european urology 53 (2008)

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1 european urology 53 (2008) available at journal homepage: Neuro-urology Histological Changes in the Urothelium and Suburothelium of Human Overactive Bladder following Intradetrusor Injections of Botulinum Neurotoxin Type A for the Treatment of Neurogenic or Idiopathic Detrusor Overactivity Apostolos Apostolidis a,b, Thomas S. Jacques c, Alex Freeman d, Vinay Kalsi a,b, Roshni Popat b, Gwendoline Gonzales b, Soumendra N. Datta a,b, Shabnam Ghazi-Noori a, Sohier Elneil b, Prokar Dasgupta b,e, Clare J. Fowler a,b, * a Institute of Neurology, UCL, London, United Kingdom b Department of Uro-Neurology, The National Hospital for Neurology and Neurosurgery, UCLH, London, United Kingdom c Neural Development Unit, Institute of Child Health, UCL, London, United Kingdom d Department of Histopathology, University College Hospital, London, United Kingdom e Department of Urology, Guys and St Thomas Hospitals, London, United Kingdom Article info Article history: Accepted February 28, 2008 Published online ahead of print on March 7, 2008 Keywords: Botulinum toxin Bladder Detrusor overactivity Histology Inflammation Abstract Background: We examined, for the first time in a prospective study, the histological changes in the urothelium and suburothelium of patients with neurogenic (NDO) or idiopathic detrusor overactivity (IDO) after one or repeat treatments with intradetrusor BoNTA. Methods: Flexible cystoscopic bladder biopsies were obtained from patients with urodynamically proven intractable spinal NDO or IDO before and 4 and 16 wk after one or repeat treatments with intradetrusor injections of BOTOX1 (NDO 300 U, IDO 200 U). Specimens were stained for haematoxylin-eosin and analysed blindly for inflammatory changes, fibrosis, hyperplasia, and dysplasia in the urothelium and suburothelium. Statistical comparisons were significant at p values less than Results: Signs of chronic inflammation were found in 59.1% of baseline biopsies (65.6% of NDO vs. 50% of IDO, p = 0.049), 67.6% of post first biopsies and 86.4% after repeat injections. The two groups were comparable for degree of baseline inflammation, which did not change significantly after first injection and up to 16 wk after a third injection. Mild fibrosis was found in 2.2% of biopsies examined, equally before and after treatment, but not after repeat injections. No dysplasia or hyperplasia was identified. Eosinophils were identified more frequently in biopsies taken after repeat injections compared with the post first injection and baseline biopsies (x 2 = 8.23, p = 0.018). No difference existed between NDO and IDO bladders. Conclusions: BoNTA injections do not appear to be producing significant inflammatory changes, fibrosis, or dysplastic changes in human bladder urothelium/suburothelium after a single injection and in a limited number of repeat treatment biopsies. The presence of eosinophils might be treatment-related, because they were mostly found in post-treatment biopsies. # 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. The National Hospital for Neurology and Neurosurgery, Department of Uro-Neurology, Mailbox 71, Queen Square, London WC1N 3BG, UK. Tel ; Fax: address: c.fowler@ion.ucl.ac.uk (C.J. Fowler) /$ see back matter # 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 1246 european urology 53 (2008) Introduction Several studies attest to the highly beneficial effect of intradetrusor injections of botulinum toxin A (BoNTA) when used to treat intractable human detrusor overactivity (DO) of either neurogenic or idiopathic origin (NDO/IDO) [1 3]. Comparable marked improvements in the symptoms of patients with NDO or IDO [4] were accompanied by equal recoveries in their quality of life [5]. The treatment s efficacy in both NDO and IDO was shown to be sustained with repeat injections [6,7]. After 8 yr of use, the few reported clinical adverse events were generally mild and transient in nature [8]. Few studies have examined the histopathological effect of BoNTA injections, either in the skeletal or smooth muscle; existing studies have reported conflicting results. Bladders of spinal cord injured rats treated with BoNTA showed significantly less fibrosis and hyperplasia compared with untreated ones, independent of the time interval between spinal cord transection and BoNTA treatment [9]. Pretreatment with BoNTA also reduced inflammation-associated oedema in the hind paw of a rat model of formalin-induced inflammatory pain [10]. In contrast, no change was found in bladder mast cell counts and leucocyte infiltration following BoNTA treatment in a rat model of chemical cystitis [11], whereas post-bonta severe inflammation and mild fibrosis were shown in animal oesophagus [12]. In humans, a retrospective study has investigated excised neurogenic overactive bladders for histological changes that could be associated with intradetrusor BoNTA injections versus no treatment [13]. Specimens from bladders previously treated with BoNTA showed significantly less fibrosis, but no differences in inflammation and oedema compared with untreated ones, despite comparable degrees of inflammation, oedema, and fibrosis in the two groups. A trend for less fibrosis and oedema was noticed in bladder specimens from patients who were considered to have responded to BoNTA treatment compared with non-responders [13]. An earlier ultrastructural study of human detrusor biopsies from patients with neurogenic bladders successfully treated with BoNTA had shown no significant changes in the structure of muscle cell fascicles, width of intercellular space, or number or type of muscle cell junctions or collagen deposit. These results were maintained until the time of relapse from a single treatment [14]. To date, no study has reported on the effect of BoNTA on the histological features in patients with IDO, and histological safety data on repeat injections are very limited. We examined, for the first time in a prospective study, the histological changes in the urothelium and suburothelium after one or repeat treatments with intradetrusor BoNTA in patients with IDO, compared with patients with spinal NDO. 2. Methods 2.1. Patients Flexible cystoscopic bladder biopsies were obtained from patients with intractable spinal NDO or IDO before and after one or repeat treatments with intradetrusor injections of BoNTA, as part of an ethically approved, single-centre, openlabel study designed to look into the mechanism of action of BoNTA when used to treat human DO. All patients had had a history of urgency, frequency and/or urgency incontinence for more than a year and could not be managed by oral medication with or without use of clean intermittent selfcatheterisation, or suffered intolerable adverse events from therapeutic doses of anticholinergics. Symptom severity was documented in 4-d bladder diaries [4]. The presence of phasic or terminal DO [15] was shown by standard subtraction cystometry performed in the absence of oral medication that could affect bladder function. Patients with reduced bladder compliance (increased detrusor pressure at low cystometric capacity) [15] in the absence of obvious DO were excluded from the study. Neurogenic patients were treated with 300 units BOTOX 1 (Allergan Pharmaceuticals, Ireland) and idiopathic patients with 200 units injected at 30 and 20 sites respectively, with the use of a minimally invasive outpatient technique avoiding the trigone [16]. Follow-up biopsies were obtained during check flexible cystoscopy, 4 and 16 wk after each treatment session, and during clinical relapse in patients receiving repeat treatment. Relapse was confirmed by resurgence of DO upon standard voiding cystometry and symptom deterioration documented in 4-d bladder diaries. All biopsies were taken from a consistent bladder area, 2 cm above and lateral to the ureteric orifices, as previously described [4]. A dipstick-clear (negative for leucocytes and nitrates) urine specimen had to be established before every cystoscopy, to minimise the possibility of an active urinary tract infection Histology Four biopsies were obtained per patient during each cystoscopy performed at baseline, 4 wk, and 16 wk. Two biopsies were used for electron microscopy, one for immunohistochemistry [4] and one for histology. All histology biopsy specimens were snap-frozen in liquid nitrogen, embedded in optimal cutting temperature compound, and kept at 60 8C, until frozen 10-mm-thick sections were cut in a cryostat and collected on superfrost aminopropyltriethoxysilane-coated slides. Sections were post-fixed in 4% w/v paraformaldehyde in phosphate-buffered saline (0.05 mol/l phosphate; 0.9% w/v saline; ph 7.2) for 30 min and stained for haematoxylin-eosin. Two pathologists (T.J. and A.F.) who were blinded to all clinical treatment related information (NDO or IDO, pre- or posttreatment) examined the sections for inflammatory changes

3 european urology 53 (2008) (acute or chronic); site of inflammation; and presence of fibrosis, hyperplasia and dysplasia in the urothelium and suburothelium. The degree of inflammation was assessed with the use of a qualitative grading scale (0 = none, 1 = mild, 2 = moderate, 3 = severe) as used in previous studies [17,18]. An assessment of fibrosis of the lamina propria was made on haematoxylin-eosin stained sections Statistics For statistical purposes results were expressed as mean stanstandard error; the Wilcoxon matched pairs test was used for intragroup comparisons of pre- with post-treatment values, whereas the nonparametric Mann-Whitney U test was used for baseline intergroup comparisons. The chi-square and Fisher exact tests were used to assess differences between quality variables. p values less than 0.05 were considered significant. 3. Results Fig. 1 Percentage of inflammation-positive biopsies before treatment and after one or repeat injections in the whole study population. A trend for increased evidence of inflammatory signs was noted in biopsies taken after the first and repeat injections compared with those obtained before treatment Biopsy data A total of 252 biopsies from 88 treated patients were examined; 73 biopsies were considered inadequate for histological assessment because of problems related to the size and processing of the tissue and were excluded from results analysis. The remaining 179 biopsies obtained from 79 patients, 123 (63.1%) from 51 NDO patients and 56 (26.9%) from 28 IDO patients, constituted the study sample. The majority of adequate specimens (n = 157, 87.7%) were taken before (n = 49, 27.4%) and after the first injection (n = 108, 60.3%). The remaining 22 (12.3%) specimens were taken after repeat injections. Urothelium was present in all but 2 biopsies (98.9%), whereas smooth muscle could be found in only 33 of 179 (18.4%) specimens Histological findings in total DO population Inflammation Signs of chronic inflammation were present in 125 of 179 (69.8%) biopsies, whereas mixed acute and chronic inflammation was present in 1 (0.5%). The inflammatory cells were in the lamina propria in 123 of 125 (98.4%) biopsies and the urothelium in 22 of 125 (17.6%). Chronic inflammation was identified in 29 of 49 (59.1%) baseline biopsies, 73 of 108 (67.6%) biopsies after the first injection, and 19 of 22 (86.4%) biopsies after repeat injections (Fig. 1). A linear trend was identified for increasing frequency of inflammatory findings in biopsies after the first and repeat injections compared with baseline ones (x 2 = 4.69, p = 0.03). However, the degree of inflammation (inflammation score) did not change at 4 wk, 16 wk, and relapse time post first injection ( vs vs vs ; Wilcoxon matched pairs test p = 0.72, 0.13 and 0.25, respectively) despite the increase seen in mean values (Table 1, Fig. 2). No change in inflammatory score was found in six patients with available biopsies after a second injection and in four patients with available biopsies post third injection compared with baseline (Table 1). Only one patient had an Table 1 Changes in mean inflammatory score at various time points after BoNTA treatment and comparisons with baseline values First injection Second injection Third injection Pre 4 wk post 16 wk post Pre 4 wk post 16 wk post Pre 4 wk post 16 wk post Mean SEM No of biopsies p value NA Pre, pre-injection; post, post-injection; SEM, standard error of the mean; NA, not applicable. No significant change was seen in degree of inflammation in biopsies taken from the whole study population after the first and repeat injections compared with those obtained before treatment.

4 1248 european urology 53 (2008) Fig. 2 No significant change was seen in degree of inflammation in biopsies taken from the whole study population after the first and repeat injections compared with those obtained before treatment. adequate set of biopsies taken after a fourth injection Eosinophils Eosinophils were found in 20 biopsies from 18 patients; 18 were follow-up biopsies (18 of 130, 13.8%); only 2 had been obtained at baseline (2 of 49, 4.1%). Eosinophils were seen more often after repeat injections (6 of 22 biopsies, 27.3%) than either after the first injection (12 of 108, 11.1%) or in baseline biopsies (x 2 = 8.23, 2 degrees of freedom, p = 0.018; Fig. 3). However, in 6 patients no eosinophils were found in further follow-up biopsies, whereas in another 10 patients further biopsies were unavailable Fibrosis Mild fibrosis was found in 4 of 179 (2.2%) of all biopsies examined. No difference was seen in the incidence of fibrosis between baseline (1 of 49 biopsies, 2%; NDO patient) and follow-up biopsies Fig. 3 Percentage of eosinophil-positive biopsies before treatment and after one or repeat injections in the whole study population. There was increased evidence of eosinophils in biopsies taken after the first and repeat injections compared with those obtained before treatment. Table 2 Changes in mean inflammatory score at 4 wk, 16 wk, and relapse time after a single BoNTA treatment, and comparisons with baseline values in NDO versus IDO study subgroups NDO IDO Pre first injection 4 wk post 16 wk post Pre second injection Pre first injection 4 wk post 16 wk post Pre second injection Mean SEM No of biopsies p value NA (Pre vs. 4 wk, 16 wk) p value (NDO vs. IDO) NDO, neurogenic detrusor overactivity; IDO, idiopathic detrusor overactivity; post, post-injection; SEM, standard error of the mean; Pre, preinjection; NA, not applicable. A progressive increase was noted in absolute values in the NDO population, which became significant only at 16 wk after treatment. No change was seen in the IDO population at either 4 or 16 wk post-bonta. Numbers of evaluable biopsies at relapse time were too small for safe statistical comparisons.

5 european urology 53 (2008) Fig. 4 Mean inflammatory score did not change in biopsies obtained after the first injection in either the NDO or IDO subgroup of patients, although a progressive increase was noted in absolute values in the NDO population. (3 of 130, 2.3%; 2 IDO and 1 NDO patient). The presence of fibrosis was not related to reduced compliance; 3 of 4 patients had an increase in extrapolated compliance after treatment. In addition, no fibrosis was found in biopsies taken after repeat injections Dyplasia or hyperplasia No definitive evidence of urothelial dysplasia and hyperplasia was found Histological findings in NDO versus IDO Chronic inflammation was seen more commonly in NDO patients (65.6% vs. 50%, p = Fisher exact test), but no difference existed between NDO and IDO in degree of baseline inflammation ( vs , p = 0.59; Table 2; Figs. 4 and 5). No significant change was seen in inflammatory score at 4 wk, 16 wk, and relapse time post first injection in either NDO (Wilcoxon matched pairs test, p = 0.49, 0.11 and 0.25, respectively) or IDO group ( p = 0.84 and 1.00, respectively); numbers were inadequate for analysis at relapse time) (Table 2; Fig. 4). Absolute mean values of inflammatory score were higher in NDO biopsies at all time points, but statistical comparison between NDO and IDO scores at 4 wk, 16 wk, and relapse time after the first injection showed no difference at 4 wk and relapse time. Inflammatory score was significantly higher in NDO bladders at 16 wk (Table 2, Figs. 4 and 5). The number of evaluable biopsies after the second and third injection was inadequate for a subgroup NDO versus IDO comparison. Eosinophils were seen in 17 of 123 (13.8%) NDO biopsies as opposed to 3 of 56 (5.3%) IDO biopsies. The difference, however, was not significant ( p = 0.12, Fisher exact test). 4. Discussion This is the first study, to our knowledge, to prospectively examine the histopathological changes associated with intradetrusor BoNTA treatment in patients with IDO and in comparison with those with spinal NDO. No significant fibrotic, dysplastic, hyperplastic, or inflammatory changes were seen after one injection in either patient group and in a limited number of biopsies examined after repeat treatments, although different doses were used in the two patient groups, suggesting that the effect of BoNTA is independent of the underlying pathophysiology of DO and also of the dose of the toxin used, at least for these two particular doses. A significant proportion of baseline biopsies had signs of chronic inflammation in both patient groups, although this finding was somewhat more common in patients with NDO. A possible explanation for this difference might be the use of clean intermittent self-catheterisations in the majority of patients with NDO, whereas patients with IDO were not using any at baseline. Previous histopathological studies had already shown that presence of histological inflammation is common at least in patients with NDO [13,17], especially those with spinal cord injury [19,20]. It has been previously hypothesised that increased expression of sensory receptors, such as the vanilloid receptor TRPV1 and the purinergic receptor P2X 3, in the human neurogenic overactive bladder is associated with an increase in histological inflammation [17,18]. Both animal and human studies [21 23] support an aetiological connection between inflammation and TRPV1 or P2X 3 expression in peripheral organs. Because intradetrusor BoNTA injections have been shown to drastically reduce the levels of both TRPV1 and P2X 3 in the suburothelium of human bladders with DO [24], our

6 1250 european urology 53 (2008) Fig. 5 Top panel: representative photographs from (a) NDO specimen and (b) IDO specimen at baseline. No significant difference existed between NDO and IDO bladders in degree of baseline inflammation. Photographs in the bottom panel show the presence of inflammatory findings in NDO specimens after BoNTA treatment. Arrows indicate increased numbers of lymphocytes at 4 wk post-bonta (c), and a larger number of lymphocytes forming a well-circumscribed node at 16 wk post-bonta (d). Despite the increase in mean values, the inflammatory score remained unchanged compared with baseline. All photos were taken at T40 magnification. findings imply that the mechanism of action of BoNTA on these receptors is unrelated to an effect on degree of cellular-mediated inflammation, but more possibly related to a reduction in neurogenic inflammation associated with release of neuropeptides such as substance P and Calcitonin Gene Related Peptide (CGRP) [10,25,26]. However, although severity of inflammation remained unchanged even after three repeat injections, there was a trend for increasing frequency of inflammatory findings in biopsies taken from patients who were repeatedly treated with the toxin. Such findings could be partly explained by the mechanical damage at the multiple injection sites, expected to be higher in patients who had repeat treatments. Mean values of the inflammatory score were also higher in NDO patients who received injections at more sites than IDO patients, although the differences were not statistically significant. It is uncertain whether post-treatment inflammation is a drug-related reaction, especially since there were no obvious differences between different drug doses used in the two patient groups. There are diverse reports about the inflammatory response generated by the toxin when it is injected in the smooth or skeletal muscle, or the effect of the drug on existing inflammation. In neurogenic human bladders BoNTA had no effect on the degree of inflammation, independent of response to treatment [13]. In animal models of chronic bladder inflammation, BoNTA has been reported to reduce inflammatory mediators or markers both peripherally and centrally; in cyclophosphamide-induced rat bladder chronic cystitis BoNTA dramatically reduced the enhanced release of adenosine triphosphate from the urothelium [27] and the c-fos expression in the L6 and S1 segments of the spinal cord [28], suggesting an inhibitory neural afferent effect. The effect on cellular-mediated, as opposed to neurogenic inflammation, however, was not examined. No additional inflammatory changes were induced by BoNTA in human internal anal sphincters of children with Hirschprung s disease [29]. Similarly, in a human model of cutaneous inflammation, BoNTA injec-

7 european urology 53 (2008) tions showed no anti-inflammatory effect [30]. Contradicting these reports, a study of animal oesophagus treated with BoNTA displayed severe post-treatment inflammation [12]. There was a clear increase in the presence of eosinophils in post-treatment biopsies, particularly in those obtained from patients who had had repeat BoNTA injections. It is thus valid to hypothesise that increasing identification of eosinophils in biopsies after treatment is drug-related. Although it is possible that these observations are part of a transient response to the injection of a foreign protein into the detrusor/urothelium in a small subset of our NDO/IDO patient population (eg, to a component of the botulinum toxin protein complex or its excipients), allergic reactions have been very rarely reported in the literature for other methods of botulinum toxin administration (ie, intramuscular or intradermal injection) [31]. Eosinophils are thought to be secreting vesicularly stored inflammatory mediators via SNARE-dependent exocytosis, but the proteins of the SNARE-complex responsible for vesicle docking in eosinophils (SNAP-23 and VAMP-2) are not a substrate for BoNTA s action [32]. The clinical significance of eosinophil response in our study remains uncertain; in the majority of patients with one eosinophil-positive biopsy, this finding could not be confirmed at further followups, at which times biopsies were either negative for eosinophils or missing for those patients. Fibrosis was not common in our set of patients and BoNTA treatment did not induce any significant de novo fibrosis. This finding is complementary to a previous ultrastructural study of the detrusor muscle showing lack of collagen deposit after BoNTA injections [14]. In contrast, a recent histopathological study of full-thickness specimens from neurogenic human bladders showed significantly less fibrosis in those previously treated with BoNTA compared with untreated ones [13]. In this study, all examined bladders displayed some degree of fibrosis as opposed to only 2.2% of all biopsies analysed in our study. However, muscle was present in less than 20% of our biopsies; thus, the two studies are not fully comparable in this aspect. We did not find any definitive evidence of hyperplasia or dysplasia in either pre- or posttreatment biopsies. However, the value of this finding may be limited by the size and processing characteristics of the biopsies and the small number of evaluable specimens after repeat treatments. Further studies may be required to exclude the possibility of such changes in the long term. No post- BoNTA malignancy was identified in the previous histopathological study by Comperat et al [13] in neurogenic bladders only. The findings of both studies support the safety of the treatment when used repeatedly. Earlier studies in spinal-cord injured rats had displayed a reduced degree of urothelial hyperplasia following treatment of the bladder with BoNTA [9]. 5. Conclusions BoNTA injections did not induce significant histopathological changes in the urothelium and suburothelium of the human overactive bladder after one or repeat treatments. No significant fibrosis, or hyperplastic or dysplastic changes were seen post-treatment, and severity of histological inflammation remained unchanged in both NDO and IDO bladders. Because eosinophils were found mainly in post-treatment biopsies, the presence of eosinophils might be drug-related, although they were not seen consistently in the further followup biopsies, when available. Thus, despite the relatively small number of biopsies examined after a second, third, and fourth injection, BoNTA appears to be a safe treatment for human detrusor overactivity, complementing previous ultrastructural detrusor findings. Further long-term studies are needed to confirm these findings. Author contributions: Clare J. Fowler had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Financial disclosures: C.J. Fowler, A. Apostolidis, S. Ghazi-Noori, and P. Dasgupta are recipients of unrestricted educational grants from Allergan Ltd. C.J. Fowler, A. Apostolidis, and P. Dasgupta have also acted as consultants for Allergan Ltd. Allergan Ltd was the gratis provider of BOTOX 1 used in this study for research purposes. C.J. Fowler, A. Apostolidis, and S. Datta were recipients of an Independent Research Grant from Pfizer Inc. C.J. Fowler, P. Dasgupta, and V. Kalsi were the recipients of The Multiple Sclerosis Society of Great Britain and Northern Ireland Project Grant. Funding/Support and role of the sponsor: Pfizer Inc Independent Research Grant (C.J. Fowler, A. Apostolidis. and S. Datta); Allergan Ltd Unrestricted Educational Grant and consultations (C.J. Fowler, A. Apostolidis, S. Ghazi-Noori, and P. Dasgupta); Allergan Ltd, gratis provider of BOTOX 1 for Research; The Multiple Sclerosis Society of Great Britain and Northern Ireland Project Grant (C.J. Fowler, P. Dasgupta and V. Kalsi). This work was undertaken at UCLH/UCL who received a proportion funding from the Department of Health s NIHR Biomedical Research Centres funding scheme. Acknowledgement statement: We would like to thank Ms Aviva Petrie, Head of Biostatistics Unit and Senior Lecturer at UCL Eastman Dental Institute, for her invaluable contribution in the statistical analysis of the data presented in this manuscript.

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P2X3-immunoreactive nerve fibres in neurogenic detrusor overactivity and the effect of intravesical resiniferatoxin. Eur Urol 2004;46: [19] Janzen J, Vuong PN, Bersch U, Michel D, Zaech GA. Bladder tissue biopsies in spinal cord injured patients: histopathologic aspects of 61 cases. Neurourol Urodyn 1998;17: [20] Janzen J, Bersch U, Pietsch-Breitfeld B, et al. Urinary bladder biopsies in spinal cord injured patients. Spinal Cord 2001;39: [21] Ji RR, Samad TA, Jin SX, Schmoll R. Woolf CJ. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 2002;36: [22] Yiangou Y, Facer P, Baecker PA, et al. ATP-gated ion channel P2X(3) is increased in human inflammatory bowel disease. Neurogastroenterol Motil 2001;13: [23] Yiangou Y, Facer P, Dyer NH, et al. Vanilloid receptor 1 immunoreactivity in inflamed human bowel. Lancet 2001;357: [24] 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: [25] Welch MJ, Purkiss JR, Foster KA. Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins. Toxicon 2000;38: [26] 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(4 pt 1): [27] Smith CP, Vemulakonda VM, Kiss S, Boone TB, Somogyi GT. Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: Effect of botulinum toxin A. Neurochem Int 2005;47: [28] Vemulakonda VM, Somogyi GT, Kiss S, et al. Inhibitory effect of intravesically applied botulinum toxin A in chronic bladder inflammation. J Urol 2005;173:621 4.

9 european urology 53 (2008) [29] Langer JC, Birnbaum EE, Schmidt RE. Histology and function of the internal anal sphincter after injection of botulinum toxin. J Surg Res 1997;73: [30] Sycha T, Samal D, Chizh B, et al. A lack of antinociceptive or antiinflammatory effect of botulinum toxin A in an inflammatory human pain model. Anesth Analg 2006;102: [31] Mezaki T, Sakai R. Botulinum toxin and skin rash reaction. Mov Disord 2005;20:770. [32] Logan MR, Odemuyiwa SO, Moqbel R. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion. J Allergy Clin Immunol 2003;111:923 32, quiz 933. Editorial Comment on: Histological Changes in the Urothelium and Suburothelium of Human Overactive Bladder Following Intradetrusor Injections of Botulinum Neurotoxin Type A for the Treatment of Neurogenic or Idiopathic Detrusor Overactivity Axel Haferkamp Department of Urology, University of Heidelberg, Germany Axel_Haferkamp@med.uni-heidelberg.de Apostolidis and co-authors [1] evaluated bladder biopsies from patients with neurogenic and idiopathic detrusor overactivity for histological changes caused by the injection of botulinum toxin type A. They demonstrated that the toxin does not produce significant inflammatory changes, fibrosis, or dysplastic changes in the human urothelium or suburothelium after a single injection and even after repeated injections, although the number of patients in the latter group is limited. Why is this study important and worth reading? When searching the Medline for botulinum toxin and detrusor overactivity, I found 110 papers, 39 of which were reviews. Most of the others were papers with level 4 evidence dealing with the clinical efficacy of the toxin. Almost nothing substantial has been reported on safety data, especially on structural changes of the bladder wall caused by the toxin itself or by the injection technique (possible microscaring). Because the toxin is not approved by the U.S. Food and Drug Administration or the European Medicines Agency for use in bladder dysfunction and because long-term follow-up evaluations, especially after repeated injections, are limited, basic research to evaluate the safety of the drug is needed. Only three papers evaluated structural bladder changes caused by the toxin, but two of those evaluated only bladder specimens from patients with neurogenic detrusor overactivity [2 4]. Apostolidis et al s study is the first to evaluate structural changes of the urothelium and suburothelium in patients with idiopathic detrusor overactivity treated with botulinum toxin type A. Their results are in accordance with the studies of Comperat et al and Haferkamp et al, who also did not observe histological changes, either inflammatory or fibrotic. It seems that a single intradetrusor injection of the toxin causes only a minor trauma and does not result in a permanent structural change of the bladder wall. Whether repeated injections cause bladder wall changes remains to be seen because, even in the present study, the patient numbers are too small from which to draw definitive conclusions. References [1] Apostolidis A, Jaques TS, Freeman A, et al. Histological changes in the urothelium and suburothelium of human overactive bladder following intradetrusor injections of botulinum neurotoxin type A for the treatment of neurogenic or idiopathic detrusor overactivity. Eur Urol 2008;53: [2] 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: [3] 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 82, discussion [4] Compérat E, Reitz A, Delcourt A, Capron F, Denys P, Chartier-Kastler E. Histologic features in the urinary bladder wall affected from neurogenic overactivity a comparison of inflammation, oedema and fibrosis with and without injection of botulinum toxin type A. Eur Urol 2006;50: DOI: /j.eururo DOI of original article: /j.eururo