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In the Human Urothelium and Suburothelium, Intradetrusor Botulinum Neurotoxin Type A Does Not Induce Apoptosis: Preliminary Results
EUROPEAN UROLOGY 57 (2010) 879–883
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Neuro-urology
In the Human Urothelium and Suburothelium, Intradetrusor Botulinum Neurotoxin Type A Does Not Induce Apoptosis: Preliminary Results Thomas M. Kessler a,b,*, Shahid Khan a, Jalesh N. Panicker a, Sohier Elneil a, Sebastian Brandner c, Clare J. Fowler a, Alexander Roosen a,d a
Department of Uro-Neurology, The National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust,
and University College London Institute of Neurology, Queen Square, London, UK b
Department of Urology, University of Bern, Bern, Switzerland
c
Division of Neuropathology and Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK
d
Department of Urology, Ludwig-Maximilians-University, Mu¨nchen, Germany
Article info
Abstract
Article history: Accepted September 3, 2009 Published online ahead of print on September 11, 2009
Background: Intradetrusor injections of botulinum neurotoxin type A (BoNTA) are emerging as the preferred second-line treatment for neurogenic and idiopathic overactive bladder (OAB). In animal experiments, intradetrusor BoNTA injections have been shown to cause apoptosis in the bladder urothelium and suburothelium but not the detrusor. Objective: To investigate BoNTA-induced apoptosis in patients with refractory neurogenic OAB. Design, setting, and participants: Twelve refractory OAB patients with neurogenic detrusor overactivity resulting from multiple sclerosis (MS) and seven controls were included prospectively. Measurements: The number of apoptotic cells before and 4 wk after first intradetrusor BoNTA (300 U of BOTOX [Allergan, Irvine, CA, USA]) injections were estimated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. Results and limitations: Comparison of TUNEL-positive cells (yes vs no) in the bladder urothelium and suburothelium revealed no significant differences in OAB patients before (4 of 12, 33%) versus after (3 of 12, 25%) BoNTA treatment ( p = 0.99). In addition, no significant differences ( p = 0.99) were found in OAB patients versus controls. Because our findings are based on first intradetrusor BoNTA injections only, it is unclear whether the results could be extrapolated to repeat injections. Conclusions: In contrast to preliminary animal experiments, first intradetrusor BoNTA injections for treating refractory neurogenic OAB—a highly effective treatment—did not induce apoptosis in the bladder urothelium and suburothelium.
Keywords: Overactive bladder Detrusor overactivity Multiple sclerosis Botulinum neurotoxin type A Apoptosis
# 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Department of Urology, University of Bern, 3010 Bern, Switzerland. E-mail address: [email protected] (T.M. Kessler).
0302-2838/$ – see back matter # 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2009.09.023
880
1.
EUROPEAN UROLOGY 57 (2010) 879–883
Introduction
Detrusor injections of botulinum neurotoxin type A (BoNTA) were introduced to treat intractable bladder symptoms resulting from neurogenic detrusor overactivity [1] on the premise that the cholinergic innervation of the detrusor would be blocked [2]. Studies of patients with various causes of spinal cord dysfunction and multiple sclerosis (MS) in particular [3] have demonstrated the efficacy of this intervention [4], and placebocontrolled trials have confirmed a real effect [5,6]. Indeed, its efficacy has been so remarkable that a hypothesis has been proposed that it may not only be acting on the efferent but also on the afferent innervation, because its effect on urgency is so early, profound, and long lasting [7]. There is now also emerging evidence that prostatic injections of BoNTA have a marked effect of improving urinary flow and lower urinary tract symptoms (LUTS) in men who are poor candidates for prostate surgery [8–10]. It is far too early to know if this will ever become a licensed treatment for benign prostatic hyperplasia, but some interesting experimental studies in dogs and most recently rats have shown that the observed atrophy that affects the gland is largely the result of apoptosis, a process that appears to be related to sympathetic chemodenervation [11]. So far, the changes in human bladder following BoNTA injections that can be associated with a successful outcome are a reduction in TRPV1 and P2X expression [12] in the suburothelial innervation. However, from the sparse data available, no definitive conclusions can be drawn about degeneration or reinnervation phenomena in the human bladder wall following BoNTA injections as of yet. The only data existing are those by Haferkamp et al [13], who showed nearly no structural differences of the detrusor before and after BoNTA injections. Contrary to studies on striated muscle, no increase in axonal sprouting after BoNTA injections was observed, indicating pathophysiologically different reactions to the toxin either between striated muscle and smooth muscle or between different treated diseases [13]. Based on laboratory experiments, it seems highly probable that the toxin blocks release of various N-ethylmaleimidesensitive factor attachment protein receptor–dependent neurotransmitters at least in the short term, but an explanation for the effects of BoNTA, which have been observed to last between 9 and 10 mo, is still awaited. In keeping with the evidence of extensive apoptosis that underlies the mechanism of action in the prostate, a recent rat study could demonstrate (repeated) injections 10 U of BoNTA into the bladder wall to induce marked apoptotic cell losses in the bladder urothelium and suburothelium but not the detrusor [14]. We set out to address the question of whether a comparable apoptotic cell loss could be observed in the human urothelial and suburothelial layer after intradetrusor BoNTA injections.
2.
Patients and methods
2.1.
Patients and injection technique
In this prospective study, bladder biopsies were taken from 12 overactive bladder (OAB) patients with neurogenic detrusor overactivity resulting from MS (eight women, four men; median age: 45 yr [interquartile range (IQR): 42–50]) and seven controls (all women; median age: 56 yr [IQR: 43–64]). The 12 patients were part of the group of 43 whose excellent clinical response to detrusor injections of BoNTA has already been reported [3]. Briefly, detrusor overactivity was proven urodynamically in all, and treatment with more than one antimuscarinic drug for at least 3 mo failed. Patients were treated according to a research protocol approved by the local research ethics committee. Intradetrusor BoNTA injections were performed in an outpatient setting using the previously described minimally invasive technique [15], injecting 300 U of BoNTA (BOTOX; Allergan, Irvine, CA, USA) at 30 different sites in the detrusor, sparing the trigone [1]. Flexible cystoscopic bladder biopsies were obtained at baseline pretreatment and during check flexible cystoscopy at 4 and 16 wk after each treatment session. Biopsies were obtained from a consistent bladder area 2 cm above and lateral to the ureteric orifices. Control tissue was obtained endoscopically from seven patients (all women; median age: 56 yr) being examined under anaesthesia prior to pelvic floor repair procedures. They had macroscopically normal bladders, no symptoms of OAB, and sterile urine at the time of endoscopy.
2.2.
Outcome measures
Outcome measures were the number of apoptotic cells in the bladder of OAB patients before and 4 wk after first intradetrusor BoNTA injections and apoptotic cells in the bladder of controls.
2.3.
Immunoreaction
Biopsy specimens were snap frozen in liquid nitrogen, embedded in optimal cutting temperature medium, and stored at 60 8C. Three sections per specimen were cut in a cryostat at 10 mm thickness and collected on 3-aminopropyltriethoxysilane-coated superfrost slides. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining, a well-established method for the rapid identification of apoptotic cells, was performed using the In Situ Cell Death Detection Kit, Fluorescein (FITC; Roche Diagnostics, Burgess Hill, UK). Frozen sections were fixed, permeabilised, and labelled following the manufacturer’s instructions using a 1:2 TUNEL dilution. Nuclei were counterstained with 40 , 6-diamidino-2-phenylindole (DAPI; Invitrogen, D1306, 1:50 000) during incubation in a humidified atmosphere in the dark. One negative control (a section without terminal deoxynucleotidyl transferase) was included in each experimental set. Human glioblastoma multiforme tissue (malignant glial brain tumour) was used as a positive control. Slides were cover slipped using Citifluor mounting medium (Agar Scientific, Stansted, UK). Immunolabelled sections were examined using a laser scanning confocal microscope (Zeiss LSM-510 META, Germany) equipped with an argon laser (458 nm, 488 nm, 514 nm), a helium-neon laser (543 nm, 633 nm), and a 405-nm diode laser. Fluorescence was excited at 488 nm (FITC) and 405 nm (DAPI) and 543 nm (Cy3) and recorded with separate detectors. Only those sections that displayed a complete and strictly transversal cross-section of the bladder urothelium and suburothelium were chosen for numeric determination of apoptotic cells. Whole-section images were taken with a 20 objective blinded to the sample. We first calibrated the detection system on a reference section and reused the
EUROPEAN UROLOGY 57 (2010) 879–883
881
Fig. 1 – (a) Suburothelial terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)–positive cell 4 wk after intradetrusor botulinum neurotoxin type A injections; (b) TUNEL-positive cells in a glioblastoma multiforme patient (positive control). Bar = 50 mm.
parameter setting on the LSM confocal microscope (pinhole, optical slice thickness; detector gain) for all images. Given the sparse distribution of TUNEL-positive cells within the bladder urothelium and suburothelium, the entire section was searched for TUNEL-positive cells.
2.4.
Statistical analysis
The McNemar test and the Fisher exact test were used to compare related and unrelated samples, respectively. A p value <0.05 was considered significant. Statistical analysis was performed using SPSS v.16.0 (SPSS, Chicago, IL, USA).
3.
Results
Apoptotic cells were found sparsely distributed exclusively in the suburothelial space (Fig. 1). The number of TUNELpositive cells was extremely low, with a maximum of two per section. Of the 12 OAB patients, 4 (33%) had TUNELpositive cells before and 3 (25%) after BoNTA treatment. Two of the seven controls (29%) showed TUNEL-positive cells. Comparison of TUNEL-positive cells (present vs absent) revealed no significant differences in OAB patients before (4 of 12, 33%) and after (3 of 12, 25%) BoNTA treatment ( p = 0.99). In addition, no significant differences were found in OAB patients compared to controls (OAB before BoNTA treatment [4 of 12, 33%] vs controls [2 of 7, 29%], p = 0.99; OAB after BoNTA [3 of 12, 25%] treatment vs controls [2 of 7, 29%], p = 0.99). 4.
Discussion
To our knowledge, this is the first study attempting to investigate BoNTA-induced apoptosis in the human bladder urothelium and suburothelium. The number of TUNELpositive cells in OAB patients with neurogenic detrusor overactivity resulting from MS before and after BoNTA treatment as well as in controls were similarly low, indicating that intradetrusor BoNTA injections do not cause apoptosis in the bladder layers we have studied.
The classical concept of the pharmacologic effect of BoNTA is that it 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 paralysis, however, and its effect of reducing urgency is especially welcomed by patients [16], a sensation believed to be afferently mediated. This notion has started an ongoing debate as to whether BoNTA has additional inhibitory effects on neuropeptides, neurotransmitters, receptors, and even structural surrogates in the afferent bladder pathway. In this context, rapid diffusion of the drug into the whole bladder wall from the injection site has been demonstrated [17]. In fact, several subcellular structures were shown to be numerically altered in conditions of OAB and to be eventually restored to normal levels upon BoNTA treatment. These include P2X3 and TRPV1 receptors on suburothelial afferent nerve fibres [12,18,19] as well as gap junctions and adherens junctions in the suburothelial myofibroblast layer [20,21]. Over the past decade, animal experiments have been performed to assess a putative apoptotic effect of BoNTA injections into various tissues of the lower urinary tract. Apoptosis is the process of programmed cell death that occurs in multicellular organisms. It involves a series of biochemical events that lead to a variety of morphologic changes, including cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. TUNEL has become one of the main methods for detecting apoptotic programmed cell death [22]. The assay relies on the presence of nicks in the DNA, which can be identified by terminal deoxynucleotidyl transferase, an enzyme that will catalyse the addition of dUTPs that are secondarily labelled with a marker. Mitochondria-associated apoptotic signalling has been shown to be upregulated during denervation in skeletal muscle of rats, suggesting that apoptosis has a physiologically important role in regulating denervation-induced muscle atrophy
882
EUROPEAN UROLOGY 57 (2010) 879–883
[23]. The magnitude of muscle fibre apoptosis is increased both by denervation and muscle paralysis and can be prevented by administration of trophic factors [24]. Doggweiler et al [25] were the first to inject rat prostates with varying doses of BoNTA (BOTOX) for histologic examination. They found a generalised atrophy of the glands and diffuse glandular apoptosis evident with TUNEL stain [25]. These findings were corroborated by Chuang et al [26], who found a significant increase in apoptotic cells (12-, 16-, and 22-fold) and a decrease in proliferative cells (38%, 77%, and 80%) as well as a1A-adrenergic receptor (13%, 80%, and 81%) 1 wk after injecting 5, 10, and 20 U of BoNTA, respectively [26]. Another recent rat study [11] suggested prostate atrophy induced by BoNTA in the rat may be the result of sympathetic nerve impairment and decreased adrenergic stimulation of the gland. Similarly, rats (repeatedly) injected with BoNTA into the bladder wall showed significant increases of apoptotic cells in the bladder urothelium and suburothelium but not the detrusor [14]. Similar degeneration of glands and ducts as well as apoptotic nuclei on TUNEL staining have been demonstrated in the nasal mucosal tissue of guinea pigs following BoNTA [27]. The finding from the animal experiments—that intradetrusor BoNTA injections result in apoptosis in the bladder urothelium and suburothelium but not in the detrusor [14]—raised the question of whether comparable cellular losses could be noted in the human situation. Gross histologic changes in the urothelium and suburothelium of patients with detrusor overactivity after one or repeat intradetrusor BoNTA injections have been investigated previously [13,28]. BoNTA injections did not appear to produce significant inflammatory changes, fibrosis, dysplastic changes, nerve degeneration, or sprouting in the human urothelium and suburothelium. In contrast to previous animal experiments, we failed to show an increased apoptosis rate in the urothelium or suburothelium after first intradetrusor BoNTA injections. In the animal study by Watanabe et al [14], adult male Sprague Dawley rats were injected with 10 U of BoNTA into the bladder wall, and bladders were harvested after 2 wk for histologic investigation, with routine TUNEL staining being performed. However, our study population comprised male and female patients who received 300 U of BoNTA at 30 different sites (ie, 10 U per site), with biopsies taken before and 4 wk after first intradetrusor BoNTA injections. As TUNEL staining is considered a relatively robust technique in which methodologic variations have little impact on results, discrepancies between the present study and the findings by Watanabe et al [14] may be explained by different species, gender, and follow-up. The notion of absent apoptosis in the human urothelium and suburothelium is important for drug safety of intradetrusor BoNTA administration. We are aware of the limitations of our study: It was neither randomised nor placebo controlled, but it was prospective and included controls without LUTS. In addition, we analysed data from the same patients (rather than groups of different patients) before and after BoNTA
injections. Because our findings are based on first intradetrusor BoNTA injections only, it is unclear whether the results could be extrapolated to repeat injections. In the present study, apoptotic cell assessment was focused on the urothelium and suburothelium but did not include the detrusor, which is rarely obtained in flexible cystoscopy biopsies. 5.
Conclusions
First intradetrusor BoNTA injections for treating refractory neurogenic OAB in patients with MS were highly effective, but the mechanism of action does not appear to be by apoptosis, an effect that is so prominent in the prostate. Author contributions: Thomas M. Kessler had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Kessler, Fowler, Roosen. Acquisition of data: Kessler, Roosen. Analysis and interpretation of data: Kessler, Roosen. Drafting of the manuscript: Kessler, Roosen. Critical revision of the manuscript for important intellectual content: Khan, Panicker, Elneil, Brandner, Fowler. Statistical analysis: Kessler. Obtaining funding: None. Administrative, technical, or material support: Elneil, Brandner. Supervision: Brandner, Fowler. 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: Thomas M. Kessler, Sohier Elneil, and Clare J. Fowler have acted as consultants for Medtronic. Clare J. Fowler is recipient of unrestricted educational grants from Allergan Ltd and has also acted as consultant for Allergan Ltd. Clare J. Fowler was the recipient of an independent research grant from Pfizer, Inc. Funding/Support and role of the sponsor: Allergan Ltd UK was the gratis provider of BOTOX used in this study. Allergan Ltd UK was not involved in the design and conduct of the study; collection of the data; management of the data; analysis and interpretation of the data; or the preparation, review, and approval of the manuscript. Thomas M. Kessler was supported by a grant of the Swiss National Science Foundation. Alexander Roosen was supported by a grant of the German Research Foundation. This work was undertaken at the University College London Hospitals (UCLH)/University College London (UCL), which received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme.
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available at www.sciencedirect.com journal homepage: www.europeanurology.com
Neuro-urology
In the Human Urothelium and Suburothelium, Intradetrusor Botulinum Neurotoxin Type A Does Not Induce Apoptosis: Preliminary Results Thomas M. Kessler a,b,*, Shahid Khan a, Jalesh N. Panicker a, Sohier Elneil a, Sebastian Brandner c, Clare J. Fowler a, Alexander Roosen a,d a
Department of Uro-Neurology, The National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust,
and University College London Institute of Neurology, Queen Square, London, UK b
Department of Urology, University of Bern, Bern, Switzerland
c
Division of Neuropathology and Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK
d
Department of Urology, Ludwig-Maximilians-University, Mu¨nchen, Germany
Article info
Abstract
Article history: Accepted September 3, 2009 Published online ahead of print on September 11, 2009
Background: Intradetrusor injections of botulinum neurotoxin type A (BoNTA) are emerging as the preferred second-line treatment for neurogenic and idiopathic overactive bladder (OAB). In animal experiments, intradetrusor BoNTA injections have been shown to cause apoptosis in the bladder urothelium and suburothelium but not the detrusor. Objective: To investigate BoNTA-induced apoptosis in patients with refractory neurogenic OAB. Design, setting, and participants: Twelve refractory OAB patients with neurogenic detrusor overactivity resulting from multiple sclerosis (MS) and seven controls were included prospectively. Measurements: The number of apoptotic cells before and 4 wk after first intradetrusor BoNTA (300 U of BOTOX [Allergan, Irvine, CA, USA]) injections were estimated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. Results and limitations: Comparison of TUNEL-positive cells (yes vs no) in the bladder urothelium and suburothelium revealed no significant differences in OAB patients before (4 of 12, 33%) versus after (3 of 12, 25%) BoNTA treatment ( p = 0.99). In addition, no significant differences ( p = 0.99) were found in OAB patients versus controls. Because our findings are based on first intradetrusor BoNTA injections only, it is unclear whether the results could be extrapolated to repeat injections. Conclusions: In contrast to preliminary animal experiments, first intradetrusor BoNTA injections for treating refractory neurogenic OAB—a highly effective treatment—did not induce apoptosis in the bladder urothelium and suburothelium.
Keywords: Overactive bladder Detrusor overactivity Multiple sclerosis Botulinum neurotoxin type A Apoptosis
# 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Department of Urology, University of Bern, 3010 Bern, Switzerland. E-mail address: [email protected] (T.M. Kessler).
0302-2838/$ – see back matter # 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2009.09.023
880
1.
EUROPEAN UROLOGY 57 (2010) 879–883
Introduction
Detrusor injections of botulinum neurotoxin type A (BoNTA) were introduced to treat intractable bladder symptoms resulting from neurogenic detrusor overactivity [1] on the premise that the cholinergic innervation of the detrusor would be blocked [2]. Studies of patients with various causes of spinal cord dysfunction and multiple sclerosis (MS) in particular [3] have demonstrated the efficacy of this intervention [4], and placebocontrolled trials have confirmed a real effect [5,6]. Indeed, its efficacy has been so remarkable that a hypothesis has been proposed that it may not only be acting on the efferent but also on the afferent innervation, because its effect on urgency is so early, profound, and long lasting [7]. There is now also emerging evidence that prostatic injections of BoNTA have a marked effect of improving urinary flow and lower urinary tract symptoms (LUTS) in men who are poor candidates for prostate surgery [8–10]. It is far too early to know if this will ever become a licensed treatment for benign prostatic hyperplasia, but some interesting experimental studies in dogs and most recently rats have shown that the observed atrophy that affects the gland is largely the result of apoptosis, a process that appears to be related to sympathetic chemodenervation [11]. So far, the changes in human bladder following BoNTA injections that can be associated with a successful outcome are a reduction in TRPV1 and P2X expression [12] in the suburothelial innervation. However, from the sparse data available, no definitive conclusions can be drawn about degeneration or reinnervation phenomena in the human bladder wall following BoNTA injections as of yet. The only data existing are those by Haferkamp et al [13], who showed nearly no structural differences of the detrusor before and after BoNTA injections. Contrary to studies on striated muscle, no increase in axonal sprouting after BoNTA injections was observed, indicating pathophysiologically different reactions to the toxin either between striated muscle and smooth muscle or between different treated diseases [13]. Based on laboratory experiments, it seems highly probable that the toxin blocks release of various N-ethylmaleimidesensitive factor attachment protein receptor–dependent neurotransmitters at least in the short term, but an explanation for the effects of BoNTA, which have been observed to last between 9 and 10 mo, is still awaited. In keeping with the evidence of extensive apoptosis that underlies the mechanism of action in the prostate, a recent rat study could demonstrate (repeated) injections 10 U of BoNTA into the bladder wall to induce marked apoptotic cell losses in the bladder urothelium and suburothelium but not the detrusor [14]. We set out to address the question of whether a comparable apoptotic cell loss could be observed in the human urothelial and suburothelial layer after intradetrusor BoNTA injections.
2.
Patients and methods
2.1.
Patients and injection technique
In this prospective study, bladder biopsies were taken from 12 overactive bladder (OAB) patients with neurogenic detrusor overactivity resulting from MS (eight women, four men; median age: 45 yr [interquartile range (IQR): 42–50]) and seven controls (all women; median age: 56 yr [IQR: 43–64]). The 12 patients were part of the group of 43 whose excellent clinical response to detrusor injections of BoNTA has already been reported [3]. Briefly, detrusor overactivity was proven urodynamically in all, and treatment with more than one antimuscarinic drug for at least 3 mo failed. Patients were treated according to a research protocol approved by the local research ethics committee. Intradetrusor BoNTA injections were performed in an outpatient setting using the previously described minimally invasive technique [15], injecting 300 U of BoNTA (BOTOX; Allergan, Irvine, CA, USA) at 30 different sites in the detrusor, sparing the trigone [1]. Flexible cystoscopic bladder biopsies were obtained at baseline pretreatment and during check flexible cystoscopy at 4 and 16 wk after each treatment session. Biopsies were obtained from a consistent bladder area 2 cm above and lateral to the ureteric orifices. Control tissue was obtained endoscopically from seven patients (all women; median age: 56 yr) being examined under anaesthesia prior to pelvic floor repair procedures. They had macroscopically normal bladders, no symptoms of OAB, and sterile urine at the time of endoscopy.
2.2.
Outcome measures
Outcome measures were the number of apoptotic cells in the bladder of OAB patients before and 4 wk after first intradetrusor BoNTA injections and apoptotic cells in the bladder of controls.
2.3.
Immunoreaction
Biopsy specimens were snap frozen in liquid nitrogen, embedded in optimal cutting temperature medium, and stored at 60 8C. Three sections per specimen were cut in a cryostat at 10 mm thickness and collected on 3-aminopropyltriethoxysilane-coated superfrost slides. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining, a well-established method for the rapid identification of apoptotic cells, was performed using the In Situ Cell Death Detection Kit, Fluorescein (FITC; Roche Diagnostics, Burgess Hill, UK). Frozen sections were fixed, permeabilised, and labelled following the manufacturer’s instructions using a 1:2 TUNEL dilution. Nuclei were counterstained with 40 , 6-diamidino-2-phenylindole (DAPI; Invitrogen, D1306, 1:50 000) during incubation in a humidified atmosphere in the dark. One negative control (a section without terminal deoxynucleotidyl transferase) was included in each experimental set. Human glioblastoma multiforme tissue (malignant glial brain tumour) was used as a positive control. Slides were cover slipped using Citifluor mounting medium (Agar Scientific, Stansted, UK). Immunolabelled sections were examined using a laser scanning confocal microscope (Zeiss LSM-510 META, Germany) equipped with an argon laser (458 nm, 488 nm, 514 nm), a helium-neon laser (543 nm, 633 nm), and a 405-nm diode laser. Fluorescence was excited at 488 nm (FITC) and 405 nm (DAPI) and 543 nm (Cy3) and recorded with separate detectors. Only those sections that displayed a complete and strictly transversal cross-section of the bladder urothelium and suburothelium were chosen for numeric determination of apoptotic cells. Whole-section images were taken with a 20 objective blinded to the sample. We first calibrated the detection system on a reference section and reused the
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Fig. 1 – (a) Suburothelial terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)–positive cell 4 wk after intradetrusor botulinum neurotoxin type A injections; (b) TUNEL-positive cells in a glioblastoma multiforme patient (positive control). Bar = 50 mm.
parameter setting on the LSM confocal microscope (pinhole, optical slice thickness; detector gain) for all images. Given the sparse distribution of TUNEL-positive cells within the bladder urothelium and suburothelium, the entire section was searched for TUNEL-positive cells.
2.4.
Statistical analysis
The McNemar test and the Fisher exact test were used to compare related and unrelated samples, respectively. A p value <0.05 was considered significant. Statistical analysis was performed using SPSS v.16.0 (SPSS, Chicago, IL, USA).
3.
Results
Apoptotic cells were found sparsely distributed exclusively in the suburothelial space (Fig. 1). The number of TUNELpositive cells was extremely low, with a maximum of two per section. Of the 12 OAB patients, 4 (33%) had TUNELpositive cells before and 3 (25%) after BoNTA treatment. Two of the seven controls (29%) showed TUNEL-positive cells. Comparison of TUNEL-positive cells (present vs absent) revealed no significant differences in OAB patients before (4 of 12, 33%) and after (3 of 12, 25%) BoNTA treatment ( p = 0.99). In addition, no significant differences were found in OAB patients compared to controls (OAB before BoNTA treatment [4 of 12, 33%] vs controls [2 of 7, 29%], p = 0.99; OAB after BoNTA [3 of 12, 25%] treatment vs controls [2 of 7, 29%], p = 0.99). 4.
Discussion
To our knowledge, this is the first study attempting to investigate BoNTA-induced apoptosis in the human bladder urothelium and suburothelium. The number of TUNELpositive cells in OAB patients with neurogenic detrusor overactivity resulting from MS before and after BoNTA treatment as well as in controls were similarly low, indicating that intradetrusor BoNTA injections do not cause apoptosis in the bladder layers we have studied.
The classical concept of the pharmacologic effect of BoNTA is that it 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 paralysis, however, and its effect of reducing urgency is especially welcomed by patients [16], a sensation believed to be afferently mediated. This notion has started an ongoing debate as to whether BoNTA has additional inhibitory effects on neuropeptides, neurotransmitters, receptors, and even structural surrogates in the afferent bladder pathway. In this context, rapid diffusion of the drug into the whole bladder wall from the injection site has been demonstrated [17]. In fact, several subcellular structures were shown to be numerically altered in conditions of OAB and to be eventually restored to normal levels upon BoNTA treatment. These include P2X3 and TRPV1 receptors on suburothelial afferent nerve fibres [12,18,19] as well as gap junctions and adherens junctions in the suburothelial myofibroblast layer [20,21]. Over the past decade, animal experiments have been performed to assess a putative apoptotic effect of BoNTA injections into various tissues of the lower urinary tract. Apoptosis is the process of programmed cell death that occurs in multicellular organisms. It involves a series of biochemical events that lead to a variety of morphologic changes, including cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. TUNEL has become one of the main methods for detecting apoptotic programmed cell death [22]. The assay relies on the presence of nicks in the DNA, which can be identified by terminal deoxynucleotidyl transferase, an enzyme that will catalyse the addition of dUTPs that are secondarily labelled with a marker. Mitochondria-associated apoptotic signalling has been shown to be upregulated during denervation in skeletal muscle of rats, suggesting that apoptosis has a physiologically important role in regulating denervation-induced muscle atrophy
882
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[23]. The magnitude of muscle fibre apoptosis is increased both by denervation and muscle paralysis and can be prevented by administration of trophic factors [24]. Doggweiler et al [25] were the first to inject rat prostates with varying doses of BoNTA (BOTOX) for histologic examination. They found a generalised atrophy of the glands and diffuse glandular apoptosis evident with TUNEL stain [25]. These findings were corroborated by Chuang et al [26], who found a significant increase in apoptotic cells (12-, 16-, and 22-fold) and a decrease in proliferative cells (38%, 77%, and 80%) as well as a1A-adrenergic receptor (13%, 80%, and 81%) 1 wk after injecting 5, 10, and 20 U of BoNTA, respectively [26]. Another recent rat study [11] suggested prostate atrophy induced by BoNTA in the rat may be the result of sympathetic nerve impairment and decreased adrenergic stimulation of the gland. Similarly, rats (repeatedly) injected with BoNTA into the bladder wall showed significant increases of apoptotic cells in the bladder urothelium and suburothelium but not the detrusor [14]. Similar degeneration of glands and ducts as well as apoptotic nuclei on TUNEL staining have been demonstrated in the nasal mucosal tissue of guinea pigs following BoNTA [27]. The finding from the animal experiments—that intradetrusor BoNTA injections result in apoptosis in the bladder urothelium and suburothelium but not in the detrusor [14]—raised the question of whether comparable cellular losses could be noted in the human situation. Gross histologic changes in the urothelium and suburothelium of patients with detrusor overactivity after one or repeat intradetrusor BoNTA injections have been investigated previously [13,28]. BoNTA injections did not appear to produce significant inflammatory changes, fibrosis, dysplastic changes, nerve degeneration, or sprouting in the human urothelium and suburothelium. In contrast to previous animal experiments, we failed to show an increased apoptosis rate in the urothelium or suburothelium after first intradetrusor BoNTA injections. In the animal study by Watanabe et al [14], adult male Sprague Dawley rats were injected with 10 U of BoNTA into the bladder wall, and bladders were harvested after 2 wk for histologic investigation, with routine TUNEL staining being performed. However, our study population comprised male and female patients who received 300 U of BoNTA at 30 different sites (ie, 10 U per site), with biopsies taken before and 4 wk after first intradetrusor BoNTA injections. As TUNEL staining is considered a relatively robust technique in which methodologic variations have little impact on results, discrepancies between the present study and the findings by Watanabe et al [14] may be explained by different species, gender, and follow-up. The notion of absent apoptosis in the human urothelium and suburothelium is important for drug safety of intradetrusor BoNTA administration. We are aware of the limitations of our study: It was neither randomised nor placebo controlled, but it was prospective and included controls without LUTS. In addition, we analysed data from the same patients (rather than groups of different patients) before and after BoNTA
injections. Because our findings are based on first intradetrusor BoNTA injections only, it is unclear whether the results could be extrapolated to repeat injections. In the present study, apoptotic cell assessment was focused on the urothelium and suburothelium but did not include the detrusor, which is rarely obtained in flexible cystoscopy biopsies. 5.
Conclusions
First intradetrusor BoNTA injections for treating refractory neurogenic OAB in patients with MS were highly effective, but the mechanism of action does not appear to be by apoptosis, an effect that is so prominent in the prostate. Author contributions: Thomas M. Kessler had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Kessler, Fowler, Roosen. Acquisition of data: Kessler, Roosen. Analysis and interpretation of data: Kessler, Roosen. Drafting of the manuscript: Kessler, Roosen. Critical revision of the manuscript for important intellectual content: Khan, Panicker, Elneil, Brandner, Fowler. Statistical analysis: Kessler. Obtaining funding: None. Administrative, technical, or material support: Elneil, Brandner. Supervision: Brandner, Fowler. 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: Thomas M. Kessler, Sohier Elneil, and Clare J. Fowler have acted as consultants for Medtronic. Clare J. Fowler is recipient of unrestricted educational grants from Allergan Ltd and has also acted as consultant for Allergan Ltd. Clare J. Fowler was the recipient of an independent research grant from Pfizer, Inc. Funding/Support and role of the sponsor: Allergan Ltd UK was the gratis provider of BOTOX used in this study. Allergan Ltd UK was not involved in the design and conduct of the study; collection of the data; management of the data; analysis and interpretation of the data; or the preparation, review, and approval of the manuscript. Thomas M. Kessler was supported by a grant of the Swiss National Science Foundation. Alexander Roosen was supported by a grant of the German Research Foundation. This work was undertaken at the University College London Hospitals (UCLH)/University College London (UCL), which received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme.
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