In the last twenty-five years Onabotulinum Toxin-A (BTX-A) has gained increasing popularity for Neurogenic Bladder (NB) treatment. To maintain its efficacy, repeated BTX-A intradetrusor injections are required over time, with unknown effects on bladder wall in children. Aim of this paper is to report long-term effects of bladder wall in children treated with BTX-A.

Children with NB not responsive to anticholinergics were treated with BTX-A, according to our specific  protocol, including endoscopic cold cup biopsy for studying bladder wall modification. Specimens have been evaluated considering edema, chronic inflammation and fibrosis.Specimens were fixed in 10% formalin and paraffine-embedded. Tissue sections were stained with Hematoxylin and Eosin (H&E) and Masson’s trichrome to assess fibrosis.Edema, inflammation and fibrosis were classified using a qualitative grading scale (0: none, 1: mild, 2: moderate, 3: severe).Exclusion criteria: patients with less than 5 BTX-A treatments; those with an inadequate specimen for histological analysis; patients treated with BTX-A injection exclusively into the external urinary sphincter (due to detrusor external sphincter dyssynergia); patients with incomplete clinical data. All patients with symptomatic UTIs in the previous 3 months were also excluded from our analysis statistical purposes, we considered biopsies collected at T1, baseline (immediately before the first BTX-A injection) and during the 5th (T5), 6th (T6) and 7th (T7) BTX-A injections. Fisher exact test was used to perform statistical analysis between quality variables. The Wilcoxon matched pairs test and the nonparametric Mann-Whitney U test were used respectively for intragroup and intergroup comparisons. A p-value less than 0.05 was considered statistically significant.

Out of 230 patients treated from 1997 to 2022, we considered only specimens obtained in patients who had received ≥ 5 treatments (36 children), considered as threshold to evaluate clinical effectiveness on long-term treatment with BTX-A. We found 70/230 patients with a range of 5-22 BTX-A injections (treatment time-lapse: average 7.2 years, range 2.2-14.7 years). We included in the study only 36/70 children, considering only those with all specimens eligible for histological examination and all clinical data available. 
The average age at first treatment with BTX-A was 5.6 years (range: 9 months - 17.5 years). 
Most of these children (25 patients) suffered from neurogenic bladder due to a congenital disorder, the other 11 were affected by acquired neurogenic bladder. All samples included urothelium and lamina propria. Inflammatory infiltration observed was typical of chronic inflammation (lymphocytic-plasma cell infiltrates), associated with granulocytes in three 
biopsies. Three biopsies showed follicular cystitis. Fibrosis, when detected, was localized in the lamina propria . Due to the nature of the biopsy (partial thickness), it was not possible to evaluate any smooth muscle layer alterationEdema, chronic inflammation and fibrosis were identified at baseline respectively in 20 (56%), 27 (75%) and 12 (33%) out of 36 patients. We found a higher percentage of biopsies positive for edema and chronic inflammation at T5 (biopsy done immediately before the 5th injection). On the contrary, fibrosis seemed to be less frequent over time. However, these variations did not follow a linear trend and were not statistically significant . Moreover, edema, chronic inflammation and fibrosis score did not significantly vary after repeated treatments (
Histological findings were statistically compared between patients affected by congenital and acquired neurogenic bladder (Table 3, Figure 7). Intragroup comparison showed no significant difference in term of edema, inflammation and fibrosis at T1 and after repeated treatment in the two groups (Figure 7). Sample size was too small to allow a safe statistical evaluation of intragroup edema, inflammation and fibrosis score variation after repeated treatments (Wilcoxon matched pairs test). In all patients increased edema and chronic inflammation with reduced fibrosis over time have been reported; these data were not statistically significant. No difference was observed between patients with congenital and acquired disease.

Fibrosis is known as a frequent finding in neurogenic bladders, usually associated with mononuclear lymphocyte inflammation and concomitant edema. Some authors investigated before in adults patients the effects of BTX-A injections as Comperat and Apostolidis. This study evaluated long-term effects of repeated injections of BTX-A in a pediatric population. 
Repeated BTX-A injection do not lead to increased bladder fibrosis and we found a progressive reduction of fibrosis with an increased number of treatments, although not statistically significant. 
This positive data may be explained with a better management of the neurogenic bladder. On the contrary, however, a slight increase in edema and chronic inflammation emerged over time, which is also not statistically significant.  Despite its known clinical efficacy, a high discontinuation rate of BTX-A therapy is reported in literature, where treatment failure to be the first cause of discontinuation, followed by  patient's decision, increased urinary incontinence not related to drug injection, progression of neurological disease, and adverse event. 
 Several explanations were suggested for this phenomenon: preference for other forms of treatment ( common in pediatric where general anesthesia is often required for BTX-A endoscopic injections) and a progressive loss of clinical or urodynamic efficacy. Among the causes of the effectiveness reduction  has been proposed the progressive increase in bladder fibrosis due to the repeated injections, which could reduce the spread of the toxin in the bladder wall, reducing its effectiveness 
The presence of important histological alterations that could confirm this theory does not emerge from our data, and in our opinion our histological evaluation could be useful to define the long-term effectiveness  and adherence with BTX-A treatment. 
Some limitations are present in our study: first of all, this is a single center experience. Second one the study has been based on a protocol that, when established and approved, included only histological evaluation.

Conclusions: Despite the small sample examined and a retrospective nature of the study, we can conclude that in the pediatric population repeated intradetrusor BTX-A injection do not seem to lead to significant histological alterations, similarly with what has been observed in the adult population. Reduced effectiveness observed during time in some patients is not related to histological modifications.

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 Nov;50(5):1058-64.
Immunohistochemical expression of muscarinic receptors in the urothelium and suburothelium of neurogenic and idiopathic overactive human bladders, and changes with botulinum neurotoxin administration. J Urol. 2010 Dec;184(6):2578-85.

Continence 7S1 (2023) 100780
DOI: 10.1016/j.cont.2023.100780