Abstract
Background/Aim: The overactive bladder syndrome (OAB) is a bothersome condition that affects up to 33% of the population. In up to 69% of the cases, the underlying condition is an overactive detrusor (DO). Treatment options rely on behavioral changes, medical treatment, neuromodulation, and invasive treatment, such as injecting botulinum toxin (BoNT) in the detrusor or augmentation cystoplasty. The aim of this study was to evaluate, by morphological assessment on cold-cup biopsies of the bladder, the effect of botulinum toxin injections on the bladder wall, focusing on the histological structure and signs of inflammation and fibrosis. Patients and Methods: We evaluated consecutive patients with DO that received BoNT intradetrusor injections. We analyzed inflammation and fibrosis in 36 patients, divided into two groups based on their history of BoNT treatment. Our patients underwent at least one round of injections and specimens were compared individually, before and after each injection. Results: A decrease in inflammation was found in 26.3% of the cases, a reactive increase in 31.5%, and no change in 42.1%. No de novo or increase in preexisting fibrosis was found. In some cases, fibrosis diminished after a second round of BoNT. Conclusion: In most cases, BoNT intradetrusor injections in DO patients showed no effect on bladder wall inflammation and actually improved the inflammatory condition of the muscle in a significant number of samples.
The overactive bladder syndrome (OAB) is a condition defined by urinary urgency, usually with frequency and nocturia, with or without urinary incontinence, in the absence of urinary tract infection or other pathology (1). The prevalence of OAB is estimated to be in the range of 16.5%-33% (2). This medical condition, although not necessarily impairing the patients’ physical health, can have a significant impact on their quality of life and mental health, limiting their social life, travel capacity, and work activity. Stress, depression, and low self-esteem can result from this medical condition, with high costs for the patients, the health system, and the work environment. Moreover, the cost of treatments can be high, especially in cases of prolonged therapies and failure or high drop-out rates of patients due to the side effects of available drugs.
The treatment relies on a first-line life-style and behavioral change than on second-line drug treatment. The gold standard antimuscarinic therapy has shown results in multiple trials, but the incidence of the bothersome side effects leads to patients abandoning the treatment course before the 3 months recommended for symptom improvement. During the first 12 months of treatment, persistence is approximated at only 8.3-25% (3). Even newer agents, such as beta 3 mimetics, which have the advantage of less important and less frequent side effects, show persistence of only 58% at 12 weeks (4) and 38% at 12 months (5). On the other hand, highly symptomatic patients, which is the case for the vast majority of patients with a neurogenic bladder, need drug treatment to control their bladder, either as first line therapy or at a later stage, when non-invasive treatments did not provide relief.
More modern approaches rely on third-line minimally invasive therapies, recommended when medication fails or is contraindicated. In the last years, botulinum toxin injection into the bladder has proven safe and effective, with high patient acceptance rates and satisfaction. Although this treatment is not new, some patients are not willing to accept it, whereas in some countries there is a financial barrier, as botulinum toxin is not paid for by insurance companies.
In 11.8% to 69% of the OAB cases the underlying condition is the overactive detrusor (DO) (6, 7). Data are lacking on the pathological process involved in the pathogenesis of this condition. A number of theories have been proposed: the myogenic, the neurogenic, the autonomous bladder and the afferent signaling theory (8). These theories are backed up by evidence in both histology and electrophysiology areas. At present, there is a general agreement that OAB-related pathological changes include increased spontaneous myogenic activity, fused tetanic contractions, altered responsiveness to stimuli, and characteristic changes in smooth muscle ultrastructure (9). Moreover, patchy denervation of the bladder, enlarged sensory neurons, hypertrophic ganglion cells, and enhanced spinal micturition reflexes have been described (10).
Regardless of the etiology, the DO seems to exhibit exaggerated autonomous activity, generated by morphological alterations. In areas where denervation takes place, the interstitial cells proliferate, especially the N-cadherin+ type, giving the grounds for detrusor overactivity – denervated fascicles, an altered network of interstitial cells, and ultra-close abutments and protrusion junctions (11, 12).
Extensive research into a particular type of DO, the neurogenic OAB, where constantly high detrusor pressures are identified, showed even more complex detrusor alterations. The trabecular bladder is a common feature and is the result of detrusor fibrosis determined by high pressure regimens (13). The remodeling of the tissue with muscle hypertrophy and increased collagen deposits leads to bladder fibrosis and subsequent abnormal contractility, low compliance, and renal impairment. A skeletal muscle that is exercised extensively will increase in size and power – hypertrophy – however, the detrusor cannot afford to do that. An ultra-powerful detrusor will destroy the upper urinary tract and potentially even the bladder. The detrusor will develop scaring and trabeculation, limiting the force of contraction, which is kept into a safe range, and this is a very good self-protection mechanism of the urinary tract. Unfortunately, the process is irreversible, and it seems to not be limited only to the neurogenic subgroup of DO (14). Since there is yet no treatment available, efforts are made to prevent the process or to stop its evolution once initiated (15).
The aim of our study was to determine whether injecting botulinum toxin into the idiopathic DO could induce histological changes of the tissue, particularly in terms of fibrosis and inflammation.
Patients and Methods
We evaluated consecutive patients suffering from idiopathic OAB in a prospective study performed between July 2019 and December 2021. We included patients with DO confirmed by urodynamic studies, out of which we selected those exhibiting spontaneous detrusor contractions that exceeded 40 cmH2O, considering them to be at a higher risk for bladder fibrosis. We evaluated all the patients after acquiring informed consent, and they underwent either conventional medical treatment or intradetrusor injections of botulinum toxin if the drugs were contraindicated, had already a failed course of treatment, or preferred the invasive procedure upfront. Local ethics committee approval (approval no. 321/14 MAR 2019) was obtained before starting this study.
We performed cold cup bladder biopsies during exploratory cystoscopy or right before injecting the botulinum toxin, during the same procedure. We divided the specimens into two groups: Group 1, the botulinum toxin naive, and Group 2, comprised of biopsies from patients who underwent at least one round of injections in the past, before enrollment in our study. In some patients, two injections were made during the study, and biopsies were obtained after both treatments, with the second biopsy reported as BoNT retreatment. The biopsies were examined by a pathologist specialized in urologic pathology, who was not informed about the patients’ diagnoses or particularities. The procedure was performed under spinal anesthesia in most cases, and cold cup biopsy was preferred because it is considered less aggressive than resection and with lower morbidity. The catheter was removed 24 h after the procedure if there were no signs of hematuria. Detrusor muscle was present in all specimens.
The moment for initial biopsies was right before the injection, and the biopsies were repeated 6 weeks after the injections, after repeating the evaluation protocol. We defined therapy success as at least 50% improvement in urinary symptoms or urodynamic parameters (maximum cystometric capacity, maximum detrusor pressure, leak point pressure, presence of involuntary contractions) but the decision for retreatment was individualized for each patient. The samples were compared for each patient in a before/after manner.
Patients who underwent botulinum toxin injections were given a 500 U dose of onabotulinum toxin type A (Dysport®, Ipsen Biopharm Ltd., Wrexham, UK) delivered into the detrusor in a standardized manner (20 injection sites, without sparing the bladder trigone). Biopsies were obtained from the posterior bladder wall, avoiding the trigone and close to the planned injection sites.
We also defined a smaller subgroup of patients whose surgical history consisted in obstetrical and gynecological interventions, given the higher incidence of OAB in women and its link to childbirth interventions and pelvic surgery. It consisted of two patients undergoing medical treatment and another two patients that underwent one round of BoNT injection. Due to the small sample size, they were not treated as a distinct group, but their particular results were analyzed for particularities that would justify a new analysis centered on this subtype of patients, in the future.
We included 17 patients in group 1 and 19 in group 2, for a total of 36 patients analyzed by the end of the study. Out of the 36 patients, 17 (47%) had failed previous medical therapies, 7 (19.5%) had medical contraindications for antimuscarinics and 5 (33.3%) preferred the invasive approach. Five of the patients underwent a second round of BoNT injections at a mean 8.7 months due to symptom reemergence (range=7-11 months).
The biopsies were processed using hematoxylin-eosin staining (H&E, MUTO Pure Chemicals, Tokyo, Japan) and Masson trichrome, evaluating inflammation and fibrosis, respectively. An Olympus CX43 microscope (Olympus Corporation, Tokyo, Japan) was used.
The specimens obtained were analyzed in terms of morphological aspect of the urothelium, the lamina propria and partially the detrusor, and contained 1 or 2 bundles of muscles due to the extraction technique. We obtained paraffinized sections of 0.6 μm. Tissues were characterized by the extent of inflammation and fibrosis. The degree of inflammation was assessed throughout the samples, with three degrees defined as mild (+), moderate (++), and severe (+++). Mild inflammation was defined as <5 mononuclear inflammatory cells in a 10×10 grid. Moderate inflammation was defined as mononuclear inflammatory cells scattered throughout the tissue but background stromal connective tissue clearly visible. Severe inflammation was reported when mononuclear inflammatory cells densely infiltrating the tissues were observed. We considered the ‘++’ and ‘+++’ results as significant for our study. Fibrosis was reported as non-significant and significant. We defined significant fibrosis when it was present in more than 15% of the muscle depth.
Data were analyzed using Pearson’s chi-squared test, to determine whether there is a statistically significant difference in our results, looking at patterns before and after the treatment in each study group.
Results
Compared to the normal morphology of the bladder tissues, the initial specimens from group 1 exhibited a pattern of inflammation in all tissue depths in 14 (82.3%) cases, corresponding to chronic inflammation. Inflammation classified as ‘++’ was seen in 9 patients and ‘+++’ in 5 patients (Figure 1). Fibrosis was seen in 5 (29.4%) specimens; all of them underwent subsequent injection (Figure 2).
Initial inflammation patterns identified in our study population.
Mixed inflammatory infiltrate (40× magnification; left) and fibrosis (20× magnification; right).
In group 2, a slightly different distribution of initial (chronic) inflammation and fibrosis was observed, with 4 patients classified as ‘+’, 7 patients as ‘++’, and 8 cases as ‘+++’, leading to 15 specimens (78.9%) with inflammation involving the entire depth of the tissue. Fibrosis was diagnosed in 4 cases (21%).
Biopsies obtained 6 weeks after injections were analyzed using the same system and results were analyzed comparatively to previous results of the same patients. Out of the 14 patients in group 1 with significant initial inflammation, 3 had diminished tissue inflammation from ‘++’ to ‘+’ (p=0.1, i.e., not considered significant in our design), 2 patients had a decrease from ‘+++’ to ‘++’ (p<0.005), 3 patients had increased inflammation from ‘++’ to ‘+++’ (p<0.005), while the 4 patients remaining had no change in the inflammation pattern. In 1 patient, who had an initial inflammation considered ‘+’, it increased to ‘++’ after treatment (Figure 3). The 5 patients with fibrosis had no quantitative changes of more than +/−5%. No new fibrosis was developed by the other patients undergoing BoNT injections.
Inflammation pattern evolution after any round of botulinum toxin (BoNT) treatment (group 2) and after second BoNT treatment (BoNT retreatment) compared to the initial results.
For the 5 patients who underwent a second round of BoNT injections, biopsies were performed once again at 6 weeks after the second treatment. Inflammation evaluation showed no increase in inflammation compared to the first injection except for one patient who developed ‘++’ inflammation, increasing from ‘+’ as classified during the other biopsies (Figure 3). There were no cases of de novo or increase of quantity of fibrosis (+/−5% compared to the initial sample); however, there was one case where the initial fibrosis was 20%, after the first injection it remained within the accepted +/−5% range, but after the second round of treatment it decreased to 10%, i.e., not significant fibrosis according to our design. In group 2, a similar pattern of evolution was observed after injection, with data being presented in Figure 3.
The subgroup consisting of female patients with personal surgical history of obstetrical and gynecological causes showed two initial cases of inflammation and one case of fibrosis. The inflammation was identified in one of the patients undergoing medical treatment and was classified as ‘++’; the BoNT patient with inflammation was also ‘++’ but had no significant change at 6 weeks. The other patient undergoing BoNT injections advanced from ‘+’ to significant inflammation defined by ‘++’. In terms of fibrosis, the only patient in whom it was identified, showed no change at 6 weeks after BoNT injections; no de novo fibrosis developed in this subgroup.
Overall, after intradetrusor injection of botulinum toxin, we noticed a decrease in bladder tissue inflammation in 26.3% of the cases, a reactive increase in 31.5%, and no change in 42.1% of the patients. No de novo or increased fibrosis was observed and in some cases appeared to decrease after treatment. Apparently, the second injection of BoNT increases inflammation in less cases than the first dose and, in most cases determines no changes compared to the initial status of the bladder wall. As a general observation, no major complications were encountered, and the symptoms of the patients were not worsened by the biopsies.
Discussion
There is a lack of information regarding idiopathic detrusor overactivity compared to the more researched neurogenic subtype. Our study showed no significant changes in terms of inflammation and fibrosis before and after injecting botulinum toxin into the detrusor, at the selected time points.
Studies linked detrusor dysfunction and low compliance bladders in non-neurogenic patients with organic changes in the bladder tissues, rather than functional alterations. Ohnishi et al. found increased connective tissue and decreased muscle in this subtype of patients (16).
Inflammation is present in the OAB syndrome and in interstitial cystitis, proven after identifying pro-inflammatory mediators and cells that sensitize nerve afferences. Increased histamine, tissue granulation, mast cells, chemokines, and cytokines have all been described in studies (17, 18).
Increased tyrosine receptor kinase A and B and calcitonin gene-related peptide are found and are responsible for inflammation (19, 20); high concentrations of nerve growth factor in urine and serum explain mast cell proliferation and dysfunction in bladder activity and innervation (21).
Comperat et al. reported that injection of BoNT type A in patients with neurogenic overactivity is linked to significantly less fibrosis compared to patients not undergoing BoNT treatment (22). Haferkamp et al. analyzed biopsies obtained from patients with neurogenic DO before and after injecting BoNT into the detrusor. They found no structural changes of the detrusor induced by the treatment and only limited axonal sprouting compared to the effect of BoNT in striated muscles (23).
Apostolides et al. analyzed samples from patients who underwent BoNT injections for neurogenic and idiopathic DO. No changes in fibrosis, inflammation or dysplasia were observed after treatment and no differences existed between neurogenic and idiopathic DO (24). A recent study looking into the pathophysiology of urgency incontinence suggested the existence of common grounds with stress incontinence in female patients in terms of anatomical integrity of the lower urinary tract (25). Until a paradigm shift in the treatment of DO takes place, botulinum toxin will remain a powerful tool in the armamentarium of the urologist.
One limitation of our study was the small cohort we investigated. A larger number of patients in each group should be analyzed in further investigations to assess more reliably the changes induced by BoNT treatment, especially fibrosis. Also, a longer time frame and a longer follow-up would be advised. We would find it desirable to analyze patients undergoing multiple rounds of treatment, as well as having a control group with suitable follow-up to investigate the natural evolution and progress of histological processes in the idiopathic overactive detrusor.
Additionally, another important limitation of our study is the morphological approach, which is a descriptive, operator-dependent strategy. More quantitative assessments, based also on objective measurements at the molecular level regarding in particular the inflammatory reaction, should be performed on the tissues. Recently, distinctive inflammation-related molecular changes have been detected and quantified, as a response to treatments, in other benign urological conditions (26).
Although there is enough evidence in the literature to rule out any placebo effect of the botulinum toxin injection, the lack of a placebo arm in our study might be a limitation.
Conclusion
Our study shows that injecting BoNT might lead to a decrease in bladder tissue inflammation in about one fourth of the cases, a reactive increase in one third and no change for most of the patients; we found no de novo or evolution in the existing processes of fibrosis in the detrusor and our data suggest that a reversal of the fibrotic process might be seen. A second course of BoNT injections leads to an increase in inflammation in less patients and usually determines no changes compared to the initial biopsy. For the subgroup of female patients with obstetric and gynecologic history with existing inflammation we found one case of reactive inflammation after BoNT injections; there was no evolution or de novo fibrosis in our study at the observed time points. Further studies with larger sample sizes and adequate control group for this subgroup should be conducted for statistically significant conclusions.
Preserving the parameters of the urinary system is one of the prerogatives of the urologic treatment in DO, regardless of the method chosen. BoNT has already proven safe and effective, and these results show additional benefit of the therapy.
However, our data seem to show that there could be additional novel benefits, in terms of modulation of the inflammatory response and reduced fibrosis, in the field of BoNT, a therapeutic strategy already proven to be safe and effective in OAB. Further research on this topic is highly needed in the near future.
Footnotes
Authors’ Contributions
Cristian Persu: Conceptualization, Supervision, Investigation, Validation of data; Irina Ciofu: Writing of text, data analysis, correspondence; Amelia Petrescu: Methodology, pathology work; Narcis Chirca: data curation, methodology; Victor Cauni: Supervision, Project administration, oversight of paper elaboration.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received January 15, 2023.
- Revision received January 29, 2023.
- Accepted January 30, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
