2011 Toxina Botulinica y Disreflezxxia

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E L K E L I N I E T A L .

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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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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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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.

2011 Toxina Botulinica y Disreflezxxia

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