E U R O P E A N U R O L O G Y 6 1 ( 2 0 1 2 ) 1 1 8 5 – 1 1 8 71186

separate occasions. Furthermore, the image analysis should

have been conducted by two independent observers and

correlation coefficients presented. Does the AxioVision

software count nerves accurately, unlike previous grid-

counting methods? Does the software avoid accidental

double counting of nerves? Why was the statistical

significance regarded differently for the single- and multi-

ple-injection experiments? Furthermore, there are now

three-dimensional small-animal–imaging technologies that

might be more representative than the two-dimensional

images presented.

It may have been more informative if the colocalisation

studies also had included TRPV1 and P2X3, previously

shown to be downregulated by onabotulinumtoxinA in

NDO and IDO [5,6]. I know that synaptic vesicle glycopro-

tein 2, the known receptor target, remains largely elusive in

bladder experiments; however, again, staining for it may

have added to the scientific rigour.

That being said, I enjoyed reading the article, which takes

us on a journey from the patient to the laboratory and back to

the patient again. The EU INComb group, which brings

together a number of European partners, is to be congratu-

lated for producing such quality and clinically relevant

science.

Conflicts of interest: Prokar Dasgupta acknowledges financial support

from the Department of Health via the National Institute for Health

Research (NIHR) comprehensive Biomedical Research Centre award to

Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s

College London and King’s College Hospital NHS Foundation Trust. He also

acknowledges the support of the MRC Centre for Transplantation, King’s

DOIs of original articles: 10.1016/j.eururo.2012.01.046,10.1016/j.eururo.2012.02.043* Corresponding author. Faculty of Medicine of Porto, Institute of His-tology and Embryology, Alameda Hernani Monteiro, Porto, 4200Portugal. Tel. +351936046307.E-mail address: aavelino@med.up.pt (A. Avelino).

Health Partners, and unrestricted educational grants from Allergan and

The Urology Foundation. The author has been a principal investigator

for Allergan.

References

[1] Mangera A, Andersson KE, Apostolidis A, et al. Contemporary

management of lower urinary tract disease with botulinum toxin

A: a systematic review of Botox (onabotulinumtoxinA) and Dysport

(abobotulinumtoxinA). Eur Urol 2011;60:784–95.

[2] Dowson C, Watkins J, Khan MS, Dasgupta P, Sahai A. Repeated

botulinum toxin type A injections for refractory overactive bladder:

medium-term outcomes, safety profile, and discontinuation rates.

Eur Urol 2012;61:834–9.

[3] Popat R, Apostolidis A, Kalsi V, Gonzales G, Fowler CJ, Dasgupta P.

A comparison between the response of patients with idiopathic

detrusor overactivity and neurogenic detrusor overactivity to the

first intradetrusor injection of botulinum-A toxin. J Urol 2005;174:

984–9.

[4] Coelho A, Cruz F, Cruz CD, Avelino A. Spread of onabotulinumtoxinA

after bladder injection: experimental study using the distribution of

cleaved SNAP-25 as the marker of the toxin action. Eur Urol 2012;

61:1178–84.

[5] Apostolidis A, Dasgupta P, Fowler CJ. Proposed mechanism for the

efficacy of injected botulinum toxin in the treatment of human

detrusor overactivity. Eur Urol 2006;49:644–50.

[6] Apostolidis A, Popat R, Yiangou Y, et al. Decreased sensory receptors

P2X3 and TRPV1 in suburothelial nerve fibers following intra-

detrusor injections of botulinum toxin for human detrusor overac-

tivity. J Urol 2005;174:977–82.

doi:10.1016/j.eururo.2012.02.043

Platinum Priority

Reply from Authors re: Prokar Dasgupta. VolumeMatters: Bladder Injections of Botulinum Toxin Type A.

Eur Urol 2012;61:1185–6

Yes, Volume Matters: Bladder Injections of Botulinum

Toxin Type A

Ana Coelho a,b, Francisco Cruz a,b,c, Celia D. Cruz a,b, AntonioAvelino a,b,*

a Department of Experimental Biology, Faculty of Medicine, University of

Porto, Portugal; b IBMC, Institute for Molecular and Cell Biology, University

of Porto, Portugal; c Department of Urology, Hospital Sao Joao, Porto,

Portugal

We greatly appreciated the timely and detailed editorial of

Dr. Dasgupta regarding the present and putative future

applications of onabotulinumtoxinA (OnabotA) to treat

bladder pathologies [1]. Primarily focused on injection

methods, he raises very exciting questions about the

development of new methods to administer the neurotoxin.

We particularly welcome the comments made on our recent

study [2], but we think that some clarification is desirable.

It is true that some clinical trials showed greater efficacy

for higher volumes of injection [3], but we think that the

lack of clear, objective outcome measurements and the low

number of studies published justified a controlled experi-

ment. We chose to use the presence of cleaved SNAP-25

protein as a marker of the action of OnabotA, allowing the

correct identification of the affected bladder structures.

Interesting and worthy of a good scientific discussion

as they may be, we disagree with some issues raised by

Dr. Dasgupta and would like offer clarification:

� We never stated that in sensory nerves the expression of

cleaved SNAP-25 was lower at 3 d than at 1 d or 1 wk. On

the contrary, we report that there were no significant

differences (Fig. 5 in our paper [2]).

� W

e are well aware that one experiment does not provide

good science. For that reason, all experiments were

performed in quadruplicate in all cases where quantifi-

cation was performed. Fibers were not counted by

E U R O P E A N U R O L O G Y 6 1 ( 2 0 1 2 ) 1 1 8 5 – 1 1 8 7 1187

software but were hand drawn, as stated in the methods

section. This avoids artifacts and double counting. The

method is old-fashioned and time consuming but reliable.

� T

he statistical significances were regarded differently

for the single- and multiple-injection experiments

because they were different experiments, with different

objectives. This is clearly stated in the materials and

methods section.

Some of the suggestions of Dr. Dasgupta, like using TRPV1

and P2X3 immunoreactions were not relevant to the

essential issue raised in the manuscript: the spread of

OnabotA as shown by the expression of cleaved SNAP-25

immunoreactivity and the characteristics of the structures

that express the cleaved protein. Other studies will certainly

address these collateral issues in the future. Likewise, a

three-dimensional reconstruction of the whole bladder

showing the distribution of cleaved SNAP-25 would not

add any extra value to the main finding of the present study,

that the action of botulinum toxin injection is dependent on

the injection volume. That was clearly shown on a single-

plane analysis.

Finally, and again, we thank Dr. Dasgupta for his

editorial, his attention, and, last but not least, his kind

comments regarding the EU INComb group.

Conflicts of interest: Francisco Cruz is a consultant for Astellas, Allergan,

and Recordati. He is also an investigator in clinical studies for Pfizer,

Allergan, and Wieth.

References

[1] Dasgupta P. Volume matters: bladder injections of botulinum toxin

type A. Eur Urol 2012;61:1185–6.

[2] Coelho A, Cruz F, Cruz CD, Avelino A. Spread of onabotulinumtoxinA

after bladder injection. Experimental study using the distribution of

cleaved SNAP-25 as the marker of the toxin action. Eur Urol 2012;

61:1178–84.

[3] Popat R, Apostolidis A, Kalsi V, Gonzales G, Fowler CJ, Dasgupta P.

A comparison between the response of patients with idiopathic

detrusor overactivity and neurogenic detrusor overactivity to the

first intradetrusor injection of botulinum-A toxin. J Urol 2005;174:

984–9.

doi:10.1016/j.eururo.2012.03.009