Effect of Botulinum Toxin A on Neurogenic Detrusor Overactivity in Chronic Spinal Cord Injured Patients
Effect of Botulinum Toxin A on Neurogenic Detrusor Overactivity in Chronic Spinal Cord Injured Patients
This study investigated the changes in urothelial dysfunction and sensory protein expression
 in the bladder urothelium with time after onabotulinumtoxin injection in spinal cord injured
 (SCI) patients. The investigators planned to enroll 30 chronic SCI patient with neurogenic
 detrusor overactivity and urinary incontinence were treated with a single injection of 200 U
 onabotulinumtoxinA at the detrusor. Video urodynamic studies and bladder mucosal biopsies
 were performed at baseline, 3 months, and 6 months after treatment. Bladder tissues will be
 investigated for urothelial barrier and inflammation proteins as well as sensory proteins by
 Western blotting, between SCI patients and 10 controls, as well as successful and failed
 treatment groups. This study will explore the therapeutic efficacy of a single injection of
 200 U onabotulinumtoxinA on neurogenic detrusor overactivity and the changes of urothelial
 sensory proteins in SCI patients with successful and failed treatment outcome.
In recent decades, treatment of neurogenic detrusor overactivity (NDO) with
 onabotulinumtoxinA has emerged as an alternative method for the management of urological
 complications due to spinal cord injury (SCI) or multiple sclerosis. Injection of 200-300 U
 of onabotulinumtoxinA into the detrusor muscle can reduce contractility, improve bladder
 compliance, and restore urinary continence in patients with NDO. However, detrusor
 underactivity will develop after injection of 300 U of onabotulinumtoxinA and improvement of
 urodynamic and quality of life parameters lasted 9 months. Currently, a 200 U single
 injection of onabotulinumtoxinA into the detrusor has been recommended as standard treatment
 for NDO. However, the therapeutic duration of this dosage on NDO was shorter than that of a
 300 U injection.

 OnabotulinumtoxinA effectively improves lower urinary tract symptoms by inhibiting signal
 transmission at the neuromuscular and neuroglandular junctions. OnabotulinumtoxinA cleaves
 synaptosome-associated protein 25 and inhibits signal transmission by disrupting fusion of
 neurotransmitter- containing vesicles with the neuronal wall. In the urinary bladder, release
 of acetylcholine from both pre- and postganglionic parasympathetic nerves is blocked after
 onabotulinumtoxinA administration. This toxin was also found to modulate afferent activity of
 the bladder associated with reduced urgency and urgency urinary incontinence symptoms in NDO
 patients. Although the impact of onabotulinumtoxinA on the sympathetic nervous system within
 the urinary bladder is unclear, it has an inhibitory effect on release of norepinephrine to
 α- and β3-adrenoreceptors (β3-ARs) which regulate bladder neck contraction and detrusor
 relaxation, respectively.

 A previous study reported decreased expression of adhesion and junction proteins E-cadherin
 and zonula occludens-1 (ZO-1), respectively, and increased suburothelial inflammation with
 apoptosis in patients with chronic SCI bladders.The urothelial inflammation and dysfunction
 in SCI bladders might also alter sensory protein expression, such as in purinergic receptor
 P2X3, transient receptor potential vallinoid receptor subfamily 1, adenosine triphosphate,
 and nitric oxide. Urothelial dysfunction might also result in increased excitability of the
 C-fibers which become predominantly afferent nerves of the micturition reflex after SCI. In
 humans with NDO, the levels of bladder P2X2, P2X3, and muscarinic receptors M2 and M3 were
 reduced after detrusor onabotulinumtoxinA injection, suggesting this toxin inhibits DO by
 inhibiting both the sensory and motor arms of the micturition reflex. In addition, β3-ARs are
 known to promote urine storage in the bladder by inducing detrusor relaxation in animal and
 human bladders. In humans, β3-AR is the predominant β-receptor subtype in the urinary
 bladder. There has been no report on β3-AR changes in SCI bladder urothelium before or after
 onabotulinumtoxin treatment.

 After onabotulinumtoxinA injection, patients may be symptom-free for a period of 3-6 months
 before symptoms relapse. Most studies of onabotulinumtoxinA on NDO come from animal models,
 and only a few human studies have been noted. The current study investigated changes in
 urothelial dysfunction and sensory protein expression in the bladder urothelium with time
 after a single onabotulinumtoxin injection in SCI patients.

 Materials and Methods

 A total of 26 patients with chronic SCI causing NDO and urinary incontinence were treated
 with a single injection of 200 U onabotulinumtoxinA to the detrusor muscle. All patients
 presented with urinary incontinence with or without difficult bladder emptying. In patients
 who received detrusor injections, clean intermittent catheterization was suggested as a
 possible mode of urinary management after treatment. The current study was approved by the
 Institutional Review Board and Ethics Committee of Buddhist Tzu Chi General Hospital
 (IRB:098-53). Each patient was informed about the study rationale and procedures, and written
 informed consent to participate was obtained before any bladder procedure.

 Video urodynamic studies

 All patients received a video urodynamic study (VUDS) before their enrollment. Bladder and
 urethral dysfunction were classified as the presence of DO with coordinated or dyssynergic
 urethral sphincter activity. Patients with urinary tract infection were properly treated
 before they were scheduled for onabotulinumtoxinA injection. VUDS results were interpreted
 according to the recommendations of the International Continence Society. VUDS parameters
 included maximum flow rate (Qmax), detrusor voiding pressure at Qmax (Pdet), cystometric
 bladder capacity (CBC), voided volume (Vol), and post-void residual (PVR) volume.

 OnabotulinumtoxinA injection

 All patients received 200 U of onabotulinumtoxinA in 20 mL normal saline (BOTOX®, 100 U/vial,
 Allergan Inc., Irvine, USA) in the operation room by rigid cytoscopic injection (22 Fr,
 Richard Wolf, Knittlingen, Germany) into 40 sites of the bladder wall, sparing the
 trigoneAfter injection, a urethral Foley catheter was routinely inserted and removed the next
 morning before patients were discharged. Broad spectrum antibiotics were prescribed for 3 d
 after treatment.

 After onabotulinumtoxinA injection, four bladder cold-cup biopsies were taken randomly at the
 posterior wall about 2 cm above the interureteric ridge, and only the bladder mucosa and
 submucosa were taken to prevent bladder perforation; any erythematous or inflammatory bladder
 mucosa were avoided. One bladder biopsy specimen from each patient was sent to the pathology
 department to exclude the possibility of carcinoma in situ. The remaining three specimens
 from each patient were stored at optimum cutting temperature and in liquid nitrogen for
 further immunohistochemistry studies.

 Patient follow-up and outcome assessment

 Patients were followed up at OPD monthly, and VUDS and assessment of bladder and voiding
 conditions and satisfaction with treatment were performed 3 and 6 months after treatment. If
 the CBC increased by 50% from the baseline value, the treatment outcome was considered
 successful; otherwise, the outcome was considered a failure at that time-point. Ten female
 patients with stress-related urinary incontinence without frequent urgency served as
 controls. All control patients were confirmed free of bladder outlet obstruction or DO by
 VUDS. Control bladder tissue was obtained during anti-incontinence surgery and processed the
 same as SCI patients. Male patients were not selected to serve as controls because the
 prostate could cause bladder outlet obstruction and affect urothelial function.

 Immunofluorescence

 Bladder tissue samples from SCI patients and controls were examined for changes in urothelial
 E-cadherin (adhesive protein), ZO-1 (junction protein), and mast cell activation (tryptase)
 levels by immunofluorescence; cellular apoptosis was examined by terminal deoxynucleotidyl
 transferase dUTP nick end-labeling assay. These procedures were performed similar to those in
 our previous study, with 6-μm thickness tissue sections. Urinary bladder specimens were
 immersed and fixed for 1 h in ice-cold 4% formaldehyde in phosphate-buffered saline (pH 7.4)
 then rinsed with ice-cold phosphate-buffered saline containing 15% sucrose for 12 h. Biopsy
 specimens were embedded in optimum cutting temperature 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 glass slides (Muto Pure Chemicals Co., Ltd, Tokyo, Japan). Sections were
 postfixed in acetone at -20 °C and blocked with rabbit serum. The sections were incubated
 overnight at 4 °C with antihuman E-cadherin (BD Biosciences, San Jose, CA, USA) or tryptase
 (Chemicon, Temecula, CA, USA) primary antibodies. After rinsing the sections with 0.1%
 Tween-20 in BPS, rabbit antimouse-conjugated fluorescein isothiocyanate secondary antibodies
 (DakoCytomation Denmark A/S, Glostrup, Denmark) were applied and incubated on sections for 1
 h. Finally, sections were counterstained with 4',6-diamidino-2-phenylindole (Sigma Chemical
 Co., St. Louis, MO, USA). Negative controls included the isotype of the primary antibody. We
 obtained the mean, maximum, range, and standard deviation of the staining intensity and
 percent positive area measurements using three random hot spots within each specimen.

 Immunofluorescence quantification was determined in four consecutive high-power fields (400X)
 in the area with the greatest density. Immunofluorescence (tryptase and terminal
 deoxynucleotidyl transferase dUTP nick end-labeling) assays were quantified by counting the
 number of positively stained cells per unit area (4 μm2) and are shown as percentages. The
 intensity of E-cadherin (ﬂuorescence microscopy) and ZO-1 (confocal microscopy) fluorescence
 was quantified using Image J.

 Western blotting was used to assess the levels of sensory proteins in bladder mucosa
 specimens. Primary antibodies included P2X3 (1:2000), endothelial nitric oxide synthase
 [eNOS] (1:1000), M2 (1:1000), M3 (1:500), and GAPDH (1:100000; internal control) from GeneTex
 (Irvine, CA, USA); inducible NOS [iNOS] (1:1500; ThermoFisher, Rockford, IL, USA); and β3-AR
 (1:1000; Abcam, Cambridge, UK). Donkey antigoat (for β3-AR) or goat antirabbit (for all other
 proteins) IgG-horseradish peroxidase (1:3000; Santa Cruz Biotechnology, Dallas, TX, USA)
 secondary antibodies were used. The scanned ﬁlm after gel electrophoresis was quantiﬁed using
 a gel documentation system (Quantity One Version 4.6.2, Bio-Rad Laboratories, Hemel
 Hempstead, Herts, UK). Procedures were similar to those described in our previous study.

 Statistical Analysis

 Continuous variables were presented as means ± standard deviations, and categorical data were
 presented as numbers and percentages. Eligible SCI patients were grouped according to
 treatment outcome and compared with controls. Differences in the levels of functional
 proteins in the urothelium at baseline and 3 and 6 months after onabotulinumtoxinA injection
 were analyzed using a paired Student's t-test. Differences in immunofluorescence and Western
 blot of sensory proteins between successful and failed treatment groups were analyzed using
 the Kruskal-Wallis test. In order to clarify the roles of urothelial sensory proteins in NDO,
 correlation analysis using linear regression was also performed, including VUDS and
 urothelial dysfunction parameters. All calculations were done using SPSS for Windows version
 16.0 (SPSS, Chicago, IL, USA). Differences were considered statistically significant if P
 values were less than 0.05.
Inclusion Criteria:

 -  patients with chronic SCI causing NDO and urinary incontinence

 Exclusion Criteria:

 -  patients did not present with urinary incontinence

 -  patients are known to be allergic to onabotulinumtoxinA

 -  patients cannot be followed up regularly

 -  active urinary tract infection