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Keywords:

  • afferent;
  • onabotulinumtoxinA;
  • Botox;
  • urothelium;
  • sensory

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Objective

  • To investigate the direct effect of onabotulinumtoxinA (OnaBotA) on bladder afferent nerve activity and release of ATP and acetylcholine (ACh) from the urothelium.

Materials and Methods

  • Bladder afferent nerve activity was recorded using an in vitro mouse preparation enabling simultaneous recordings of afferent nerve firing and intravesical pressure during bladder distension.
  • Intraluminal and extraluminal ATP, ACh, and nitric oxide (NO) release were measured using the luciferin–luciferase and Amplex® Red assays (Molecular Probes, Carlsbad, CA, USA), and fluorometric assay kit, respectively.
  • OnaBotA (2U), was applied intraluminally, during bladder distension, and its effect was monitored for 2 h after application.
  • Whole-nerve activity was analysed to classify the single afferent units responding to physiological (low-threshold [LT] afferent <15 mmHg) and supra-physiological (high-threshold [HT] afferent >15 mmHg) distension pressures.

Results

  • Bladder distension evoked reproducible pressure-dependent increases in afferent nerve firing.
  • After exposure to OnaBotA, both LT and HT afferent units were significantly attenuated.
  • OnaBotA also significantly inhibited ATP release from the urothelium and increased NO release.

Conclusion

  • These data indicate that OnaBotA attenuates the bladder afferent nerves involved in micturition and bladder sensation, suggesting that OnaBotA may exert its clinical effects on urinary urgency and the other symptoms of overactive bladder syndrome through its marked effect on afferent nerves.

Abbreviations
OnaBotA

onabotulinumtoxinA

ACh

acetylcholine

NO

nitric oxide

LT

low threshold

HT

high threshold

OAB

overactive bladder syndrome

SNAP-25

synaptosomal-associated protein 25

SV2

synaptic vesicle protein 2

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Overactive bladder syndrome (OAB) is characterized by symptoms of urgency and frequency, which may also be associated with urgency incontinence. It is an important clinical disorder which adversely affects quality of life. Current evidence would suggest that, contrary to conventional thought, OAB and associated detrusor overactivity results from either an increase in peripheral afferent stimulation in the lower urinary tract or an altered central threshold for afferent impulses [1]. It is therefore critically important to understand the role that afferent pathways play in the function of the lower urinary tract.

OnabotulinumtoxinA (OnaBotA) is a potent neurotoxin that has been shown in striated muscle to inhibit vesicular release of neurotransmitters at cholinergic neuromuscular junctions by preventing vesicle docking and fusion at the nerve terminal. This action is conducted via the proteolytic cleavage of the SNARE protein synaptosomal-associated protein 25 (SNAP-25), and results in the inhibition of muscle contraction [2]. OnaBotA was first used in the urinary tract to treat the detrusor-sphincter dyssynergia associated with spinal cord injury [3], and it was expected that it would act in a similar fashion in the bladder for the treatment of OAB; however, OnaBotA has been shown to alleviate the sensory symptom of urgency associated with OAB. Furthermore, when OnaBotA is used clinically, it has an optimum safety/efficacy ratio at lower doses than initially thought [4]. This evidence leads one to speculate that much of the useful clinical efficacy of OnaBotA is attributable to its action on the afferent system, with higher doses acting to a greater extent on neuromuscular transmission to impair bladder contractility.

Previous functional studies in rodents have shown that OnaBotA inhibits the release of calcitonin gene-related peptide and substance P from afferent nerve terminals [5] and reduces ATP release from the urothelium in a spinal cord injury model of detrusor overactivity [6-8]. In the present study, we investigated the direct action of OnaBotA on afferent nerve responses to filling in an intact mouse bladder and measured concomitant release of ATP and acetylcholine (ACh) from the urothelium.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Animals

We humanely killed 8–12-week-old C57/BL6 male mice by cervical dislocation. The maintenance and killing of the animals followed principles of good laboratory practice in compliance with UK laws and regulations.

Preparation and Nerve Recording

Nerve recording was conducted using an in vitro model previously described [9] (Fig. 1A,B). The whole pelvic region was dissected and placed in a recording chamber (30 mL) which was continually perfused with gassed (95% O2, 5% CO2) Krebs-bicarbonate solution (NaCl 118.4 mMol/L, NaHCO3 24.9 mMol/L, CaCl2 1.9 mMol/L, MgSO4 1.2 mMol/L, KCl 4.7 mMol/L, KH2PO4 1.2 mMol/L, glucose 11.7 mMol/L) at 35oC. The urethra was catheterized and attached to a pump, enabling the controlled distension of the bladder with saline. The dome was catheterized using a dual-lumen cannula (30 G) to enable recording of intravesical pressure and allow evacuation of fluid. The multi-unit afferent nerve tracts, consisting of pelvic and hypogastric nerves, were dissected into fine branches, cut distal to the bladder and placed into a suction electrode for recording. The electrical activity was recorded by a neurologue headstage (NL100, Digitimer Ltd, Welywn Garden City, UK), amplified, filtered (NL215, band pass 300–4000 Hz) and captured by a computer via a power 1401 interface and Spike 2 software (version 7, Cambridge Electronic Design, UK).

figure

Figure 1. In vitro murine model to measure afferent nerve activity and intravesical pressure. (A) Photographs showing dissection of mouse bladder afferent nerve bundles after catheterization of the urethra and bladder dome in vitro. (B) Schematic representation of the in vitro mouse bladder model. (C) A representative trace shows the response of a multi-unit afferent nerve bundle to ramp distension of the bladder: (i) histogram of nerve firing frequency; (ii) raw nerve trace; (iii) intravesical pressure in response to ramp distension of the bladder with isotonic saline (NaCl 0.9%) at a rate of 100 μL/min. As the bladder is filled intravesical pressure and afferent nerve firing frequency rise concurrently.

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Reconstitution of OnaBotA

OnaBotA (Allergan, Irvine, CA, USA) was reconstituted using sterile saline (0.9% NaCl) to a stock concentration of 100U/mL. The solution was gently inverted, aliquoted and stored at −20°C (<2 months).

Afferent Responses to OnaBotA

Preparations were equilibrated for 30 min. Control distensions were performed using isotonic saline (0.9%) at 100 μL/min to a maximum pressure of 40 mmHg. OnaBotA (2U/bladder) was applied intraluminally during one distension phase (during filling and emptying of the bladder), and subsequent distensions were conducted using isotonic saline. Afferent responses to distension were recorded before perfusion of OnaBotA (0 min) and every 10 min for >2 h, after intraluminal perfusion with OnaBotA. Time-matched vehicle control experiments were conducted using the same protocol. Bladder compliance (used as a measure of detrusor muscle tone) was gauged by the pressure–volume relationship.

ATP and ACh Measurements

Isolated whole bladders were catheterized via the urethra, with a dual-lumen cannula (21 G), placed in a small organ bath (volume 500 μL) with fresh oxygenated Krebs solution (35°C) and distended (0.9% NaCl, buffered to pH 7.4) at a rate of 100 μL/min to a maximum pressure of 40 mmHg. After distension, both extraluminal (total bath fluid) and intraluminal fluids were immediately collected and stored at -80°C. Because of natural variation in bladder size the volume of the intraluminal samples was different for each bladder. Distensions were performed every 10 min for >2 h, after intraluminal perfusion with OnaBotA (2U/bladder) and intraluminal and extraluminal samples were collected. Time-matched vehicle control experiments were conducted using the same protocol. The amount of ATP and ACh in the samples was determined using the luciferin–luciferase ATP bioluminescent assay kit (Sigma-Aldrich, Dorset, UK) and the Amplex® Red ACh assay kit (Molecular Probes, Paisley, UK). To calculate absolute transmitter release, the amounts detected in the samples were corrected for total bladder volume and time.

Nitric Oxide Measurements

Intraluminal samples were collected using the model described above. Samples from untreated bladders and from OnaBotA-treated bladders were taken at the same timepoints (60 min after application of OnaBotA or vehicle). Nitric oxide (NO) release was assessed as the ratio of nitrite/nitrate concentration using a commercially available fluorometric assay kit and following the manufacturer's instructions. All data were corrected for bladder size (volume and filling time).

Data Analysis and Statistics

Whole-nerve multifibre afferent nerve activity was quantified using a Digitimer D130 spike processor (Digitimer Ltd), which counted the number of action potentials crossing a pre-set threshold. Single-unit discrimination was performed offline to identify the individual single units in each preparation. Low threshold (LT) afferents were defined as responding to pressures <15 mmHg, while units with thresholds >15 mmHg were considered high threshold (HT).

Data are presented as mean (sem) values. Statistical analysis was carried out using either a one- or two-way anova and Bonferroni post-test or a Student's t-test, where appropriate, and a P-value <0.05 was considered to indicate statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Ramp distension of the bladder evoked a graded increase in intravesical pressure and afferent nerve discharge (Fig. 1C). Preparations were stable for >6 h and repeated distension elicited reproducible afferent response profiles. Application of OnaBotA (2U/bladder, n = 11) caused a graded time-dependent inhibition in the afferent response to bladder filling compared with vehicle and time-matched control experiments (n = 8). At 120 min after application, afferent nerve discharge was inhibited by > 60% (Figs 2, 3).

figure

Figure 2. Intraluminal perfusion of OnaBotA significantly attenuates bladder mechanosensitivity. Representative trace showing the change in the afferent response profile after intravesical perfusion of OnaBotA (2U/bladder).

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figure

Figure 3. OnaBotA significantly inhibits both LT and HT bladder afferent nerves. (A) The afferent response to distension of the bladder was significantly inhibited, by > 60%, 2 h after application of OnaBotA (P<0.001, two-way anova and Bonferroni post-test, n = 11). (B) Time-matched vehicle control experiments showed no change in firing over a 2-h time period (n = 8). (C) The afferent–response relationship (measured as area under the curve) was significantly attenuated over time in experiments treated with OnaBotA compared with time-matched vehicle controls (P < 0.001, one-way anova and Bonferroni post-test). (D) Single-unit analysis identified 41 LT and 26 HT afferents. At 2 h after application of OnaBotA, the LT and HT populations were inhibited by 69% (P < 0.001) and 65% (P<0.004), respectively.

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Single-unit analysis was conducted to identify which sub-populations of mechanosensitive afferents were affected. 41 LT and 26 HT afferent units were confidently identified (from eight preparations). After application of OnaBotA, the afferent response profiles of both populations were significantly inhibited: by 69% (P < 0.001) and 65% (P < 0.004) for the LT and HT afferent units, respectively (Fig. 3D).

To evaluate the effect of OnaBotA on detrusor muscle tone, bladder compliance was assessed by analysing the pressure–volume relationship during distension. Bladder compliance did not significantly change over time in control preparations or in preparations treated with OnaBotA (Fig. 4).

figure

Figure 4. OnaBotA had no significant effect on bladder compliance. Bladder compliance was gauged by the pressure–volume relationship. (A) Compliance was not significantly altered by the application of OnaBotA (n = 11). (B) Time-matched control experiments also showed no change compliance over a 2-h period (n = 8).

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ATP was consistently detected in both intraluminal and extraluminal samples, with a release ranging from 2 to 75 pmole/min. In time-matched control experiments, the amount of ATP released in both luminal and extraluminal samples did not significantly change over time (Fig. 5A,D); however, in preparations treated with OnaBotA, there was a significant attenuation in intraluminal ATP release at 60 and 120 min after application (P > 0.001; Fig. 5B,C). Extraluminal ATP release was not altered after the application of OnaBotA (Fig. 5E,F).

figure

Figure 5. OnaBotA inhibits ATP release from the urothelium. Using the luciferase–luciferin assay, ATP was detected in both intraluminal and extraluminal samples. (A) In control experiments, intraluminal ATP release was not significantly altered over time (0–120 min, n = 11). (B) In preparations treated with OnaBotA (2U/bladder), ATP release was significantly inhibited at 60 and 120 min after application (P<0.001, one-way anova with Bonferroni post-test, n = 14). (C) Percentage change in intraluminal ATP release relative to time-matched vehicle control experiments. OnaBotA significantly reduced the intraluminal ATP release: by >40% (120 min after treatment, P<0.01). In contrast, extraluminal ATP release was not significantly affected by the application of OnaBotA. (D) Time-matched vehicle control experiments showing no change in ATP release over time (0–120 min, n =11). (E) In preparations treated with OnaBotA (2U/bladder) extraluminal ATP release was not significantly altered (n = 14). (F) Percentage change in extraluminal ATP release relative to time-matched vehicle control experiments. OnaBotA did not affect extraluminal ATP release relative to control experiments.

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The release of ACh was measured indirectly by measuring the breakdown product choline using the Amplex Red ACh/acetylcholinesterase assay kit. ACh was detected in both intraluminal and extraluminal samples. The amount of ACh detected was highly variable with a range of 30.83–839.40 pmole/min. In time-matched control experiments the amount of ACh released in intraluminal samples did not significantly change over time (Fig. 6A); however, in preparations treated with OnaBotA there was a significant attenuation in intraluminal ACh release at 60 and 120 min after application (P > 0.001 Fig. 6B,C). Surprisingly, when the proportional change was examined (%Δ release, treated vs untreated) no significant effect was observed between time-matched controls and experiments where OnaBotA was applied, suggesting that intraluminal ACh release was not significantly affected (Fig. 6C). Similarly, extraluminal ACh release was significantly reduced over time in both treated and untreated groups (Fig. 6D, E). Application of OnaBotA had no significant effect on extraluminal ACh compared with controls (Fig. 6F).

figure

Figure 6. OnaBotA moderately inhibited intraluminal and extraluminal ACh release. Using the Amplex Red ACh assay, ACh was detected in both intraluminal and extraluminal samples. (A) In control experiments, intraluminal ACh release was not significantly altered over time (0–120 min, n = 15). (B) In preparations treated with OnaBotA (2U/bladder) ACh release was significantly inhibited at 60 and 120 min after application (P < 0.001, one-way anova with Bonferroni post-test, n = 14). (C) Percentage change in intraluminal ACh release relative to time-matched vehicle control experiments. (D) Time-matched vehicle control experiments showing a small but significant reduction in extraluminal ACh release over time (0–120 min, one-way anova, n = 17). (E) In preparations treated with OnaBotA (2U/bladder) extraluminal ACh release was also significantly attenuated at 60 and 120 min after application (both P<0.001, n = 14). (F) Percentage change in extraluminal ACh release relative to time-matched vehicle control experiments. OnaBotA did not affect extraluminal ACh release relative to control experiments.

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Nitric oxide was detected in 5 out of 8 samples from untreated bladders and in all of the samples from OnaBotA-treated bladders at 60 min after application (n = 8). Mean (sem) NO release in samples from untreated bladders was 47.6 ± 18.26 pmole/min, but in samples from OnabotA-treated bladders NO release was significantly greater at 239.7 ± 64.81 pmole/min (Figure 7, P < 0.01).

figure

Figure 7. OnaBotA increases NO release from the urothelium. Using the BioVision assay, NO was detected in 62% samples from untreated bladders and 100% samples from OnaBotA-treated bladders. Treatment with 2U OnaBotA (1 h after application) significantly increased NO release from the urothelium compared with time-matched vehicle control experiments (P < 0.01).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Afferent information from the bladder is conveyed by the pelvic and hypogastric afferent nerves arising from the lumbrosacral and thoracolumbar dorsal root ganglia. These nerves consist of myelinated Aδ-fibres, involved in normal micturition, and unmyelinated C-fibres, responsible for painful sensations. In addition to sensory nerves, it is now clearly established that the urothelium and suburothelium actively contribute to bladder sensation. This modulation of underlying afferent nerves during filling is likely to be influenced by the non-neuronal release of neurotransmitters such as ACh, NO and ATP [10, 11].

In recent years, transurothelial injections of OnaBotA have proven to be a highly effective treatment for storage symptoms in patients with neurogenic detrusor overactivity and idiopathic OAB [12]. In the present study, we tested the hypothesis that OnaBotA in clinically effective doses may not work simply by blocking parasympathetic-mediated ACh release at the neuromuscular junction within the bladder, thereby inhibiting muscle contraction, but can also act directly on the afferent system to improve the symptoms of OAB.

Our findings suggest that OnaBotA inhibits bladder afferent firing but does not have an effect on bladder compliance. Bladder distension in this model system increased afferent nerve firing concurrently with the increases in pressure. This can be attributed to the recruitment of different populations of mechanosensitive afferent nerve units, as previously described [13, 14]. We have shown that intraluminal perfusion of the bladder by an OnaBotA-containing solution during one ramp distension resulted in a graded inhibition of firing which reached significance 2 h after application. This is in contrast to our control experiments using vehicle where reproducible distension-response profiles were obtained throughout the duration of the protocol (>2 h). Interestingly, both LT and HT afferent nerve populations were inhibited, suggesting that afferent sub-types responding to both normal bladder filling and nociceptive stimuli were affected similarly. The potential clinical implication of this is that if this finding is extrapolated to humans, then OnaBotA may have therapeutic applications in painful bladder syndrome as has been suggested in preliminary clinical studies [15].

In the present study, changes in sensory signalling were observed after a relatively short period of exposure, compared with the clinical situation where patients often report that the effects of OnaBotA may take a few days to become manifest; however, this finding could be influenced by the physiochemical factors related to penetration of the urothelium by OnaBotA, as opposed to its injection directly into the suburothelial space. In a previous study in which OnaBotA was directly injected into the mouse bladder wall, Ikeda et al. [16] also observed an attenuated afferent nerve and altered efferent nerve activity after a 48-h time period; however, their recordings were limited to pelvic nerves and failed to distinguish between different populations of mechanosensitive afferents, and the impact of OnaBotA on afferent nerves responding to bladder filling. Moreover, the marked effects on efferent function raise the possibility that the changes they observed could have been secondary to altered muscle tone and compliance. In the present study, the tone of the detrusor muscle was unaffected by OnaBotA application after 2 h.

The absence of any effect of OnaBotA on the pressure–volume relationship is consistent with experimental studies which have demonstrated that OnaBotA treatment results in a reduced expression of spinal c-Fos but had no effect on contraction amplitude in a spinal cord injury model or in muscle strip experiments [17, 18]. This suggests that OnaBotA can act on sensory transduction from the bladder with little direct effect on the detrusor muscle neuro-muscular junctions.

The existing literature shows that OnaBotA selectively binds to a high-affinity binding site, synaptic vesicle protein 2 (SV2) located on presynaptic terminals, where it is taken up and initiates proteolytic cleavage of the SNARE protein SNAP-25. This prevents docking and fusion of synaptic vesicles at the neuromuscular junction and blocks the subsequent release of ACh, inhibiting muscle contraction [2]; however, recent studies have also suggested that OnaBotA can inhibit afferent and efferent nerve activity [16, 19]. These studies, together with the results of the present study, seem to show there is little doubt that OnaBotA inhibits afferent nerve firing, but the exact mechanism by which this occurs is unclear. It is likely to be either by directly inhibiting mechanotransduction at the level of the afferent terminal, or by acting on the urothelium to modulate mediator release, which may then influence afferent firing indirectly.

SNAP-25 immunoreactive nerve fibres have been identified in the muscle and urothelial layers of the bladder, and an increase in immunostaining for cleaved SNAP-25 2 weeks after injection with OnaBotA has been reported [20]. Recent preliminary data have suggested that SV2 may be present on the plasma membranes of human urothelial cells [21], but this is still controversial as the available studies so far have been unable to identify SNAP-25 or SV2 on urothelial cells from guinea pigs and humans [20, 22], suggesting that OnaBotA targets neuronal fibres rather than the urothelium. The data we report, however, would suggest that afferent firing was inhibited as a secondary effect as a result of altered transmitter release from the urothelium. We found that the decrease in mechanosensitive nerve activity was concomitant with a reduction in ATP release and an increase in NO release from the urothelium. Whether this finding reflects a selective effect on the urothelium or is a result of the fact that the OnaBotA was applied intraluminally is still unclear. Surprisingly, OnaBotA had no significant effect on ACh release. Previous work from Hanna-Mitchell et al. [23] showed that ATP is released from the urothelium in vesicles, but that ACh release is not via a vesicular mechanism, which could explain why OnaBotA had a significant effect on ATP but not ACh release in the present study.

It has been postulated previously that excitatory and inhibitory transmitters are released from the urothelium in balance and that this balance modulates afferent activity [6]. Our data suggest that application of OnaBotA alters this balance, increasing NO (inhibitory mediator) and decreasing ATP (excitatory mediator) release from the urothelium, this shift seems to drive altered neuronal function and causes decreased mechanosensation.

Whilst in clinical practice OnaBotA is injected into the bladder wall, in the present study, OnaBotA was instilled into the bladder lumen as the bladder was distended. Surprisingly, this application was sufficient to elicit an effect, indicating that it was able to traverse the urothelial barrier in the mouse. It has been reported that distension of the bladder alters the exocytic/endocytic activities at the apical membrane of umbrella cells lining the urothelium. These dynamic changes in the surface area of the membrane are thought to help the bladder cope physiologically with increasing urine volume during the storage phase [24]. It is possible that OnaBotA uses this mechanism to cross the urothelial barrier and enter the cells. This could provide an explanation for the rapid onset of response seen in the present study compared with the latency of response observed in clinical studies where the toxin is injected transurothelially. Intravesical instillation has previously been suggested as an alternative method of delivery [25, 26], but no study has adequately investigated whether intravesical administration in humans without injection is sufficient for the agent to cross the urothelial barrier. The data presented in the present study provide evidence that this may be possible, although further experiments are required to assess the efficacy and safety of this approach before application in humans. There may also be a potential concern with this approach regarding the doses of OnaBotA needed and the lack of predictability of the dose–response curve.

In conclusion, the present study provides clear evidence that OnaBotA attenuates both the LT and HT afferent nerve populations involved in micturition and pain sensation and significantly inhibits ATP release from the urothelium. The site of action remains to be elucidated, but these data may provide an insight into an important mechanism by which OnaBotA exerts its effects on OAB symptoms in clinical practice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

We would like to thank Allergan for providing us with OnaBotA for this project. D.D. was funded by an unrestricted scientific grant from Allergan, V.C. was funded by a Pfizer research grant, M.L. was funded by a Marie Curie training network fellowship (TRUST).

Conflict of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

C.C. is an advisor, consultant and researcher for Astellas, Pfizer, Allergan and Recordati and an advisor for Lilly, ONO and Xention.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References