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2011 Toxina Botulinica y Disreflezxxia
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E L K E L I N I E T A L .
2 0 11 T H E A U T H O R S2 B J U I N T E R N A T I O N A L 2 0 11 B J U I N T E R N A T I O N A L
bladder afferent pathways reform and
induce the growth of C-fibre afferents,containing calcitonin gene-related peptide.Nerve growth factor (NGF) is a member of
the neurotrophin family and an importantregulator of neural survival, development,function and plasticity. NGF plays a major
role in the enlargement of the afferentarbour after SCI [4], and intrathecally
delivered anti-NGF has reduced AD in ratswith SCI [5]. Hence, NGF and its receptors inthe bladder and spinal cord might offer
potential targets for new therapies tocontrol AD and NDO after SCI.
Current management protocols for ADand NDO are mostly symptomatic andineffective. [6]. OnabotulinumtoxinA
(onabotA) has been used in SCI patients to
reduce detrusor overactivity via inhibition ofacetylcholine release from efferent nerve
endings [7]. There is increasing evidence thatonabotA might also affect sensory nervefibres and afferent signalling mechanisms
[7]. OnabotA also reduces NGFconcentrations in bladder tissue of patientswith detrusor overactivity [8,9]. Intravesical
onabotA administration might block AD byacting on primary afferent fibres andputative spinal neurones.
In the present study, our goal was to assessthe effect of onabotA on AD and NDO after
SCI in a clinically relevant animal model.
MATERIALS AND METHODS
Female SpragueDawley rats (n= 44),
weighing 200250 g were used in thepresent study. Rats were stratified into threegroups: sham control (laminectomy with
intact spinal cord), SCT, and spinal cordtransection (SCT) + onabotA. Each groupwas subdivided for AD and NGF assessment
(six to eight rats per subgroup). Animals
were maintained for 3 weeks after surgery.The protocol was approved by the Animal
Care Committee of University HealthNetwork/University of Toronto in accordancewith the policies established in the Guide to
Care and Use of Experimental Animalsprepared by the Canadian Council on AnimalCare.
Spinal cord transection was performed asdescribed previously [10]. Briefly, under
general anaesthesia, a limited laminectomyto the fourth and fifth vertebrae was
performed. Using a sharp microscissor, a
complete transection of the spinal cord atT4 level was performed under direct visualcontrol then aided by an operating
stereomicroscope (Spencer, American OpticalCompany, NY, USA). To ensure a completetransection of the spinal cord, the tip of
16-G needle was passed several timesaround the inner surface of the exposedvertebra. After surgery, the rat bladders were
evacuated by manual expression.
Intravesical instillation of onabotA was
performed as follows. Under generalanaesthesia using a combination of xylazine
(5 mg/kg) and ketamine (50 mg/kg), PE-50tubing (Clay-Adams, Parsippany, NJ, USA)was inserted into the bladder through the
urethra. The bladder was emptied of urine
and slowly filled with onabotA (1 mL, 20 U/mL in saline; Allergan, Irvine, CA, USA),
which was left indwelling for 30 min. Ratswere allowed to recover, and 48 h later theyeither had a suprapubic catheter implanted
for cystometrogram (CMG; AD group) orwere killed for dorsal root ganglia (DRG)retrieval (NGF group). We measured the
effect of onabotA 48 h after administrationbecause previous reports using the sameanimal model showed improved bladder
dysfunction after onabotA treatment [11,12].
Suprapubic catheter implantation and AD
assessment during CMG were carried out asfollows. Under general anaesthesia, a silicontube was implanted in the bladder via
laparotomy based on a method describedpreviously [10]. CMG was conducted onconscious rats held within a restrainer (IITC
Life Science, Woodland Hills, CA, USA). Ratswere kept in a warming chamber at 30 C tomaintain optimal ambient temperature for
blood pressure reading. A tail cuff (7/16inch) with photoelectric sensors (IITC LifeScience) measured blood pressure and heart
rate. The system also includes an automated
sphygmomanometer, amplifier and scanner(IITC Life Science) attached to a computer
interface.
Rats were acclimatized for at least 30 min.
Blood pressure measurements were initiatedthrough a computer interface and recordedwith the help of IITC BpMon software.
Measurements were taken at baseline andduring bladder dilatation via CMG. DuringCMG, the bladder was filled with sterile
saline at a rate of 0.2 mL/min using aninfusion pump (Model 2620, Harvard
Apparatus Holliston, HA, USA). At least four
micturition cycles were monitored in eachrat. The ladder pressure was recorded with aGrass Polygraph (Model 7D). Immediately
before maximum voiding pressure (whichcould be predicted after a few micturationcycles), blood pressure measurements were
initiated and recorded; the heart rate wascalculated by the software. Measurementswere taken in triplicate and the mean
calculated.
For NGF immunoassay, peptides were
extracted from DRG and the total proteincontent was determined with abicinchoninic acid assay (Pierce, Rockford, IL,
USA) for standardization. A commercial ratNGF ELISA kit (Promega, Madison, WI, USA)was used. Samples were acid-treated to
increase the amount of detectable NGF andstored at 20 C. A Nunc MaxiSorp 96-wellELISA plate was coated with polyclonal
antibody in carbonate coating buffer andincubated at 4 C for 24 h. The plate waswashed with Tris-buffered saline with Tween
20 wash buffer, blocked with buffer(Promega), and incubated at roomtemperature for 1 h. The plate was washed,
monoclonal antibodies were added to thewells and the plate was incubated at 4 Cfor 24h. The plate was washed, anti-rat
immunoglobulin G horseradish peroxidaseconjugate was added to the wells and theplate was incubated at room temperature
for 2.5 h with shaking (220 rpm). The platewas washed, TMB One solution was added
and the plate was incubated at roomtemperature for 10 min with shaking(220 rpm). The reaction was stopped with
1 N HCl and absorbance was read on a platereader at 450 nm. Using a standard curve,sample NGF concentrations were derived.
For statistical analysis, an independent t-testand one-way analysis of variance (anova)
were used to analyse the data. P< 0.05 was
considered to indicate statisticalsignificance.
RESULTS
Complete transection of the spinal cord atthe T4 segment resulted in total flaccidparalysis of the lower limbs accompanied by
bladder areflexia. Rats began regainingbladder contractility at 10 days aftersurgery as evidenced by smaller evacuated
volumes of urine during daily bladdersqueezing. By day 14, most rats were able to
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O N A B O T U L I N U MT O X I N A C O N T R O L S B L A D D E R D Y S F U N C TI O N A N D A U T O N O M I C D Y S R E F L E XI A A F T E R S P I N A L C O R D I N J U R Y
2 0 1 1 T H E A U T H O R SB J U I N T E R N A T I O N A L 2 0 1 1 B J U I N T E R N A T I O N A L 3
104 47.88 beats/min (P= 0.01) while, inthe onabotA treatment group, the heart ratedecreased by only 28.75 9.1 beats/min
(P= 0.024; Fig. 2).
Three weeks after SCT, NGF concentration
was significantly higher in the SCT T4-DRGgroup (557.66 79.54 pg/mL) than in thecontrol (105.50 33.21 pg/mL; P= 0.002),
whereas after onabotA treatment in ratswith SCT, the NGF concentration in T4-DRG(152.66 63.28 pg/mL) was significantly
lower than in the SCT study group (P=0.006). Furthermore, the bladder NGF
concentration was significantly higher afterSCT (610.33 143.20 pg/mL) than in thesham control group (11.86 1.97 pg/mL;
P= 0.01); whereas, after onabotA treatmentin rats with SCT, the NGF concentration inthe bladders was significantly lower (136.00
58.66 pg/mL) than in the SCT study group(P= 0.028; Fig. 3).
DISCUSSION
The rat is a common SCI model because itprovides an inexpensive and reliable methodto characterize complex clinical problems.
For instance, patients with SCI above T5exhibit pressor response and bradycardiaafter bladder distension, and there are
similar results in rats with SCI [13]. To assess
FIG. 1. Bladder pressure changes (CMG) in control
rats at baseline and during CMG. a, Normal rat;b,
SCT rat;c, SCT+ onabotA rat. Rats were tested 3
weeks after SCT. Asterisks indicate the voiding
contractions. Note that uninhibited contractions
occurred in the SCT rat but were not detected in
the normal rat and were significantly reduced with
onabotA treatment.
10mm/min
0.2 mL/min
0.2 mL/min
0.2 mL/min
cmH20
cmH20
cmH20
50
40
30
20
10
0
50403020100
7060
50
40
30
20
100
70
60
a
b
c
Ruler
Voiding
Uninhibited contractions
move their hips and knee joints; however, no
weight-bearing ability was evident duringthe 3-week study period.
During CMG, the control group had asmooth filling phase, with no detrusoractivity (Fig. 1). By contrast, the CMG of SCT
rats showed a filling phase with uninhibitedcontractions (8.0 0.7) and bladder pressure
reached 31.24 4.7 cmH2O. OnabotAtreatment significantly reduced the numberof uninhibited contractions (3.0 0.4, P
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AD and NDO, we developed an animal model
with a complete SCT at the T4 segment.Bladder distension is the most commoncause of AD [14], and usually occurs during
urodynamics assessment [15]. Therefore, ADproduced via bladder distension provides aconsistent and clinically relevant assessment
tool. In the present study, we measured thechanges in blood pressure and heart rate
in T4-SCT rats during urodynamics. Wealso correlated these measurements to astandard urodynamics variable (maximum
voiding pressure). Anaesthesia markedlyreduces voiding function in SCT rats; thus,we conducted CMG in conscious rats. Using
non-invasive blood pressure monitoring weassessed AD in a situation that mimicsclinical scenarios. To minimize bladder
irritation we inserted the catheter only a
few hours before urodynamics.
It is unlikely that intravesical onabotAinstillation has an effect on normal animals,as previous studies have shown that
onabotA is most effective in conditions ofincreased nerve activity [11]. Khera et al.also demonstrated no significant differences
in the bladder contraction frequency oramplitude of contractions in any animaltreated with instillation of saline alone,
which shows that the effect of theintravesical instillation procedure is notgreat enough to affect bladder function.
In the present study, there were alsouninhibited contractions in conscious SCTrats, which agree with previous findings
[16], and onabotA administration reducedthe frequency of uninhibited contractionsand significantly lowered the maximum
voiding pressure. Thus, intravesical onabotAinstillation improves bladder dysfunction.Furthermore, onabotA administered 3
weeks after SCT significantly reducedCMG-induced effects on blood pressureand heart rate, suggesting that AD
improves with onabotA treatment. This
was also associated with a decrease ofNGF concentration in T4-DRG.
Nerve growth factor regulates sensory andsympathetic neuronal growth and is known
to increase at least fourfold within a weekafter SCI [17]. Exogenous anti-NGF alsodecreased dysreflexia in rats by 30% after
SCI, suggesting that NGF plays a major rolein the pathogenesis of AD [18]. Recentstudies have shown a close relationship
between NGF and changes in the afferentarbour, which can contribute to AD [19].
FIG. 2.
Arterial pressure (a) and heart
rate (b ) changes in response
to CMG.
SCT + onabotA
P= 0.001,
P= 0.011
P= 0.01,
P= 0.024
SCTControl
SCT + onabotASCTControl
Change in arterial pressure inresponse to bladder distension
50
a
b
40
Arterialpressurechange,mmHg
Heartratechange,
beats/min
30
20
10
200
150
100
50
0
0
Change in heart rate inresponse to bladder distension
Intrathecal anti-NGF administration
improves AD in rats [5], suggesting thatdrugs with anti-NGF properties such asonabotA might have a similar effect. In the
present study, we demonstrated thatintravesical onabotA treatment reduces NGFcontent in T4-DRG after SCT in rats and also
blocked AD, possibly by lowering NGFcontent at the injury site.
Recent reports have found that increasedNGF in the spinal cord after spinal cordinjury is responsible for inducing
hyperexcitability of C-fibre bladder afferent
pathways [20], and that intrathecalapplication of NGF antibodies, which
neutralized NGF in the spinal cord,suppressed detrusor hyperreflexia anddetrusor sphincter dyssynergia in rats with
SCI [21]. In addition, intravesical onabotAinjection lowered NGF content in thebladder tissue of patients with neurogenic
detrusor overactivity [8].Together, thesereports support the findings of the presentstudy that onabotA suppressed neurogenic
bladder overactivity, lowered voidingpressure and blocked AD response during
bladder distension via urodynamics in SCI
rats. After SCI, increased NGF content in thebladder, dorsal root ganglia and spinal cordhas been reported [22]. During development,
NGF is released by the target tissue, takenup in responsive neurones by receptor-mediated endocytosis and transported
retrogradely to the cell body where it exertsits trophic/differentiative effects [23], andintrathecal administration of NGF at the
L6-S1 level of the spinal cord for 1 or 2weeks caused bladder overactivity andhyperexcitability of bladder afferent
neurones.
It has been proposed that detrusor sphincter
dyssynergia is the initial insult after SCI [3]and it leads to bladder outlet obstructionand, subsequently, to bladder hypertrophy.
Bladder smooth muscle and urotheliumwere also found to produce NGF [24].Therefore, we believe that nascent NGF in
the bladder arrives at DRG and the spinalcord via retrograde transport. At the site ofinjury, however, NGF concentration is
highest because, in addition to retrogradetransport from visceral end organs, NGF is
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O N A B O T U L I N U MT O X I N A C O N T R O L S B L A D D E R D Y S F U N C TI O N A N D A U T O N O M I C D Y S R E F L E XI A A F T E R S P I N A L C O R D I N J U R Y
2 0 1 1 T H E A U T H O R SB J U I N T E R N A T I O N A L 2 0 1 1 B J U I N T E R N A T I O N A L 5
also produced by surrounding neuroglia [4].Furthermore, the urothelium is currently
perceived as a sensing structure withsignalling properties due to neurotrophinreceptors (TrkA and p75NTR) and a number
of Transient Receptor Potential (TRP)channels, including TRPV1 [25]. NGF hasbeen shown to activate TRPV1 on small
afferent nerves, which can promote therelease of excitatory neuropeptides [25].Thus, a reduction of bladder NGF could lead
to afferent pathway desensitization. Very
recently, one study using transgenic micedemonstrated that overexpression of NGF
leads to urinary bladder enlargementcharacterized by marked nerve fibrehyperplasia in the submucosa and detrusor
smooth muscle. They also found a markedincrease in the density of calcitoningene-related peptide and substance
P-positive C-fibre sensory afferents,neurofilament 200-positive myelinated
sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibres in thesub-urothelial nerve plexus [24].
In conclusion, in the present studywe showed that intravesical onabotA
treatment blocks AD in rats withT4-SCT. This reduced dysreflexia responsewas associated with a decrease NGF
concentrations at the bladder and dorsalroot ganglia T4 segment, which suggestan afferent pathway modulation by
intravesical onabotA treatment. Thefindings of the present study highlight thepotential benefits of intravesical onabotA
treatment in patients with SCI, and also
provide a novel mechanism for the controlof AD via a minimally invasive treatment
modality.
ACKNOWLEDGEMENTS
Financial assistance (postdoctoral fellowshipin the area of incontinence) was provided toM.S.E. through a partnership programme of
the Canadian Foundation for Research onIncontinence and the Canadian Institutes ofHealth Research.
CONFLICT OF INTEREST
None declared.
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FIG. 3.
NGF concentration at T4-DRG (a)
and bladder (b) in control, SCT
and SCT+ onabotA groups.
SCT
SCT
Control
800
a
b
600
400
T4-NGF,pg/m
l
BladderNGF,pg/ml
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1000
800
600
400
200
0
Sham control0
P= 0.002,
P= 0.006
P= 0.01,
P= 0.028
SCT + onabotA
SCT + BTX-A
SCT
SCT
Control
Sham control
SCT + onabotA
SCT + BTX-A
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Correspondence: Mohamed S. Elkelini MDMSc, 399 Bathurst Street, MP 8-306,
Toronto, Ontario, M5T 2S8, Canada.
e-mail: [email protected]
Abbreviations:AD, autonomic dysreflexia;CMG, cystometrogram; DRG, dorsal rootganglia; NDO, neurogenic detrusor
overactivity; NGF, nerve growth factor;onabotA, onabotulinumtoxinA onabotA,onabotulinumtoxinA; SCI, spinal cord injury;
SCT, spinal cord transection.