of a weekly oral cyclic antibiotic (WOCA) programme with a 2 year follow-up an observational prospective study. J Antimicrob Chemother 2006;57: [17] Sapru HN, Krieger AJ. Procedure for the decerebration of the rat. Brain Res Bull 1978;3: [18] Yoshiyama M, de Groat WC. Role of spinal metabotropic glutamate receptors in regulation of lower urinary tract function in the decerebrate unanesthetized rat. Neurosci Lett 2007;420: [19] Gillespie JI, van Koeveringe GA, de Wachter SG, de Vente J. On the origins of the sensory output from the bladder: the concept of afferent noise. BJU Int 2009;103: [20] Uemura E, Fletcher TF, Bradley WE. Distribution of lumbar and sacral afferent axons in submucosa of cat urinary bladder. Anat Rec 1975;183: [21] Mitsui T, Kakizaki H, Matsuura S, et al. Afferent fibers of the hypogastric nerves are involved in the facilitating effects of chemical bladder irritation in rats. J Neurophysiol 2001;86: [22] Moss NG, Harrington WW, Tucker MS. Pressure, volume, and chemosensitivity in afferent innervation of urinary bladder in rats. Am J Physiol 1997;272:R [23] Vera PL, Nadelhaft I. Conduction velocity distribution of afferent fibers innervating the rat urinary bladder. Brain Res 1990;520: [24] Evans JP. Observations on the nerves of supply to the bladder and urethra of the cat, with a study of their action potentials. J Physiol 1936;86: [25] Talaat M. Afferent impulses in the nerves supplying the urinary bladder. J Physiol 1937;89:1 13. [26] Downie JW, Armour JA. Mechanoreceptor afferent activity compared with receptor field dimensions and pressure changes in feline urinary bladder. Can J Physiol Pharmacol 1992;70: [27] Sengupta JN, Gebhart GF. Mechanosensitive properties of pelvic nerve afferent fibers innervating the urinary bladder of the rat. J Neurophysiol 1994;72: [28] Filippi S, Morelli A, Sandner P, et al. Characterization and functional role of androgen-dependent PDE5 activity in the bladder. Endocrinology 2007;148: [29] Tinel H, Stelte-Ludwig B, Hutter J, Sandner P. Pre-clinical evidence for the use of phosphodiesterase-5 inhibitors for treating benign prostatic hyperplasia and lower urinary tract symptoms. BJU Int 2006;98: [30] Greenland JE, Brading AF. The effect of bladder outflow obstruction on detrusor blood flow changes during the voiding cycle in conscious pigs. J Urol 2001;165: [31] Morelli A, Filippi S, Comeglio P, et al. Acute vardenafil administration improves bladder oxygenation in spontaneously hypertensive rats. J Sex Med 2010;7: