Central inhibitory effect of intravesically applied botulinum toxin A in chronic spinal cord injury

Neurourology and Urodynamics 30:1376–1381 (2011)

Central Inhibitory Effect of Intravesically AppliedBotulinum Toxin A in Chronic Spinal Cord Injury

Alvaro Munoz,1 George T. Somogyi,1 Timothy B. Boone,1,2 and Christopher P. Smith1,3*1Baylor College of Medicine, Houston, Texas2The Methodist Hospital, Houston, Texas

3Michael E. DeBakey VAMC, Houston, Texas

Aims: We evaluated a putative central inhibitory effect of intravesical botulinum toxin A (BoNT-A) on the activity oflumbosacralspinalneurons inachronicspinalcord injury (SCI)modelofbladderoveractivity.Methods: FemaleSprague-Dawley rats underwent T8 spinal cord transection. Fourweeks later, once overactive neuropathic detrusor pathways haddeveloped,theanimalsunderwentintravesicalinstillationwitheithersaline(1 ml)orBoNT-A(Botox1,20 U/1 ml)for1 hr.Twodays later, therats thencompletedacystometricevaluationprior tospinal cordharvest.Sections fromtheL4-S1spinalcord segments were examined for the total number of c-fos immunoreactive cells.Results: Comparison of the saline andBoNT-AtreatedgroupsshowedasignificantdecreaseinL6(i.e.,67%,P < 0.001)andS1(i.e.,47%,P < 0.01)c-fosexpression(43%) in BoNT-A treated rats compared to saline controls. Cystometrogram studies revealed that the frequency of non-voiding bladder contractions was reduced by 73% (P < 0.05) in BoNT-A compared to saline treated rats. No change in thefrequencyofvoidingbladdercontractionsoramplitudeofbladdercontractionwasobservedbetweenthesalineandBoNT-Atreated groups. Conclusion: In a SCI model of bladder overactivity, intravesical BoNT-A significantly inhibits theresponse of bladder afferent activated lumbosacral neurons without significantly impairing efferent bladder function.Neurourol. Urodynam. 30:1376–1381, 2011. � 2011 Wiley-Liss, Inc.

Key words: botulinum toxin-A; c-fos; overactive bladder; spinal cord injury

INTRODUCTION

Spinalcordinjury (i.e., SCI) inducesneuroplasticityresultinginthe development of pathologic bladder reflexmechanisms lead-ing toneurogenic detrusor overactivity (NDO). Studies inanimalmodels suggest that one possible mechanism for bladder over-activity is theemergenceofC-fiber inducednon-voidingbladdercontractions.1,2 In fact, desensitization of C-fibers using capsai-cin eliminates non-voiding contractions in SCI rats withoutaffecting A-d afferent driven voiding bladder contractions.

Botulinum toxin A (BoNT-A) injection has been used to suc-cessfullytreatNDOresultingfromSCI.3Invariousanimalmodelsof bladder overactivity (i.e., SCI and chronic bladder inflam-mation), intravesical instillation of BoNT-A has been shown toselectively reduce thebladder afferent responsewithout signifi-cantly affecting bladder efferent function (i.e., bladder contrac-tility).4,5 In fact, SCI animal studies suggest that BoNT-A has aprominent effect in inhibiting C-fiber mediated non-voidingbladder contractions.6 Animal and human studies have docu-mented a potential role for sensory purinergic pathways in thedevelopment ofbladder overactivity.7–10Moreover, aperipheraleffect of BoNT-A in reducing purinergic transmission is evidentfrom clinical and laboratory research studies.6–8,11

Activationofafferentnerves inthe lowerurinarytract leads toimmediate early gene expression of c-fos in postsynaptic spinalcordneurons.12–14 In fact,noxiousornon-noxious stimulationofsensory nerves in the rat lower urinary tract has been shown toincrease Fos immunoreactivity in specific areas of the L6-S1spinal cord in neurally intact and SCI rats.12–14 Our laboratoryhas previously shown that BoNT-A bladder instillation signifi-cantly reduces c-fos expression to bladder stimulation in ananimal model of chronic inflammation.4 However, no one hasevaluatedwhether bladder application of BoNT-A alters centralneuronal response inananimalmodel of bladder overactivity inwhichthespinalcordhasbeentransected (i.e., SCI). Inthecurrent

study,we evaluated the impact of BoNT-A treatment onbladderafferent responses in an animal model of SCI induced bladderoveractivity by (1)measuring the lumbosacral spinal cord levelsofc-fosevokedbynonnoxiousbladder stimulationand (2) inves-tigating the functional consequences of intravesical BoNT-Aapplication on cystometric parameters of bladder afferent andefferent function.

MATERIALS AND METHODS

FemaleSprague-Dawleyratsweighing250–300 gwereusedforthis study. Six animals per group were divided by intravesicalinstillation into two groups: group 1-saline (intravesical saline,n ¼ 6), group 2-BoNT-A (intravesical BoNT-A, n ¼ 6). All animalexperiments were performed in accordance with the NationalInstitutesofHealthguideforthecareanduseoflaboratoryanimalsand the research protocol was approved by the InstitutionalAnimal Care and Use Committee of Baylor College of Medicine.

Experimental Protocol

All animals underwent total T8 spinal cord transection.Four weeks later, after bladder hyperreflexic pathways havedeveloped,1 animals underwent intravesical instillation with

Conflict of interest: none.Dirk De Ridder led the peer review process.Grant sponsor: USDepartment of Veterans Affairs; Grant number: CDA 200600158;Grant sponsor: AUA Foundation.*Correspondence to: Christopher P. Smith, MD, MBA, Scott Department of Urology,Baylor College of Medicine, 1709 Dryden St, #1610, Houston, TX 77030.E-mail: [email protected] 21 October 2010; Accepted 6 January 2010Published online 20 April 2011 in Wiley Online Library

(wileyonlinelibrary.com).

DOI 10.1002/nau.21068

� 2011 Wiley-Liss, Inc.

either saline (1 ml) or BoNT-A (Botox1; Allergan, Inc., Irvine, CA,20 U/1 ml) under isoflurane anesthesia (2%) for 1 hr (i.e., day 0)per prior protocol.5,15 After 40 hr, the saline or BoNT-A instilledratsunderwentacystometrogramevaluation (i.e., day2) for2 hrprior to spinal cord harvest.

Cystometry and c-fos Experiments

On day 2, animals were anesthetized with 0.9 g/kg urethane(Sigma, St Louis,MO) subcutaneously and a suprapubic catheterwas placed through the bladder dome. The catheter was con-nected to a syringe pump and saline was infused at a rate of0.12 mlperminutefor2 hr. Infusedsalinewasexpelledbyreflexbladder contractions. Reflex bladder contractions and voidedurine were measured with a pressure and force transducerconnected to a computerized data acquisition system (DataQ,Akron, OH). Bladder contractions were designated as voiding ornon-voiding contractions depending on whether urine wasexpelled with each contraction. The frequency of voiding(VCF) and non-voiding contractions (NVCF), and the amplitudeof voiding bladder contractions were calculated during the last60 minofbladderperfusionusingtheWindaqplaybackprogram(WindaqEx; DataQ).

Following bladder infusion for 2 hr, the rats underwent intra-cardiac perfusion with ice-cold Krebs solution followed by ice-cold 4% paraformaldehyde. After fixation and dissection thespinal cords were stored overnight and then transferred tophosphate buffer solution. Sections from the L4-S1 spinal cordsegments were identified after following the nerve rootscorresponding to the L4-S1 dorsal root ganglions. The L4-S1 seg-ments of the spinal cord were excised, placed in 30% sucrosefor 24 hr, embedded in Tissue-Tek compound (Sakura Finetek,Torrance, CA) and flash frozen. Frozen segments were sectionedat 40–50 mm transversely using a cryostat (Microm HM 505E;Ted Pella, Redding, CA).

Spinal sections were incubated for 72 hr with Fos antiserumdiluted inpotassiumphosphatebufferedsalinewith0.4%TritonX-100.4 Sections were treated with avidin-biotin peroxidasecomplex,mountedongelatincoatedslidesanddehydratedusinggraded ethanol, followed by xylene. Tissue from the treatmentand control groups were processed simultaneously. Slides werethen examined using light microscopy in a blinded fashion andc-fos immunoreactive cells were counted for each spinal cord.A total of 10 sections were evaluated per spinal cord segmentusing an Eclipse 50i microscope and the NIS-Elements software(both from Nikon Instruments, Melville, NY). Immunoreactivecells were counted in four areas: dorsal commissure (DCM),sacral parasympathetic nucleus (SPN), medial dorsal horn(MDH), and lateral dorsal horn (LDH).

Statistical Analysis

Statistical analysis was performed using Prism4 (GraphPad,San Diego, CA). The mean � SEMwas calculated for each seriesofmeasurements. c-fos Immunoreactivity and cystometrogrammeasurements were compared among the groups using one-way ANOVA with Bonferonni’s multiple comparison testand unpaired t-test, respectively, with P < 0.05 consideredsignificant.

RESULTS

Fos Immunoreactivity

Figure 1 shows c-fos immunoreactivity in SCI animals thatwere treated with intravesical saline or BoNT-A.

Total c-fos

Saline application: Figure 2 shows that after 2 hr of salinebladder perfusion, mean total c-fos levels in the L6 spinal cordsegments was increased by 217% and 195% over the total c-foslevels in the L4 and L5 segments, respectively (P < 0.001). Sim-ilarly,mean total c-fos levelswere increased in theS1spinal cordsegment by 156% and 138%, respectively, when compared tolevels found in the L4 and L5 spinal cord segments (P < 0.01).BoNT-Aapplication: As shown inFigure 2, the increase in c-fos

immunoreactivity induced by saline in the L6 and S1 spinal cordsegments was markedly decreased by 67% (P < 0.001) and 47%(P < 0.01), respectively, inanimals pre-treatedwith intravesicalBoNT-A compared to controls (i.e., intravesical saline). However,BoNT-A did not affect c-fos expression in the L4 or L5 spinalsegments.

Regional c-fos Expression

Similar patterns of c-fos immunoreactivity were observedwhen all four regions of the dorsal hornwere examined individ-ually. Significant increases in c-fos levels were demonstrated inL6-S1 segments compared to L4-L5 segments and BoNT-A pre-treatment markedly reduced c-fos levels but only in the L6-S1spinal cord (Fig. 3). Interestingly, the highest c-fos levels wereobserved in the MDH region. In addition, BoNT-A’s inhibitoryeffect on c-fos expression was greatest in the L6 spinal MDHand LDH regions (i.e., 72% and 73% decrease, respectively,P < 0.001).

Cystometrogram Evaluation

Intravesical instillation of BoNT-A to rats with SCI signifi-cantly reduced the number of non-voiding contractions by73% when compared to saline treated animals (P < 0.05;Figs. 4 and 5). However, no significant changes in voiding con-traction frequency or the amplitude of voiding contractionweredetected when comparing saline treated with BoNT-A treatedanimals.

DISCUSSION

WeusedaSCImodelofbladderoveractivitytoinduceNDOandevaluated central spinal pathway responses to bladder stimu-lation. BoNT-Awas tested to determine its local impact on reflexbladder activity as well as its distant effect on lumbosacralneuronal c-fos response. Our experiments demonstrate that:(1) the activity of central spinal pathways projecting from theL6-S1 rat spinal cord is significantly enhanced inchronic SCI rats;(2) the exaggerated c-fos activity in chronic SCI rats coincideswith bladder overactivity observed during cystometrogramstudies; (3) intravesical administration of BoNT-A significantlyreduces bladder hyperactivity, in particular, by reducing thenumber of non-voiding bladder contractions without signifi-cantly impairing efferent bladder function (i.e., no effect oncontractile amplitude and voiding contraction frequency); and(4)thereductioninbladderoveractivitycorrelateswithamarkeddecrease in c-fos levels in the L6-S1 dorsal horn region, wherebladder afferent nerves are known to project.Birder and de Groat16 first described changes in the lumbo-

sacral spinal expression of the proto-oncogene c-fos followinglower urinary tract irritationwith 1%acetic acid andafter appli-cation of glutamate antagonists. The increases in c-fos expres-sion were most pronounced in the L5-S1 spinal segments. Theinvestigators found that the inhibitory effects of glutamateantagonists on c-fos expression did not differmuchwhen using

BoNT-A Reduces c-fos Expression in SCI Rats 1377

Neurourology and Urodynamics DOI 10.1002/nau

intact versus acute spinal transected rats. They suggested thatmostspinalc-fospositiveneuronsarearesultofdirectactivationof spinal neurons by primary afferents rather than throughsupraspinal reflex pathways.Further investigations have demonstrated enhanced c-fos

expression in chronic SCI animals from non-noxious bladderstimulation. Vizzard12 found that chronic SCI altered both thesegmentalandtheregionalexpressionofc-fosproteintobladderafferent stimulation. For starters, she found that both L1-L2(i.e., sympathetic afferents) and L6-S1 (i.e., pelvic afferents)c-fos immunoreactivity was significantly increased in chronicSCI compared to normal control rats. In addition, the DCM dem-onstrated the greatest increase in c-fos positivity followed bythe LDH and MDH regions, respectively. No significant changein SPN c-fos expression was found in chronic SCI rats. Most ofthe increase in c-fos signaling could be attributed to enhancedactivity of C-fiber pathways as evidenced by the dramaticreduction in c-fos expression in SCI animals pretreated withcapsaicin.In contrast, other investigators have found that SCI induces

a more widespread increase in c-fos expression to activationof bladder afferents. For example, one study found bladderperfusion of SCI rats enhanced c-fos signaling in L3-S1 spinalsegments compared to neurally intact rats.17Moreover, as com-pared toVizzard’s study, these investigators showeddifferencesin c-fos expression in the LDH, MDH, or SPN regions of L6 andS1 while no change in DCM c-fos immunoreactivity wasobserved.12,17

Our results lie somewhere between both investigators.While increases in c-fos immunoreactivity with bladder stimu-lation were localized to the L6-S1 spinal cord, the increasedexpression was widespread across all four dorsal horn regions.

Fig. 2. The total number of c-fos positive cells in the L4-S1 spinal cord seg-

ments of BoNT-A treated versus saline treated rats. Note the significant

decrease in c-fos expression in the L6-S1 segments of BoNT-A treated rats.###Denotes P < 0.001 versus L4 and L5 saline, ##denotes P < 0.01 versus L4

and L5 saline, ��denotes P < 0.001 versus L6 saline, ��denotes P < 0.01

versus S1 saline.

Fig. 1. Microscopic views of immunostained L6 sections from saline (A,C) or BoNT-A (B,D) The clear squares in (A)

and (B) indicated the regions shown in (C) and (D). Objective magnification in (A,B) was 4� and 40� in (C,D). Note

the reduction in c-fos expression in BoNT-A treated rats (B,D).

1378 Munoz et al.


In addition, although BoNT-A inhibited c-fos expression to thegreatest extent in the LDH and MDH dorsal horn regions, it’seffects were, in general, widespread and indiscriminate. Viz-zard12 demonstrated that desensitizing capsaicin-sensitiveafferents reduced c-fos expression to bladder stimulation butonly in the DCM dorsal horn area. This brings to questionwhether BoNT-A also inhibits non-capsaicin-sensitive bladderafferent pathways (i.e., Adbladder afferents). On the other hand,our cystometrogramstudies showed thatbladder BoNT-A instil-lation only inhibits non-voiding but not voiding bladder con-tractions. As non-voiding bladder contractions in SCI rats aredriven by capsaicin-sensitive bladder afferents,1,2 the currentresults suggest that sprouting of capsaicin-sensitive bladderafferents may be more widespread in SCI rats, at least underour experimental conditions. Future experiments will evaluatethe effects of pretreatment with capsaicin in BoNT-A and non-BoNT-A treated SCI rats.

BirderanddeGroat13 foundregionaldifferences indorsalhornc-fos expression in neurally intact rats dependent on the type ofbladder stimulation used (i.e., nociceptive vs. non-nociceptive).For example,while theDCMhad similar c-fos expression to bothsaline and acetic acid stimulation, the SPN had much greater c-fos immunoreactivity to saline infusion (i.e., non-nociceptive

stimulation) while nociceptive stimulation (i.e., acetic acid)induced much greater c-fos expression in the LDH and MDHareas. The investigators also performed experiments in animalsthat had previously undergone pelvic or pudendal nerve tran-section.13 There results suggest that c-fos expression to lowerurinary tract stimulation is the result of activation of both para-sympathetic and somatic afferent pathways. Noxious pelvicnerve stimulation predominantly activates c-fos expression inthe DCM, LDH, and SPN areas while noxious pudendal nervestimulation induces c-fos immunoreactivitymainly in theDCMandMDHareas. There resultswere consistentwithprior studiesthatshowedthatthemajorityofpudendalafferentsterminateinthe MDH region while pelvic nerve afferents mainly project tothe LDH area of the L6-S1 spinal cord.18–20 It is possible thatpudendal afferents could have been stimulated in our animalmodel on day 0 by urethral catheterization and/or temporaryclosure of the urethral outlet during saline or BoNT-A bladderinstillation. Inaddition,bladder instillationofBoNT-Acouldalsotheoretically have impaired pudendal nerve activity if thesolutionmigrated alongside the transurethral catheter to bathethe urethra.Previous investigations in rats have demonstrated that blad-

derBoNT-Ainstillationreducesbladdercontractionfrequencyin

Fig. 3. The number of c-fos positive cells in specific regions of the L4-S1 spinal segments in control (i.e., saline)

and BoNT-A treated rats: (A) DCM, (B) SPN, (C) MDH, and (D) LDH. ###Denotes P < 0.001 versus L4 and L5 saline,##denotes P < 0.01 versus L4 and L5 saline, #denotes P < 0.05 versus saline, ��denotes P < 0.001 versus L6 saline,��denotes P < 0.01 versus either L6 or S1 saline, �denotes P < 0.05 versus S1 saline.



several models of NDO (i.e., SCI and chronic bladder inflam-mation).4–6 Furthermore, each study showed that BoNT-A treat-ment reverses alterations in bladder urothelial transmitterrelease (i.e., ATP � nitric oxide) induced by bladder overactivityindirect concertwithrestoringamorenormalvoidingpattern.Aseparate study found that SCI induces enhanced release of ATPfrom afferent terminalswithin the spinal cord aswell aswithinthe bladder.21 It remains to be determined whether BoNT-A’sinhibition of bladder urothelial ATP release translates intoreduced ATP release from central afferent terminals. However,the significance of elevatedATP levels in conditions of enhancedbladdersensoryactivationandtheeffectofBoNT-Aonimpairingsensory purinergic transmission is highlighted by recent immu-nohistochemical studies in human patients with bladderoveractivity.10,11

BoNT-A’s inhibitory effects on bladder sensory pathways fol-lowing SCI are not limited to the purinergic system but mayinclude reductions in release ofmultiple transmitters, peptides,

and, even growth factors. Nerve growth factor (NGF) release isenhanced within the bladder and spinal cord after SCI, andcontributes to the sprouting of afferent nerve fibers and theresultant NDO that develops.22–24 The sprouting of afferentnervefibersprobablyaccounts for theenhancedc-fosexpressiondetected in SCI compared to neurally intact rats. Clinical studieshave demonstrated significant reductions in bladder NGF con-tent in SCI patients responding to bladder BoNT-A injection.25

Future studies will address whether BoNT-A instillation in ratsalters peripheral and central NGF release in both acute andchronic SCI rats.

CONCLUSION

BoNT-Abladder instillation significantly reduces lumbosacralspinal neuronal activity in chronic SCI rats, as evidenced by thediminishedc-fosexpressiontonon-noxiousbladderstimulation.Theeffect of BoNT-Aon c-fos expressionappear to result, at least

Fig. 4. Characteristic cystometrograms from rats with SCI after intravesical instillation with saline (A,C) or

BoNT-A (B,D). Note the marked decrease in non-voiding bladder contractions following treatment with BoNT-A

(B,D).

Fig. 5. Intravesical instillation of BoNT-A in SCI rats significantly reduces the number of non-voiding contractions

without impairing voiding contraction frequency or contractile amplitude.�Denotes P < 0.05 versus saline

treated rats using Student’s t-test.

1380 Munoz et al.


inpart, frominhibitionofcapsaicin-sensitiveC-fibers,consistentwith the reduction in non-voiding bladder contractionsobserved. Future studies will address the degree to whichBoNT-A acts through nociceptive bladder afferents and thespecific transmitter, peptide, or growth factor systems involved.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Erika M. Munch for helping toperform some of the preliminary experiments.

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Central inhibitory effect of intravesically applied botulinum toxin A in chronic spinal cord injury

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