Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT03063827 |
| Other study ID # |
BuddhistTCGH 098-53 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
February 21, 2017 |
| Last updated |
March 9, 2017 |
| Start date |
January 2010 |
| Est. completion date |
December 2012 |
Study information
| Verified date |
March 2017 |
| Source |
Buddhist Tzu Chi General Hospital |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
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.
Description:
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.
