Botulinum toxin A in the treatment of bladder dysfunction after spinal cord injury: beyond SNAP 25 cleavage

Oliveira R1, Sílvia C1, Cavaleiro H1, Cruz F2, Cruz C1

Research Type

Basic Science / Translational

Abstract Category

Neurourology

Abstract 371
E-Poster 2
Scientific Open Discussion ePoster Session 18
Thursday 5th September 2019
13:45 - 13:50 (ePoster Station 8)
Exhibition Hall
Animal Study Basic Science Biochemistry Spinal Cord Injury
1. Dept. Biomedicine - Experimental Biology Unit, Faculdade de Medicina da Universidade do Porto, Portugal / Translational Neurourology Group - IBMC - Institute for Innovation and Health Research, Porto, Portugal, 2. Translational Neurourology Group - IBMC - Institute for Innovation and Health Research, Porto, Portugal / Dept. Urology - Hospital São João, Porto, Portugal
Presenter
R

Raquel Oliveira

Links

Poster

Abstract

Hypothesis / aims of study
Bladder  wall injections of botulinum toxin A (BoNT/A) is the gold-standard treatment for neurogenic detrusor overactivity (NDO), leading to marked improvements on bladder function. BoNT/A acts  on  motor,  autonomic  and  sensory bladder fibres,  cleaving the  synaptic  protein SNAP-25  and impairing vesicle-mediated neurotransmission. The beneficial effects of BoNT/A are long-lasting but many patients require re-injections as improvement of lower urinary tract function subsides over time. Understading the fine mechanisms of action of BoNT/A opperating at the intracellular level could help improve treatment and potentiate the duration of the effects of this neurotoxin. In the present study, we are investigating the intracellular events occuring in bladder afferents to clarify the mechanisms of action of BoNT/A. We hypothesize that this toxin may produce neuronal injury and cellular stress. Thus, we evaluated effects of NDO treatment with BoNT/A in neuronal growth patterns and expression of cell stress markers of bladder afferent neurons.
Study design, materials and methods
Female rats were habituated to handling for at least 1 week prior to all expriments. Indwelling intravesical catheteres were placed before they were submitted to T8/T9 largely incomplete spinal cord transection (SCT) or sham surgery. Awake cystometries were performed at baseline, 1 week and 4 weeks after SCT to evaluate the development  of  urinary  dysfunction.  
At  4  weeks  post-SCT,  when  NDO  is  typically  present,  rats  received  10 bladder-wall injections of BoNT/A 10U diluted in 50uL of saline. Control rats received only saline.Three days after bladder injections, awake cystometries were performed and rats were euthanized, followed by bladder and DRG L5-S1 collection. Bladders were processed for immunohistochemistry to evaluate the catalytic activity of BoNT/A and levels of cleaved SNAP25 were measured. DRG were processed for western blotting (n=4/group) to measure levels of cell stress markers, namely ATF3 (a well established marker of neuronal injury) and PERK (a marker of reticulum stress). In some animals (n=3/group), DRG were collected and digested with collagenase. Cells were mechanically dissociated and plated in NGF-supplemented medium. Cells were allowed to attach and grow for 14 hours. After this, cells were fixated with ice-cold PFA and immunostained against beta-3 tubulin. The lenght and branching of neurites were analysed to evaluate the intrinsic growth ability.
Results
Four weeks after SCT, saline-treataed rats presented increased bladder pressure and frequent bladder contractions associated with high voided  volumes.  We found that BoNT/A  treatment  reduced  maximal  bladder  pressure  and  frequency  of bladder contractions. This was accompanied by increased expression of cleaved SNAP-25 cleavage in the bladder wall, confirming the effects of BoNT/A. At the cellular level, we found that improvement of bladder function was accompanied by upregulation of cellular stress  markers, such as ATF3 and PERK, in the cell body of bladder afferents. In vitro  assays, used to  evaluate intrinsic ability of terminal growth, suggested a tendency for reduced dendritic length and branching inBoNT/A-treated SCTrats in comparison with saline-injected SCT animals.
Interpretation of results
Preliminary results suggest that urodynamic improvements following bladder treatment with BoNT/A, known to reflect SNAP-25 cleavage, may reflect the occurrence of metabolic injury in affected bladder afferent neurons. This is supported not only by increased expression of ATF3 and PERK but also by impairment of neurite emission and growth in in vitro condition. These data indicates that BoNT/A induces cellular stress, which could underlie the prolonged duration of the beneficial effects of BoNT/A in SCI-induced bladder dysfunction.
Concluding message
A better understanding of the fine molecular mechanisms of action of BoNT/A is crucial to explain the long duration of effects of treatment. In addition, the identification of cellular events induced by BoNT/A could contribute to modification of the toxin to improve its efficacy, by increasing the specificity and/or the duration of BoNT/A-induced improvement of lower urinary tract function. These results could expand and potentiate the therapeutic use of BoNT/A and support protocols adjusting and optimizing treatment to different pathologies of the lower urinary tract.
Disclosures
Funding Prémio Melo e Castro Neurociências 2016 ,Santa Casa da Misericórdia de Lisboa –INSPIReD; Raquel Oliveira PhD fellowship: NORTE-08-5369-FSE-000026 -Programas Doutorais, FSE –Fundo Social Europeu –NORTE2020 –Programa Operacional Regional do Norte; Sílvia Chambel PhD fellowship: SFRH/BD/135868/2018, FCT-Fundação para a Ciência e Tecnologia Clinical Trial No Subjects Animal Species Rat Ethics Committee Ethics committee at the Faculty of Medicine of the University of Porto