Proposed Mechanism for the Efficacy of Injected Botulinum Toxin in the Treatment of Human Detrusor Overactivity

european urology 49 (2006) 644–650

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Review - Neuro-urology – Voiding Dysfunction

Proposed Mechanism for the Efficacy of Injected Botulinum Toxin in the Treatment of Human Detrusor Overactivity Apostolos Apostolidis a, Prokar Dasgupta a,b, Clare J. Fowler a,* a b

Department of Uro-Neurology, the National Hospital for Neurology and Neurosurgery, London, UK Department of Urology, Guys’ and St. Thomas’ Hospitals and GKT Medical School, London, UK

Article info

Abstract

Article history: Accepted December 5, 2005 Published online ahead of print on January 4, 2006

Background: Treatment of human bladder overactivity with intradetrusor Botulinum-A neurotoxin (BoNT/A) injections temporarily blocks the presynaptic release of acetylcholine from the parasympathetic innervation and produces a paralysis of the detrusor smooth muscle. The efficacy of the treatment exceeds that expected from simple detrusor muscle paralysis, however, and its effect of reducing urgency is greatly welcomed by patients. Objectives: To examine whether BoNT/A has a complex effect on sensory mechanisms by inhibiting vesicular release of multiple excitatory neurotransmitters by urothelial and suburothelial nerves and reducing axonal expression of SNARE-complex dependant proteins. Methods: A literature review. Conclusions: We propose that a primary peripheral effect of BoNT/A is the inhibition of release of acetylcholine, ATP, substance P, and reduction in the axonal expression of the capsaicin and purinergic receptors. This may be followed by central desensitization through a decrease in central uptake of substance P and neurotrophic factors. The summation of these effects is a profound and long-lasting inhibition of those afferent and efferent mechanisms that are thought to be the pathophysiological basis for DO. # 2005 Elsevier B.V. All rights reserved.

Keywords: Botulinum toxin Capsaicin Cholinergic Detrusor ovaractivity Overactive bladder Purinergic Receptor Suburothelium Urothelium

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* Corresponding author. The National Hospital for Neurology and Neurosurgery, Department of Uro-Neurology, Internal Mailbox 71, Queen Square, London WC1N 3BG, UK. Tel. +44 20 7837 3611x3418; Fax: +44 20 7813 4587. E-mail address: [email protected] (C.J. Fowler).

1. Botulinum toxin injections in the overactive human bladder: clinical aspects Over the past 6 years, the use of Botulinum-A neurotoxin (BoNT/A), the most potent poison known

to man, has been pioneered in the treatment of lower urinary tract symptoms (LUTS) such as frequency and urgency incontinence due to intractable neurogenic (NDO) or idiopathic (IDO) overactivity of the detrusor smooth muscle of the

0302-2838/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2005.12.010

european urology 49 (2006) 644–650

bladder. The treatment was introduced on the theory that BoNT/A would temporarily block the presynaptic release of acetylcholine (ACh) from the parasympathetic innervation and produce a paralysis of the detrusor smooth muscle [1], comparable to its mode of action in skeletal muscle [2]. However, BoNT/A injections have been shown to increase bladder capacity, volume at first reflex detrusor contraction, and bladder compliance [3,4] as well as to induce changes in detrusor function with decreases in detrusor pressures during bladder filling and voiding [3–5]. These urodynamic changes underlie the remarkable symptomatic improvements in frequency and incontinence that patients are reporting [3,5]. Prominent in patients’ clinical responses to BoNT/A is an amelioration in their pathological sensation of urgency [5], a sensation believed to be afferently mediated [6]. These benefits are maintained for a mean of 9–11 mo after treatment [3,7]. Comparable responses have been demonstrated in patients with intractable NDO of various spinal etiologies and in patients with IDO [5,8].

2. Role of the sensory urothelium/ suburothelium in the pathophysiology of detrusor overactivity Novel findings support a role for the suburothelial innervation expressing the capsaicin receptor TRPV1 [9], the purinergic receptor P2X3 [10], and/or the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) [11] in the pathophysiology of human detrusor overactivity (DO). Patients with NDO and, to a lesser degree, those with IDO were found to have increased TRPV1and P2X3-immunoreactive suburothelial innervation compared to controls [12]. Intravesical instillations of the C-fiber toxin resiniferatoxin in NDO patients resulted in dramatic decreases of TRPV1and P2X3-immunoreactive fibers in clinical responders only [9,10] and produced significant improvements in LUTS and urodynamic parameters in patients with IDO [13]. Women with IDO were found to have increased density of suburothelial SP- and CGRP-immunoreactive fibers compared to controls [11]. Similarly, urothelial release of neurotransmitters (e.g., ACh, ATP) [14] and neuropeptides including SP [15,16] and the expression of receptors (TRPV1, P2X3) [17,18] strongly imply a role for the urothelium in human bladder mechanosensation. The discovery of a suburothelial nexus of myofibroblasts or interstitial cells [19,20], which are extensively

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linked by gap junctions [20,21] and may respond to ATP in a mode similar to the activation of ATPgated P2Y receptors [22], led to the proposal that they may be the substrate for a stretch-receptor organ. This new knowledge and recent clinical and laboratory observations suggest that the mechanism of action of BoNT/A in the overactive bladder may be more complicated than originally believed.

3. Evidence for an effect of BoNT/A on afferent pathways Despite the long established view that the effect of BoNT/A is selective for ACh-containing nerves inhibiting the presynaptic vesicular ACh release after cleavage of the synaptosomal-associated protein with a molecular weight of 25 KDa (SNAP-25) [2], in vivo and in vitro evidence is now emerging that BoNT/A has additional inhibitory effects on neuropeptides, neurotransmitters, and receptors mediating sensory neurotransmission. In cultured rat dorsal root ganglion (DRG) cells, BoNT/A was shown to induce a delayed, long-lasting inhibition of the release of SP, following the initial cleavage of SNAP25 [23]. In another study of primary DRG cells, BoNT/ A inhibited the protein kinase C (PKC) mediated SNARE-dependent exocytosis of TRPV1 to the plasma membrane [24]. BoNT/A also blocked the release of glutamate in an animal model of inflammatory pain [25], affecting specifically neurogenic inflammation mediated by the release of SP, a neurotransmitter known to be colocalized with glutamate.

4. Evidence for an effect of BoNT/A on bladder afferent pathways Animal studies first provided evidence for bladder afferent neuromodulation by BoNT/A via the purinergic or TRPV1-mediated pathways. In a rat model of spinal NDO, BoNT/A significantly reduced the abnormal distension-evoked urothelial release of ATP [26]. BoNT/A has also been reported to reduce DO evoked by application of ATP or capsaicin in rat bladders in vivo [27]. In a rat bladder pain model, intravesical BoNT/A administration produced significant improvement in the intervals between bladder contractions in parallel with a reduction of the release of CGRP from the superficial bladder layers [28]. A recent study of biopsies from a mixed population of NDO and IDO patients treated with intradetrusor BoNT/A showed no change in the overall

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suburothelial neuronal population both during clinical response [12] and clinical relapse time [29], but it did show progressive decrease and ultimate normalization of P2X3 and TRPV1 suburothelial nerve immunoreactivity, suggesting that BoNT/A affects sensory receptors’ expression in suburothelial fibers [12]. The fastest change was in P2X3immunoreactivity, which correlated with improvements in patients’ sensation of urgency [12]. In support of a possible BoNT/A-induced phenotypic change of bladder afferents, bladder levels of neurotrophic growth factor (NGF) in patients with NDO were reported to be reduced significantly after BoNT/A treatment [30]. Such effect may also explain the absence of significant nerve sprouting in the detrusor after successful BoNT/A treatment [31], unlike that seen in skeletal muscle.

5. Mechanism of BoNT/A’s action in the overactive human bladder Diffusion of the toxin solution from the detrusor injection sites into the suburothelium has already been demonstrated [32]. Therefore, we propose that BoNT/A injected in the overactive human bladder has a complex inhibitory effect on vesicular release of excitatory neurotransmitters and on the axonal expression of other SNARE-complex-dependent proteins in the urothelium/suburothelium, which are important in mediating those intrinsic or spinal reflexes thought to cause DO. We propose that the immediate effect of BoNT/A is due to peripheral afferent desensitization and involves inhibition of the vesicular release of ACh, ATP, and SP and of the axonal expression of TRPV1 from the urothelium [14] and suburothelial nerve endings [19,33] (Fig. 1). Furthermore extending the findings that the recently described suburothelial myofibroblasts act as an integrating stretch-receptor organ [19], we propose that BoNT/A induces phenotypic changes on these cells including inhibition of expression of purinergic and proposed SP receptors—assuming a homology of myofibroblasts with the interstitial cells of Cajal [34]—and a reduction in the expression of contractile and gap junction proteins. The urothelial release of ACh and ATP on bladder filling has been found to increase with aging [14] and in spinal NDO [26], implicating an abnormal release of these neurotransmitters in the pathophysiology of DO. Prevention of SNAP-25 phosphorylation via PKC-mediated pathways could explain the inhibitory effect of BoNT/A on the release of ACh from the urothelium and suburothelial nerve endings, similar

to the mechanism proposed for the detrusor [35] based on earlier studies of the effect of BoNT/A on PKC facilitatory pathways of transmitter release [36,37]. By inhibiting urothelial ACh release, BoNT/A may block its proposed excitatory effect on suburothelial afferent and detrusor parasympathetic nerve endings during urine storage, thus affecting the pathophysiology of DO by a mechanism similar to that suggested for oral anticholinergics [38]. Inhibition of the increased urothelial ATP release would reduce its proposed excitatory effect on suburothelial and urothelial P2X3 receptors and on the P2Y receptors on the myofibroblast network. ATP has been shown to potentiate TRPV1 activity via a PKC-dependent pathway involving metabotropic P2Y receptors [39]. Thus, decreased release of ATP after BoNT/A injection would also minimize potentiation of the TRPV1 receptor. BoNT/A may also inhibit vesicular SP release from suburothelial nerves. There is evidence that SP shares a common pathway with the muscarinic and purinergic transmitters to activate Ca+2-dependent nonselective cation channels (including TRP channels) [40]. SP may also potentiate susceptibility of P2X3 to ATP via a pathway involving PKC [41]. Inhibition of SP release would reduce activation of TRPV1 and P2X3 receptors in suburothelial afferents and detrusor muscle, as well as the responsiveness of the myofibroblast network to afferent signaling via decreased activation of proposed NK1 receptors on these cells [34]. Because the axonal expression of TRPV1 depends on interactions with proteins of the SNARE complex [24], destabilization of the complex following cleavage of SNAP-25 by BoNT/A will affect TRPV1 expression in peripheral nerve axons. A similar effect on P2X3-receptor expression may occur: the presence of P2X3 in terminal afferent boutons with complex synaptic properties [42] and its colocalization with TRPV1 in the CNS have been shown [43], and there is indirect evidence for colocalization with TRPV1 in bladder suburothelial afferents [9,10]. Were human bladder myofibroblasts demonstrated to express metabotropic purinergic and SP receptors, as has been shown in animals [22], and possess the intermediate filaments alpha smooth muscle actin (a-SMA) and vimentin, as myofibroblasts elsewhere in the body do [44,45], BoNT/Ainduced inhibition of P2Y/SP receptor expression and decrease in contractile filaments and gap junctions in these cells [46–49] would result in their reduced activation, contractility, and electrical coupling. Mediation of afferent signaling between the urothelium and the closely apposed nerve

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endings would thus be reduced, achieving maximization of the BoNT/A-induced peripheral desensitization. A secondary effect involves further peripheral inhibition at the synaptic level. Immunofluorescence has demonstrated the long-lasting persistence of BoNT/A-cleaved SNAP-25 (SNAP-25A), which acts as exocytosis inhibitor [2]. SNAP-25A immunofluorescence has been detected up to 40 d after BoNT/A-induced paralysis of mouse motor nerve terminals [2]. Hence, we propose that jamming of the SNARE complex by a persisting presence of SNAP-25A continues for that or an even longer

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period in humans, further inhibiting the release of SP and the axonal expression of TRPV1- and P2X3receptor. Furthermore, the decrease in SP-release would result in attenuation of central sensitization leading to further peripheral desensitization. BoNT/Ainduced inhibition of rapid afferent firing has been demonstrated by a reduction of Fos-positive cells in the dorsal horn of formalin-challenged rat models [25]. Increased central c-fos expression has been demonstrated in animal models of NDO [50] and chronic bladder inflammation [51]. A decrease in central SP-receptors has been shown to reduce

Fig. 1 – A schematic diagram of identifiable ultrastructural components of the human bladder wall (bl, basal lamina of urothelium; mf, myofibroblast layer; det, detrusor muscle; see [60] for more detailed explanation of ultrastructural components). Superimposed are the known or proposed location of receptors and sites of release of neuropeptides and growth factors involved in bladder mechanosensation. A complex interaction between the release of neurotransmitters and actions on respective receptors located on structural constituents is proposed. All connections identified by arrows are thought to be upregulated in detrusor overactivity and may be reduced with the peripheral afferent desensitization that follows injections of BoNT/A. Thin solid arrows, the proposed pathways whereby vesicular ATP release from the urothelium activates urothelial or suburothelial receptors (P2X3-P2Y) or potentiates the response of urothelial/suburothelial receptors (TRPV1) to irritative stimuli; thin dashed arrows, proposed pathways whereby vesicular ACh release from the urothelium or suburothelial nerves activates suburothelial or detrusor muscle muscarinic ACh receptors; thick dashed arrows, proposed pathways whereby suburothelially released SP acts on NK1 receptors on the myofibroblast cell layer or potentiates the activation of TRPV1-P2X3 receptors in suburothelial afferents (SP is also known to affect the expression of NGF and vice versa); thick solid arrows, NGF is thought to affect the expression of urothelial and/or suburothelial TRPV1.

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spinal c-fos expression and bladder overactivity [52]. In addition, consequent on the postulated decrease in SP-induced neurogenic inflammation, reduced peripheral production and retrograde uptake of neurotrophic factors to the DRG/spinal cord would result in decreased production of TRPV1/P2X3 in DRGs and their antegrade transport to bladder suburothelial afferents. Because NGF also may activate TRPV1 on small afferent nerves [53], which can in turn promote the vesicular release of SP via an ATP-P2Y mediated pathway [54], a reduction in NGF production, already shown in BoNT/A treated human bladders [30], could lead to further peripheral desensitization. Such proposed cascade mechanisms of neural plasticity might explain the longer-lasting effect of BoNT/A in the overactive bladder compared to its duration of action at other sites. C-fiber afferents constitute the majority (twothirds) of bladder afferents. In addition, experimental findings suggest that it is the neuroplasticity of these afferents that is involved in both the reorganization of the micturition reflex in NDO [55,56] and the intrinsic bladder wall reflexes in IDO. However, the role of Ad-fibers cannot be ignored. Following injections of a BoNT/A endopeptidase conjugate into the intrathecal L4–L5 space of anaesthetized rats, recordings from C-fibers from DRG neurons closest to the zone of the spinal cord injection showed a reduction in activity by 75.8% [57]. The Ad-fiberevoked activity was also reduced, although to a lesser extent (42.3%), whereas the Ab-fiber-evoked responses were unchanged. Following intradetrusor BoNT/A injection patients experience a reduction in sensation of urgency but retain sensation of bladder filling, which could possibly be attributed to a relative sparing of Ad-fiber activity.

6.

from bladder parasympathetic motor innervation [35]. In clinical studies, the post-BoNT/A decreases in detrusor pressures during both the filling and voiding phases, in conjunction with post-treatment increases in post-void residual urine volumes [3–5] suggest an inhibitory effect of BoNT/A on the motor innervation of the detrusor smooth muscle. A study of the sensory immunohistochemical changes following BoNT/A injection in the human DO bladder found no correlation between the changes in suburothelial P2X3- and TRPV1-immunoreactivity and the changes in maximum filling detrusor pressures or maximum cystometric capacity [12]. A significant correlation with changes in urgency, however, suggested an independent effect [12]. Thus, a synergistic effect of BoNT/A on afferent and efferent pathways is proposed. Several nonadrenergic noncholinergic (NANC) neurotransmitters, such as the vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and leucine enkephalin (ENK), have been identified in peripheral efferent bladder pathways, and it has been postulated that these may modulate efferent inputs to the lower urinary tract, but with a mainly inhibitory effect [56]. Evidence of a significant effect of BoNT/A on the release of these transmitters is inconclusive, however, and their putative role in detrusor function remains largely unclear [59]. Further research is needed to determine the effect of BoNT/A on the balance of modulatory transmitter actions on both the afferent and efferent (cholinergic and noncholinergic) pathways of the micturition reflex. Funding: The Multiple Sclerosis Society of Great Britain and Northern Ireland Project Grant (C.J. Fowler, P. Dasgupta); Pfizer, Inc. (A. Apostolidis); Allergan Ltd. UK (gratis provider of BOTOX1 for research purposes).

BoNT/A and the efferent bladder pathways

Although the emphasis of this proposed mechanism has been that the bladder afferent signaling is affected by BoNT/A, it seems highly likely that its effect on efferent pathways is also considerable. Under high frequencies of electrical field stimulation (EFS) thought to mimic the effect of the parasympathetic nerve overactivity that may occur in patients with DO, detrusor strips removed from rat bladders that had been treated with BoNT/A displayed a significant transient decrease in the neuronal release of ACh compared to strips from sham-treated animals [58]. A subsequent in vitro study using similarly high EFS frequencies concluded a dual inhibition of ACh and ATP release

References [1] Schurch B, Stohrer M, Kramer G, Schmid DM, Gaul G, Hauri D. Botulinum-A toxin for treating detrusor hyperreflexia in spinal cord injured patients: A new alternative to anticholinergic drugs? Preliminary results. J Urol 2000;164: 692–7. [2] Dolly O. Synaptic transmission: inhibition of neurotransmitter release by Botulinum toxins. Headache 2003;43(Suppl 1):S16–24. [3] Reitz A, Stohrer M, Kramer G, et al. European experience of 200 cases treated with Botulinum-A toxin injections into the detrusor muscle for urinary incontinence due to neurogenic detrusor overactivity. Eur Urol 2004; 45:510–5.

european urology 49 (2006) 644–650

[4] Schurch B, de Seze M, Denys P, et al. Botulinum toxin type a is a safe and effective treatment for neurogenic urinary incontinence: results of a single treatment, randomized, placebo controlled 6-month study. J Urol 2005;174:196– 200. [5] Popat R, Apostolidis A, Kalsi V, Gonzales G, Fowler CJ, Dasgupta P. A comparison between the response of patients with idiopathic detrusor overactivity and neurogenic detrusor overactivity to the first intradetrusor injection of Botulinum-A toxin. J Urol 2005;174:984–8. [6] Oliver S, Fowler CJ, Mundy A, Craggs M. Measuring the sensations of urge and bladder filling during cystometry in urge incontinence and the effects of neuromodulation. Neurourol Urodyn 2003;22:7–16. [7] Grosse J, Kramer G, Stohrer M. Success of repeat detrusor injections of Botulinum A toxin in patients with severe neurogenic detrusor overactivity and incontinence. Eur Urol 2005;47:653–9. [8] Kessler TM, Danuser H, Schumacher M, Studer UE, Burkhard FC. Botulinum A toxin injections into the detrusor: An effective treatment in idiopathic and neurogenic detrusor overactivity? Neurourol Urodyn 2005;24:231–6. [9] Brady CM, Apostolidis A, Harper M, et al. Parallel changes in bladder suburothelial vanilloid receptor TRPV1 (VR1) and pan-neuronal marker PGP9.5 immunoreactivity in patients with neurogenic detrusor overactivity (NDO) following intravesical resiniferatoxin treatment. BJU Int 2004;93:770–6. [10] Brady C, Apostolidis A, Yiangou Y, et al. P2X3-immunoreactive nerve fibres in neurogenic detrusor overactivity and the effect of intravesical resiniferatoxin (RTX). Eur Urol 2004;46:247–53. [11] Smet PJ, Moore KH, Jonavicius J. Distribution and colocalization of calcitonin gene-related peptide, tachykinins, and vasoactive intestinal peptide in normal and idiopathic unstable human urinary bladder. Lab Invest 1997;77:37–49. [12] Apostolidis A, Popat R, Yiangou Y, et al. Decreased sensory receptors P2X3 and TRPV1 in suburothelial nerve fibers following intradetrusor injections of Botulinum toxin for human detrusor overactivity. J Urol 2005;174: 977–83. [13] Silva C, Ribeiro MJ, Cruz F. The effect of intravesical resiniferatoxin in patients with idiopathic detrusor instability suggests that involuntary detrusor contractions are triggered by C-fiber input. J Urol 2002;168:575–9. [14] Yoshida M, Miyamae K, Iwashita H, Otani M, Inadome A. Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging. Urology 2004;63:17–23. [15] Kushner L, Chiu PY, Brettschneider N, et al. Urinary substance P concentration correlates with urinary frequency and urgency in interstitial cystitis patients treated with intravesical dimethyl sulfoxide and not intravesical anesthetic cocktail. Urology 2001;57:129. [16] Sukiennik A, Carr DB, Bonney I, Marchand JE, Wurm H, Sant GR. The effect of short-term epidural local anesthetic blockade on urinary levels of substance P in interstitial cystitis. Anesth Analg 2004;98:846–50.

649

[17] Sun Y, Chai TC. Up-regulation of P2X3 receptor during stretch of bladder urothelial cells from patients with interstitial cystitis. J Urol 2004;171:448–52. [18] Apostolidis A, Brady CM, Yiangou Y, Davis J, Fowler CJ, Anand P. Capsaicin receptor TRPV1 in the urothelium of neurogenic human bladders and the effect of intravesical resiniferatoxin. Urology 2005;65:400–5. [19] Wiseman OJ, Fowler CJ, Landon DN. The role of the human bladder lamina propria myofibroblast. BJU Int 2003;91:89–93. [20] Sui GP, Rothery S, Dupont E, Fry CH, Severs NJ. Gap junctions and connexin expression in human suburothelial interstitial cells. BJU Int 2002;90:118–29. [21] Sui GP, Wu C, Fry CH. Electrical characteristics of suburothelial cells isolated from the human bladder. J Urol 2004;171:938–43. [22] Wu C, Sui GP, Fry CH. Purinergic regulation of guinea pig suburothelial myofibroblasts. J Physiol 2004;559:231–43. [23] Welch MJ, Purkiss JR, Foster KA. Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins. Toxicon 2000;38:245–58. [24] Morenilla-Palao C, Planells-Cases R, Garcia-Sanz N, Ferrer-Montiel A. Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity. J Biol Chem 2004;279:25665–72. [25] Cui M, Khanijou S, Rubino J, Aoki KR. Subcutaneous administration of Botulinum toxin A reduces formalininduced pain. Pain 2004;107:125–33. [26] Khera M, Somogyi GT, Kiss S, Boone TB, Smith CP. Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury. Neurochem Int 2004;45:987–93. [27] Atiemo H, Wynes J, Chuo J, Nipkow L, Sklar GN, Chai TC. Effect of Botulinum toxin on detrusor overactivity induced by intravesical adenosine triphosphate and capsaicin in a rat model. Urology 2005;65:622–6. [28] Chuang YC, Yoshimura N, Huang CC, Chiang PH, Chancellor MB. Intravesical Botulinum toxin A administration produces analgesia against acetic acid induced bladder pain responses in rats. J Urol 2004;172:1529–32. [29] Apostolidis A, Popat R, Yiangou Y, Fowler CJ, Dasgupta P, Anand P. Successful clinical outcome of intra-detrusor Botulinum toxin for human detrusor overactivity is not associated with suburothelial neuronal or cholinergic fibre degeneration or sprouting. J Endourol 2004;18:A108, (MP18/12). [30] Giannantoni A, Di Stasi SM, Nardicchi V, et al. BotulinumA toxin injections into the detrusor muscle reduce nerve growth factor bladder tissue levels in patients affected by neurogenic detrusor overactivity. Eur Urol Suppl 2005;4(3):69, (A268). [31] Haferkamp A, Schurch B, Reitz A, et al. Lack of ultrastructural detrusor changes following endoscopic injection of Botulinum toxin type A in overactive neurogenic bladder. Eur Urol 2004;46:784–91. [32] Schulte-Baukloh H, Knispel HH. A minimally invasive technique for outpatient local anaesthetic administration of intradetrusor Botulinum toxin in intractable detrusor overactivity. BJU Int 2005;95:454.

650

european urology 49 (2006) 644–650

[33] Khullar V, Nadler R, Chaliha C, Yeo L, Underwood J. Muscarinic type 2 receptors on bladder sensory nerves: A new site of drug action for detrusor overactivity? Abstracts of the 33rd Annual Meeting of the International Continence Society 2003:A146. [34] Lavin ST, Southwell BR, Murphy R, Jenkinson KM, Furness JB. Activation of neurokinin 1 receptors on interstitial cells of Cajal of the guinea-pig small intestine by substance P. Histochem Cell Biol 1998;110:263–71. [35] Smith CP, Boone TB, de Groat WC, Chancellor MB, Somogyi GT. Effect of stimulation intensity and Botulinum toxin isoform on rat bladder strip contractions. Brain Res Bull 2003;61:165–71. [36] Nakov R, Habermann E, Hertting G, Wurster S, Allgaier C. Effects of Botulinum A toxin on presynaptic modulation of evoked transmitter release. Eur J Pharmacol 1989;164: 45–53. [37] Somogyi GT, Tanowitz M, Zernova G, de Groat WC. M1 muscarinic receptor-induced facilitation of ACh and noradrenaline release in the rat bladder is mediated by protein kinase C. J Physiol 1996;496:245–54. [38] Andersson KE. Antimuscarinics for treatment of overactive bladder. Lancet Neurol 2004;3:47–53. [39] Tominaga M, Wada M, Masu M. Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia. Proc Natl Acad Sci USA 2001;98:6951–6. [40] Ito K, Rome C, Bouleau Y, Dulon D. Substance P mobilizes intracellular calcium and activates a nonselective cation conductance in rat spiral ganglion neurons. Eur J Neurosci 2002;16:2095–102. [41] Paukert M, Osteroth R, Geisler HS, et al. Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit. J Biol Chem 2001;276:21077–82. [42] Burnstock G. Purine-mediated signalling in pain and visceral perception. Trends Pharmacol Sci 2001;22:182–8. [43] Guo A, Vulchanova L, Wang J, Li X, Elde R. Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. Eur J Neurosci 1999;11:946–58. [44] Desmouliere A. Factors influencing myofibroblast differentiation during wound healing and fibrosis. Cell Biol Int 1995;19:471–6. [45] Tomasek JJ, McRae J, Owens GK, Haaksma CJ. Regulation of alpha-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-beta1 control element. Am J Pathol 2005;166:1343–51. [46] Wiegers W, Just I, Muller H, Hellwig A, Traub P, Aktories K. Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADP-ribosyltransferase. Eur J Cell Biol 1991; 54:237–45.

[47] Anderson S, DiCesare L, Tan I, Leung T, Sundar Raj N. Rhomediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts. Exp Cell Res 2004;298:574–83. [48] Anderson SC, Stone C, Tkach L, Sundar Raj N. Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium. Invest Ophthalmol Vis Sci 2002;43:978–86. [49] Saint-Amant L, Drapeau P. Synchronization of an embryonic network of identified spinal interneurons solely by electrical coupling. Neuron 2001;31:1035–46. [50] Yotsuyanagi S, Yokoyama O, Komatsu K, Kodama K, Niikura S, Namiki M. Expression of neural plasticity related gene in the pontine tegmental area of rats with overactive bladder after cerebral infarction. J Urol 2001; 166:1148–55. [51] Vemulakonda VM, Somogyi GT, Kiss S, Salas NA, Boone TB, Smith CP. Inhibitory effect of intravesically applied Botulinum toxin A in chronic bladder inflammation. J Urol 2005;173:621–4. [52] Seki S, Erickson KA, Seki M, et al. Elimination of rat spinal neurons expressing neurokinin 1 receptors reduces bladder overactivity and spinal c-fos expression induced by bladder irritation. Am J Physiol Renal Physiol 2005;288: F466–73. [53] Chuang HH, Prescott ED, Kong H, et al. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature 2001;411:957– 62. [54] Huang H, Wu X, Nicol GD, Meller S, Vasko MR. ATP augments peptide release from rat sensory neurons in culture through activation of P2Y receptors. J Pharmacol Exp Ther 2003;306:1137–44. [55] de Groat WC, Kawatani M, Hisamitsu T, et al. Mechanisms underlying the recovery of urinary bladder function following spinal cord injury. J Auton Nerv Syst 1990;30:S71–7. [56] Yoshimura N. Bladder afferent pathway and spinal cord injury: possible mechanisms inducing hyperreflexia of the urinary bladder. Prog Neurobiol 1999;57:583–606. [57] Duggan MJ, Quinn CP, Chaddock JA, et al. Inhibition of release of neurotransmitters from rat dorsal root ganglia by a novel conjugate of a Clostridium botulinum toxin A endopeptidase fragment and Erythrina cristagalli lectin. J Biol Chem 2002;277:34846–52. [58] Smith CP, Franks ME, McNeil BK, et al. Effect of Botulinum toxin A on the autonomic nervous system of the rat lower urinary tract. J Urol 2003;169:1896–900. [59] Andersson KE, Wein AJ. Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence. Pharmacol Rev 2004;56:581–631. [60] Fowler CJ, Harper M, Fry CH. Voiding and the sacral reflex arc: lessons from capsaicin instillation. Scand J Urol Nephrol (Suppl 210):2002;46–50.