What's known on the subject? and What does the study add?
A single set of botulinum toxin A (BoNT-A) injections relieves clinical symptoms of interstitial cystitis/bladder pain syndrome (IC/BPS), but lacks long-term effect. An inadequate anti-inflammatory effect is likely to cause treatment failure.
The study shows that chronic inflammation and apoptotic signalling molecules are significantly reduced after repeated intravesical BoNT-A injection in patients with IC/BPS. It also shows that repeated BoNT-A injections are necessary to achieve greater success in the treatment of IC/BPS.
To investigate the mechanisms of action of botulinum toxin A (BoNT-A) treatment on interstitial cystitis/bladder pain syndrome (IC/BPS).
Patients and Methods
A total of 23 women with IC/BPS who received single intravesical BoNT-A injection were studied. Among them, 11 received three repeated injections every 6 months to improve their symptoms.
Bladder biopsy was obtained before each BoNT-A injection and the clinical symptoms and urodynamic variables were recorded.
Immunohistochemical (IHC) staining for TUNEL and mast cell activity, and western blotting analysis of tryptase, cytokines, Bax and phospho-p38 (p-p38) were carried out. We compared the clinical results and IHC data among baseline, single or repeated BoNT-A treatments.
Single BoNT-A injection improved clinical symptoms, pain score and daytime urinary frequency.
Mast cell activity and apoptotic cell count did not decrease significantly, while Bax and p-p38, but not tryptase, decreased significantly after a single BoNT-A injection.
The 11 patients who received three repeated BoNT-A injections had significantly lower pain scores than the remaining patients (mean [SD]: 5.80 [2.27] vs. 3.03 [2.30], P = 0), glomerulation degree (mean [SD]: 1.80 [1.06] vs. 1.20 [1.06], P = 0.026) and global response scores (mean [SD]: 0.30 [0.92] vs. 1.20 [1.06], P = 0) after treatment.
Tryptase, Bax, p-p38 and apoptotic cell counts all decreased significantly.
25-kD synaptosomal-associated protein also decreased after BoNT-A treatments, which confirmed the therapeutic effect of repeated BoNT-A injections.
Chronic inflammation and apoptotic signalling molecules were significantly reduced after repeated BoNT-A injections in patients with IC/BPS.
The IHC improvement was associated with clinical symptom improvement.
Repeated BoNT-A injections are necessary to achieve a greater success rate in the treatment of IC/BPS.
Interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating, chronic disease characterized by urinary urgency, frequency, bladder and pelvic pain, has a negative impact on the quality of life of patients . Some investigators believe that IC/BPS is caused by chronic inflammation of the bladder [2-4]; however, the actual pathophysiology remains unclear. The most common clinical presentations are bladder and pelvic pain, glomerulations under cystoscopic hydrodistention and denudation or thinning of the bladder epithelium, suggesting that bladder inflammation and urothelial dysfunction occur in IC/BPS [1, 3].
Recent studies in rats showed that botulinum toxin type A (BoNT-A) inhibited sensory neuropeptide release, including substance P and calcitonin gene-related peptide, from isolated rat bladder preparations after cyclophosphamide-induced inflammation, suggesting that BoNT-A has a potential clinical benefit in the treatment of neurogenic inflammation . Intravesical injection of BoNT-A effectively improved frequency urgency and bladder pain symptoms in patients with IC/BPS . BoNT-A injection decreases bladder tissue nerve growth factor production and urinary nerve growth factor level, suggesting an anti-inflammatory response is achieved after intravesical BoNT-A treatment , but a single BoNT-A injection is effective only in the short-term management of IC/BPS . The beneficial effects of BoNT-A decrease progressively within a few months of treatment, and a long-term therapeutic effect of BoNT-A on IC/BPS has not been shown. Repeated injections of BoNT-A are required for efficacious treatment [8, 9].
We hypothesize that inhibition of neuroplasticity of the sensory fibres by repeated BoNT-A injections might have a beneficial therapeutic effect on the long-term relief of bladder pain and sensory urgency in patients with IC/BPS. However, the underlying mechanisms of action are not yet elucidated. The present preliminary study explored the mechanisms of action of BoNT-A treatment in patients with IC/BPS. We investigated the association between the clinical and immunohistochemical (IHC) characteristics of BoNT-A treatment in these patients, analysing inflammation and apoptotic signalling molecules after single and repeated BoNT-A injections.
Botulinum toxin type A acts by cleaving the 25-kD synaptosomal-associated protein (SNAP-25) complex in the presynaptic terminal, which prevents formation of the soluble N-ethyl malemide sensitive factor-attachment protein-receptor (SNARE) system. By this mechanism, the neurotransmitter vesicles cannot work at the presynaptic membrane, which decreases the release of neurotransmitter at the synaptic cleft and reduces nociceptive fibre discharge. We also investigated the changes in SNAP-25 expression after BoNT-A treatment to confirm the therapeutic effect in the bladder tissue.
Patients and Methods
Twenty-three women with characteristic symptoms of IC/BPS and glomerulations after cystoscopic hydrodistention were consecutively enrolled in the present study from January 2008 to December 2009. All patients had non-ulcerative type IC, were free of UTIs and were untreated for IC/BPS before undergoing cystoscopic hydrodistention. The O'Leary-Sant symptom score , a visual analogue score (VAS) for pain, data from a 3-day voiding diary of daytime frequency, nocturia and functional bladder capacity (FBC), urodynamic study and cystoscopic hydrodistention findings, such as maximum bladder capacity (MBC) and degree of glomerulation, were recorded at baseline and 6 months after treatment. The degree of glomerulation after hydrodistention was graded as 0, 1, 2 or 3, representing none, mild, moderate or severe glomerulations .
The study was approved by the Institutional Review Board and Ethics Committee of the Buddhist Tzu Chi General Hospital. Each patient was informed about the study rationale and procedures and written informed consent to participate in the study was obtained before the bladder procedures.
All patients underwent intravesical BoNT-A injection after failure of conventional therapy. They received intravesical injection of 100 U of BoNT-A (onabotulinumtoxin-A, Allergan, Irvine, CA, USA) under i.v. general anaesthesia in the operating room. Each vial of BoNT-A was diluted with 20 mL normal saline and 40 suburothelial injections were given. The injection needle was inserted into the urothelium at the posterior and lateral walls of the bladder, using a 23-gauge needle and a rigid cystoscopic injection instrument (22 Fr, Richard Wolf, Knittlingen, Germany). After BoNT-A injection, cystoscopic hydrodistention was performed to an intravesical pressure of 80 cm of water for 15 min and the MBC under hydrodistention was recorded .
After BoNT-A injection, patients were treated with antibiotics and analgesics for 1 week and monitored at the outpatient clinic. They received repeated BoNT-A injection every 6 months regardless of bladder symptom improvement. Before repeated BoNT-A injection, all patients reported their subjective treatment outcome using the global response assessment (GRA). A GRA increase of ≥2 or more indicated successful treatment; otherwise treatment was regarded as a failure . The present study was designed to investigate the therapeutic efficacy and the desensitization effect of four repeated BoNT-A injections on IC/BPS and to identify patients who might have a successful result or not when they were retreated.
Urodynamic studies were performed at baseline and every 6 months before the next BoNT-A injection. The urodynamic variables measured included the cystometric bladder capacity (CBC), maximum flow rate (Qmax), detrusor pressure (Pdet) and postvoid residual urine volume (PVR), O'Leary-Sant score and pain VAS. The MBC during hydrodistention, and degree of glomerulation were recorded at baseline and at each repeated BoNT-A injection.
The patients underwent bladder biopsies −2 cm above the interureteric ridge on the lateral and posterior wall and only bladder mucosa was obtained. The specimens with grade 1–3 glomerulations were used in this study. The bladder biopsy specimens were sent to the pathology department for haematoxylin and eosin staining to exclude the possibility of carcinoma in situ. Specimens were also stored, frozen in liquid nitrogen, for further investigation. The IHC studies, including TUNEL, tryptase, phospho-p38 (p-p38) and Bax, were also compared between baseline and 6 months after the first BoNT-A injection and after three repeated BoNT-A injections. In addition, western blotting for Synaptic Vesicle Protein-2 (SV-2) and SNAP-25 were performed to confirm the therapeutic effects of BoNT-A on the bladder tissues.
IHC Staining for Mast Cell Activity
The urinary bladder specimens were immersed and fixed for 1 h in an ice-cold solution of 4% formaldehyde in PBS (pH 7.4). Next, they were rinsed with ice-cold PBS containing 15% sucrose for 12 h. Biopsy specimens were embedded in optimum cutting temperature (OCT) medium and stored at −80 °C. Four sections per specimen were cut using a cryostat at a thickness of 8 μm and collected on new silane III-coated slides (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). Sections were post-fixed in acetone at −20 °C and blocked with rabbit serum. The sections were incubated overnight at 4 °C with primary antibodies to anti-human p-p38 antibodies (Cell Signalling Technology, Danvers, MA, USA) and anti-human tryptase (Chemicon USA & Canada, Temecula, CA, USA). After rinsing the sections with 0.1% Tween-20 in PBS, rabbit anti-mouse conjugated fluorescein isothiocyanate secondary antibodies (DakoCytomation Denmark A/S, Glostrup, Denmark) were applied to the sections and incubated for 1 h. Then the sections were stained using the TUNEL assay kit as described below. Finally, the sections were counterstained with DAPI (Sigma Chemical Company, St. Louis, MO, USA). Negative controls included the isotype of the primary antibody. We obtained the mean, maximum, range and SD of staining intensity and percent positive area measurements using three randomly selected hot spots within each specimen.
The urinary bladder specimens were fixed by immersion for 1 h in an ice-cold solution of 4% formaldehyde in PBS (pH 7.4) and rinsed with ice-cold PBS containing 15% sucrose for 12 h. The specimens were embedded in OCT medium (Miles, Elkhart, Indiana) and then stored at −80 °C. The tissues were cut into 8-μm thick slices and were collected onto new silaneIII-coated slides. The sections were incubated with 100 μL of 20 μg/mL proteinase K (Calbiochem, Darmstadt, Germany) at room temperature for 20 min and washed with PBS. The sections were covered with 100 μL of terminal deoxynucleotidyl transferase (TdT) equilibration buffer (Calbiochem) and incubated at room temperature for 30 min. After carefully blotting the 1X TdT equilibration buffer from the specimens, we applied TdT labelling reaction mixture (Calbiochem) onto the specimens and incubated them for 90 min at 37 °C. The positive control, apoptotic HL-60 cells (Calbiochem) were treated with 1 μg/μL of DNase I in TBS (1 mM MgSO4) at room temperature for 20 min (Promega Corp., Madision, WI, USA). The negative control was generated by substituting dH2O for the TdT enzyme in the reaction mixture. After washing with PBS, the cells were mounted using Fluorescein-FragELTM mounting media (Calbiochem). The total cell population was visualized using a 330–380-nm filter for DAPI, while the labelled nuclei were visualized using a standard fluorescein filter (465–495 nm).
Human bladder biopsy specimens were homogenized in liquid nitrogen and the human primary urothelial cells were washed twice in ice-cold PBS and then lysed for 10 min on ice using PRO-PREPTM Protein Extraction Solution (iNtRON Biotechnology, Inc., Seongnam, South Korea) supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche Diagnostics). Proteins were separated on SDS-PAGE, and Bax, p-p38, tryptase (Cell Signaling Technology, Danvers, MA, USA) and SV-2, SNAP-25 were evaluated via western blotting using α-tubulin (Cell Signaling Technology) as a loading control.
The intensities of proteins in the western blots were quantified using Image J processing . Differences in expressions of proteins in IC/BPS patients at baseline and 6 months after the first BoNT-A injection or three repeated injections were analysed using the paired t-test. The Kruskal–Wallis test was used for comparison of clinical and IHC data between successful and failed treatment subgroups. All calculations were performed using SPSS for Windows, version 10.0. A P value of <0.05 was considered to indicate statistical significance.
A total of 23 women were enrolled in the present study. Their mean (SD) age was 46.6 (2.2) years. At 6 months after the first BoNT-A injection, 10 reported having a successful treatment (GRA ≥ 2), while 13 reported failed treatment (GRA < 2). The mean age of the women in the two groups was similar. A total of 11 patients received three repeated BoNT-A injections and had corresponding bladder biopsies 6 months after the third injection (before the fourth injection).
Table 1 shows the clinical and IHC data in patients with IC/BPS at baseline and 6 months after the first BoNT-A injection. The O'Leary-Sant score, VAS pain score and daytime frequency decreased significantly after BoNT-A injection, but the urodynamic variables, degree of glomerulation and MBC did not improve significantly. Although the tryptase content and apoptotic cell count did not significantly decrease after a single BoNT-A injection, the apoptotic signalling molecules Bax and p-p38 decreased significantly.
Table 1. The clinical, IHC and western blotting characteristics at baseline and 6 months after single BoNT-A injection in 23 patients with IC/BPS.
When we compared the clinical and IHC data after the first BoNT-A injection between the successful and failed groups, the difference in clinical and IHC variables did not differ significantly, except for clinical symptom score and GRA (Table 2). The mast cell and apoptotic cell counts did not decrease significantly after a single BoNT-A injection, but the reductions in these cell counts were remarkable after three repeated BoNT-A injections (Fig. 1A) Co-localization of p-p38 and mast cell activity was also noted (Fig. 1B). After a single BoNT-A injection, tryptase expression was not significantly decreased, while p-p38 and Bax expression decreased significantly (Fig. 2). Further analysis showed that the decrease in p-p38 was significantly correlated with the MBC increase (P = 0.042) and the tryptase activity decrease was associated with the improvement in GRA (P = 0.071). There was no significant correlation between other variables clinical and IHC results.
Table 2. Changes in clinical and IHC and western blotting variables after single BoNT-A injection between patients with IC/BPS with successful (GRA ≥ 2) and failed (GRA < 2) treatment outcomes.
Among the 11 patients who received three repeated BoNT-A injections, six patients had a GRA ≥ 2 and five had a GRA <2 after the first injection and all 11 patients had a successful result at 6 months after the 3rd injection. The mean [SD] apoptotic cell count (2.30 [1.83] vs. 0.86 [1.00], P = 0.026), mast cell activity tryptase (64.2 [17.4]%, P = 0.001) and apoptotic proteins Bax (84.3 [21.9]%, P = 0.050) and p-p38 (66.3 [15.9]%, P = 0) all decreased significantly compared with baseline levels. The clinical symptoms, VAS pain score, functional bladder capacity and degree of glomerulation also improved significantly 6 months after three repeated BoNT-A injections (Table 3). All of the bladder tissues showed high SV-2 expression, while the SNAP-25 intensity decreased significantly after three repeated BoNT-A injections (53.1 ([11.7%], P= 0.001), suggesting the BoNT-A had a beneficial effect on the bladder (Fig. 3).
Table 3. Clinical and IHC variables at baseline and 6 months after three repeated BoNT-A injections in 11 patients with IC/BPS.
The present study showed that a single intravesical BoNT-A injection reduced pain, clinical symptoms and apoptotic signalling proteins in patients with IC/BPS; however, urothelial apoptosis, degree of glomerulation and mast cell activity did not improve, suggesting inadequate treatment after a single BoNT-A injection. After three repeated BoNT-A injections, both clinical symptoms and IHC results improved significantly, suggesting chronic inflammation and urothelial apoptosis of IC/BPS require repeated BoNT-A injections to achieve better results.
Some authors have considered IC/BPS to be an inflammatory disease [2-4]. In addition, IC/BPS involves an aberrant differentiation programme in the bladder urothelium that leads to altered synthesis of several proteoglycans, cell adhesion and tight junction proteins, and bacterial defence molecules . Antiproliferative factor (APF), vascular endothelial growth factor (VEGF), barrier and junction proteins, such as zonula occludens-1, uroplakins and E-cadherin, are defective and are associated with increased permeability of the urothelium and clinical pain symptoms in patients with IC/BPS [15-17]. Furthermore, APF regulates the expression of other cytokines, including up-regulating heparin-binding epidermal growth factor-like growth factor and down-regulating epidermal growth factor. These cytokine abnormalities are also related to increases in purinergic signalling, which mediate increased bladder sensation .
The apoptotic process of IC/BPS was found in bladder microvascular endothelial cells and was accompanied by bladder pain . Our recent study  showed that abnormal urothelial function was significantly associated with chronic inflammation. The junctions between urothelial cells in IC/BPS bladders were abnormal and were associated with pain symptoms . The apoptotic signalling molecules, including Bad, Bax, and cleaved caspase-3 were increased in the bladder tissues of patients with IC/BPS. Those findings suggest that apoptosis of bladder tissue in IC/BPS is attributable to the up-regulation of inflammatory signals, and that the molecular mechanism of apoptosis in IC/BPS specimens is mediated by TNF-α and p38-mitogen- activated protein kinase (MAPK) .
Decreased Pain VAS and Clinical Symptoms
The most dramatic changes after single intravesical BoNT-A injection were the decrease in clinical symptoms and pain VAS among the patients with IC/BPS. About half of the patients were satisfied with the treatment. A previous study showed that bladder pain reduction was also associated with nerve growth factor mRNA production in the bladder tissues of patients with IC/BPS . Pain VAS was also significantly correlated with apoptosis cell count and VEGF expression in the lamina propria, suggesting that a barrier deficit is responsible for the pain elicited during bladder filling in patients with IC/BPS [16, 19]. Several bladder epithelial abnormalities are associated with IC/BPS, including abnormal expression of epithelial differentiation markers, growth factors, cell membrane proteins, neurotransmitters and other cytokines . These urothelial abnormalities could be attributable to suburothelial inflammation. Intravesical BoNT-A injection could reduce nerve growth factor mRNA production in human bladders and could decrease the expression of calcitonin gene-related peptide and substance P expression in rats with acute or chronic inflammation [5, 6]. The results of the present study confirmed that the apoptotic signalling proteins Bax and p-p38, and mast cell activity tryptase expression were suppressed by repeated BoNT-A injections, suggesting that BoNT-A injection had an anti-inflammatory effect on IC/BPS bladders, and thus, improved the clinical symptoms such as pain and frequency urgency. However, a single BoNT-A injection could not relieve the symptoms and reverse the increased inflammation and apoptosis in part of the IC/BPS patients; in these patients, repeated injections were necessary to achieve complete IHC resolution.
Mast Cell Activity and Apoptosis
Mast cells are considered to be the main inflammatory infiltrate. An increased number of activated mast cells are often seen in the urothelium, lamina propria and in the detrusor of patients with IC/BPS, which results in an inflammatory response in the bladder, including tissue changes and remodelling, increased vascular permeability and apoptosis . Previous research indicated that tryptase induced phosphorylation of p38-MAPK, causing cytokine production and release . In the present study, we showed that phosphorylation of p38 was induced by tryptase in IC/BPS bladder samples and intravesical BoNT-A injection decreased this mast cell activity and led to p-p38 down-regulation. However, the results of the present study showed that a single BoNT-A injection only decreased p-p38 and Bax expression, but not tryptase expression, indicating a reduction of inflammation-induced apoptosis after a single BoNT-A injection, but without achievement of complete resolution. The IHC results are compatible with clinical findings that single BoNT-A injection does not have a long-term therapeutic effect in patients with IC/BPS and repeated injections are necessary for long-term cure.
BoNT-A Effect on Glomerulations
In patients with IC/BPS, the degree of glomerulation decreases after BoNT-A injection , and glomerulations during hydrodistention are highly associated with the overexpression of angiogenic growth factor . Although the actual mechanism for this BoNT-A effect on angiogenesis is unknown, it is important to elucidate which signal molecules are responsible for the regulation of angiogenesis after BoNT-A treatment. The glomerulation and vascular permeability of IC/BPS bladder tissues seems to be attributable to inflammatory stimulation. Tryptase and TNF-α induced endothelial expression of intracellular adhesion molecules, suggesting mast cells play an important role in blood vessel permeability and glomerulations in patients with IC/BPS. We suggest that the glomerulations and increased vascular permeability are the result of inflammatory stimulation and that BoNT-A blocks the expression of angiogenic markers; additionally, the degree of glomerulation decreased owing to inflammatory suppression after repeated BoNT-A injections. However, the degree of glomerulation did not decrease in all patients after only one BoNT-A injection, suggesting that angiogenesis is more complicated in bladders with IC/BPS. Therefore, repeated BoNT-A injections are necessary for complete resolution of glomerulations.
Effect of BoNT-A on Bladder Capacity
The actual mechanism of reduced FBC or MBC in IC/BPS has not been clearly elucidated. It is possible that the tight junction protein of the urothelial cells and smooth muscle cells are changed with the chronic inflammation of IC/BPS. The angiogenic factors transforming growth factor-β and VEGF are important regulators of fibrosis, which leads to substantial deposition of extracellular matrix components, connective or scar tissue . In the past, several studies have indicated that p38-MAPK activation was involved in inflammation, fibrosis and mediating apoptosis in different cell types in various species [24, 25]. P38-MAPK was recently found to be an important mediator of the effects of APF in bladder epithelial cells . In the present study, the p-p38 decrease was significantly correlated with the MBC increase after BoNT-A injection. The FBC did not increase after the single BoNT-A injection, but increased significantly after the three repeated BoNT-A injections. This evidence suggests that the restricted bladder capacity found in chronic IC/BPS is closely associated with inflammation.
We also measured the intensity of SV-2 and SNAP-25 expression in IC/BPS bladder tissues at baseline and after repeated BoNT-A injections. SNAP-25 protein is a component of the SNARE complex, which is proposed to account for the specificity of membrane fusion and to directly execute fusion by forming a tight complex that brings the synaptic vesicle and plasma membranes together. After the three BoNT-A injections, SNAP-25 expression decreased to 53% of baseline, indicating that BoNT-A indeed affected bladder tissue.
A limitation of the present study is lack of a control arm. Because we cannot exclude the natural course of IC/BPS and the effects of other factors, such as hydrodistention, analgesics or antibiotic treatment used after the injections, the direct association between BoNT-A and molecular changes found in bladder tissue cannot be completely established. In addition, the limited correlation between molecule changes and clinical improvement is possibly attributable to the small number of patients studied. However, the study demonstrated, for the first time, improved efficacy after repeated BoNT-A injections and the molecular changes after BoNT-A injection in patients with IC/BPS.
In conclusion, the results of the present study provide evidence that apoptotic signal molecules in IC/BPS are reduced by a single intravesical BoNT-A injection, but chronic inflammation and glomerulations are not. This improvement is more pronounced in patients who receive three repeated BoNT-A injections. Based on our results, we suggest that patients who fail an initial trial of BoNT-A might benefit from repeat application.