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Keywords:

  • botulinum toxin type A;
  • overactive bladder;
  • SNAP-25

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

Background

Botulinum toxin type A (BoNT/A), one of the seven subtypes of Botulinum toxin, is commercially available naked or complexed to other proteins. Onabotulinum toxin type A is the most extensively studied BoNT/A brand. Dose equivalence studies between the different brands have never been carried out. BoNT/A is internalized by nerve fibers after binding synaptic vesicle proteins, and the final target of action is synaptosome-associated protein 25 kDa (SNAP-25), a membrane protein essential for synaptic vesicle fusion with the neuronal membrane.

Methods

The current literature about botulinum toxin mechanisms was reviewed to provide an up to date knowledge about the topic.

Results

Immunoreactivity to cleaved SNAP-25, the end product of BoNT/A activity, has been identified in parasympathetic (pre- and postganglionic), sympathetic, and afferent fibers. A consistent decrease in the release of acetylcholine from parasympathetic, norepinephrine from sympathetic, and glutamate and neuropeptides from sensory neurons follows BoNT/A administration. Immunoreactivity to cleaved SNAP-25 was not identified in the urothelium or in myofibroblasts. Nevertheless, a decreased release of ATP and neurotrophins from the urothelial cells has been consistently observed after BoNT/A. The toxin does not cause apoptosis in the bladder. However, injection in rat and dog prostates was shown to induce apoptosis in acinar and stromal cells.

Conclusion

There is now robust information to support that the mechanism of action of BoNT/A in the bladder involves neurotransmitter release from nerve fibers and urothelial cells. Which neurotransmitter is more relevant is, however, unclear. Likewise, the long duration of effect, the importance of the volume of vehicle injected and the selection of specific injection sites, like the trigone, needs further evaluation. Neurourol. Urodynam. 33:31–38, 2014. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

Historical Perspective

Botulinum toxin is the most potent biological poison known to humankind; a few grams in its purified crystalline form is enough to kill millions of people. It is therefore fascinating how a potent poison, even considered for biological warfare, is now among the most promising and innovative therapeutic agents. Botulinum toxin is already approved for the treatment of strabismus, blepharospasm, muscular dystonias, hyperhidrosis, and migraine and is widely used for cosmetic purposes. By the end of 2011, following the conclusion of regulatory phase 3 trials, botulinum toxin was finally approved for the treatment of urinary incontinence secondary to neurogenic detrusor overactivity (NDO).[1] Pivotal studies in overactive bladder (OAB) have been concluded with very positive results,[2] and botulinum toxin is already approved by the US Food and Drug Administration for the treatment of OAB. Product approval is pending in other countries, and new indications for botulinum toxin are in development. Studies in bladder pain syndrome (BPS)/interstitial cystitis (IC) and male lower urinary tract symptoms associated with benign prostatic enlargement are underway.

Three men contributed decisively to the development of botulinum toxin as a therapeutic agent. Christian Kerner 1786–1862, a German physician, was the first to isolate a fatty, highly toxic substance from poisoned sausages. In 1897, Emile van Ermengem 1851–1932, a Belgian bacteriologist, isolated the bacterium producing the toxin, and by the end of the 1970s, Alan B Scott, an American ophthalmologist, successfully injected one of his patients with botulinum toxin as a treatment for strabism. The use of botulinum toxin in the lower urinary tract was initiated by Dykstra and Sidi[3] who, in 1990, pioneered toxin injection into the urethral sphincter to relieve detrusor-sphincter dyssynergia. Schurch et al.[4] reported in March 2000 the results of botulinum toxin injection into the bladder to treat urinary incontinence in patients with NDO due to spinal cord injury.

Types of Botulinum Toxin and Mechanism of Neuronal Internalization

Excellent reviews on the mechanism of action of botulinum toxin have been published previously.[5-7] Botulinum toxin is a neurotoxic protein produced by a great variety of Gram-positive, spore-producing bacteria that form Clostridium botulinum.[5-7] Clostridium botulinum bacteria produce one or more of the seven different botulinum toxin serotypes (A–G).[5-7] Subtype A (BoNT/A) has the longest duration of action, making it the most attractive from a clinical point of view.

BoNT/A is synthesized as a single polypeptide chain (≈150 kDa) which is cleaved into a light chain (≈50 kDa) and a heavy chain (≈100 kDa) held together by a fragile disulphide bond and noncovalent bonds. This structure subsequently forms a complex with other proteins, which provides additional characteristics, including resistance to proteolysis and denaturation.[5-7] As the BoNT/A gene may differ in nucleotide sequence, four A subtypes have been classified based on up to 15% variation in the amino acid composition.[7]

The amino acid sequence of the BoNT/A light chain constitutes a catalytic Zn-dependent endopeptidase domain. The heavy chain is subdivided into three portions (HN, HCN, and HCC), but only two have clear functions. The HCC is associated both with the recognition of neuronal-specific areas and toxin internalization. The HN is responsible for translocation of the light chain from synaptic vesicles into the neuronal cytoplasm.[5-7]

BoNT/A is available in different commercial forms which have diverse relative potency. This is the reason for the legal requirement of each brand to have a nonproprietary name in addition to its commercial name. In Botox (onabotulinum toxin A; OnabotA) and Dysport (abobotulinum toxin A; AbobotA), the toxin is complexed with several proteins that provide distinct molecular weights of ≈900 and ≈400 kDa, respectively. A third brand, Xeomin (incobotulinum toxin A; IncobotA), is basically a naked form of the toxin, as its molecular weight, ≈150 kDa, is the same as the toxin alone. Prosigne is the proprietary name of a BoNT/A produced in China that does not yet have a nonproprietary name. For lower urinary tract indications, NDO and OAB, only onabotulinum toxin—in doses of 200 and 100 U, respectively—has received approval up to now.

The current approved method to estimate the potency of a BoNT/A brand is the mouse LD50 (lethal dose 50%); that is, the mass of toxin (expressed in ng/kg of body weight) that kills 50% of mice. More recently, a cell-based potency assay was approved specifically for onabotulinum toxin assay which uses differentiated human neuroblastoma SiMa cells and replicates all steps in BoNT/A mechanism of action.[8] The assay measures the BoNT/A-dependent intracellular increase of cleaved SNAP-25. The EC50, that is, the concentration of toxin required to provoke a response halfway between the baseline and maximum response for OnabotA, is about 1–0.4 U per well. Despite these tests, clinical dose conversion studies of the different BoNT/A brands for use in lower urinary tract dysfunctions have not been carried out. Thus, at present, doses of each brand should be considered as nonexchangeable. Most of the experimental and clinical data available on BoNT/A have been generated from studies utilizing OnabotA.

As the strains producing BoNT/A may differ in gene nucleotide sequence, considerable variation may occur in the amino acid sequence of the toxin.[7] This may have considerable impact on developing strategies for producing neutralizing antibodies required for the treatment of botulism. In addition, such variation may suggest dissimilarities regarding binding-site affinity and relative potency for SNAP-25 cleavage. Four A subtypes have been identified. Smith et al.[9] showed large differences in monoclonal antibody-binding affinity between two BoNT/A toxins despite having 89% sequence identity, leading to the identification of the subtypes A1 and A2. Later, two other subtypes were sequenced the A3 (or Loch Maree which was responsible for a botulism outbreak in Scotland) and the A4, produced by a strain that also produces BoNT/B.[10]

DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

The heavy chain of BoNT/A binds to low-affinity sites, like polysialogangliosides, and to protein receptors that form high-affinity complexes on the neuronal surface, such as synaptic vesicle protein (SV2). The latter is exposed in the synaptic cleft when the synaptic vesicles open on the neuronal surface to release neurotransmitters.[11] The toxin is, therefore, internalized inside the synaptic vesicles. The two chains are then separated, and the light chain is translocated into the cytosol through a pore formed by the HN fragment of the heavy chain. There, the light chain cleaves the attachment proteins that induce fusion of synaptic vesicles to the cytoplasmatic membrane, thereby impeding neurotransmitter release (Fig. 1). Attachment proteins, or soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs), include synaptosome-associated protein 25 kDa (SNAP-25), vesicle-associated membrane protein (VAMP)/synaptobrevin, and syntaxin. BoNT/A cleaves SNAP-25 in contrast with subtype B, which acts preferentially on VAMP.[5-7, 12]

image

Figure 1. Mechanism of action of BoNT/A in a neuronal ending leading to the impairment of the release of neurotransmitters. BoNT/A, botulinum toxin type A; SNAP-25, synaptosome-associated protein of 25 kDa; SV2, synaptic vesicle protein.

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In the human bladder, SV2 and SNAP-25 expression has been demonstrated by immunohistochemistry in parasympathetic, sympathetic, and sensory fibers.[13] Almost all parasympathetic nerves express SV2 and SNAP-25, whereas only half the peptidergic sensory and sympathetic fibers express the two proteins.[13]

Cleaved, inactive SNAP-25 appears rapidly after BoNT/A injection. In the guinea-pig, a robust immunolabeling for cleaved SNAP-25 could be detected in the bladder wall as early as 12 hr after OnabotA injection; maximum intensity was observed at 24 hr, and the intensity remained stable up to 7 days[14] (Fig. 2). In other guinea-pig studies, cleaved SNAP-25 expression was found to be restricted to nerve fibers. Almost all parasympathetic fibers, while less than half the sensory and sympathetic fibers, expressed the cleaved protein.[14, 15] In the human urinary bladder, cleaved SNAP-25 could be detected in patients with NDO up to 11 months after BoNT/A injection.[16]

image

Figure 2. Percentage of (A) parasympathetic, (B) sympathetic, or (C) sensory fibers that also express cSNAP-25 at 24 hr, 3 days, or 7 days after OnabotA intramural injection throughout the whole bladder. The proportion of parasympathetic fibers is statistically higher than sympathetic or sensory fibers (P < 0.0001). However for each type of fiber there, the proportion of positive fibers at the three time points is similar. CGRP, calcitonin gene-related peptide; cSNAP-25, cleaved synaptosome-associated protein of 25 kDa; OnabotA, onabotulinum toxin A; TH, tyrosine hydroxylase; VAChT, vesicular acetylcholine transporter. From Coelho et al.[14]

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PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

In the human bladder, the parasympathetic system comprises preganglionic and postganglionic neurons. Preganglionic parasympathetic axons make synaptic contact with postganglionic parasympathetic neurons in parasympathetic ganglia embedded in the bladder wall. Here, synaptic transmission is also modulated via sympathetic and afferent fibers spread through the ganglia. Preganglionic parasympathetic axons present in the bladder wall are also impaired by BoNT/A injection. Experiments carried out on the guinea-pig bladder, which unlike the rat bladder contains parasympathetic ganglia embedded in its wall, concluded that the preganglionic parasympathetic neurons expressed intact SNAP-25.[14] In fact, after OnabotA injection, these preganglionic neurons exhibited a strong immunolabeling for the cleaved SNAP-25[14] (Fig. 3).

image

Figure 3. Intramural parasympathetic ganglions in the guinea-pig urinary bladder. A: After immunoreaction to cSNAP-25 preganglionic parasympathetic endings appear labeled as red dots encircling unlabeled cell bodies of postganglionic neurons (dark, unlabeled circles). B: The parasympathetic ganglion is now double immunoreacted against NF200 that labels the cell bodies of postganglionic neurons in red-brown, and against VAChT that labels preganglionic parasympathetic endings in green. cSNAP-25, cleaved synaptosome associated protein of 25 kDa. VAChT, vesicular acetyl-choline transporter. Magnification bar indicates 50 µm for both images. Courtesy of Ana Coelho and Antonio Avelino.

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The duration of clinical effect of OnabotA in the human bladder is roughly double that observed in skeletal muscle.[1] Skeletal muscle is innervated by lower motor neurons that lack a synaptic relay station between the spinal cord and the muscle. It is, therefore, tempting to suggest that the longer duration of action of BoNT/A in the bladder results from the impairment of both pre- and postganglionic neurons,[15] which increases the probability of maintaining inhibition of parasympathetic function.

DISTRIBUTION OF OnabotA IN THE BLADDER

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

BoNT/A is delivered by injection into the bladder wall. BoNT/A has not been found to cross the intact urothelium[14, 15] unless it is encapsulated in liposomal formulations[17] or carried by an electric current.[18] However, electromotive administration has been tested successfully only in a pediatric population, and the injection technique has not been evaluated in systematic studies. Variables like number, location or volume of injection, in addition to technical details, such as depth of injection or the manner in which the toxin is reconstituted, may have a profound influence on the final clinical outcome.

Distribution of the toxin after injection is difficult to ascertain. Efforts to carry out direct labeling of the BoNT/A molecule have not yet been successful. Thus, spread of BoNT/A after bladder injection was ascertained indirectly by labeling the fluid used to dissolve the toxin. Patients receiving 300 U of OnabotA, containing gadolinium in the injecting fluid for identification by magnetic resonance imaging, achieved a similar distribution of the fluid whether administered over 30 injection sites (30 ml of fluid in total) or 10 injection sites (10 ml of fluid in total) above the trigone. About ⅓ and ¼ of the total detrusor volume was covered by 10 and 30 ml of injected fluid, respectively.[19]

The ideal method to identify sites of BoNT/A action is the identification of cleaved SNAP-25, the end product of the enzymatic activity of the BoNT/A light chain. For obvious reasons, such study cannot be easily carried out in humans. However, a recent study using guinea-pig bladder, which shares with human bladder a similar parasympathetic innervation, has yielded important findings. The amount of cleaved SNAP-25 induced by a fixed quantity of OnabotA was directly related with the volume of the injection.[14] A volume of 2 U of OnabotA, diluted in 20 µl of fluid and injected in one single point of the bladder wall, generated approximately two-times more cleaved SNAP-25 than the same dose of toxin diluted in 2 µl of fluid. Also, the high diluting volume, in contrast with the low one, generated a robust immunolabeling for cleaved SNAP-25 in bladder areas distal to the injection point (Fig. 4). Injection in the bladder wall remains the recommended method to deliver the toxin, but future clinical studies are necessary to validate the ideal volume of saline in which to dilute BoNT/A.

image

Figure 4. Guinea-pig bladder diagrams showing the distribution of cSNAP-25 immunoreactive fibers (dark lines). A total of 2 U of OnabotA diluted in 2 ml (upper diagrams) or 20 ml (lower diagrams) of saline were administered in the indicated area (syringes). cSNAP-25, cleaved synaptosome-associated protein of 25 kDa; OnabotA, onabotulinum toxin A. From Coelho et al.[14]

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SITES OF ACTION OF BoNT/A IN THE BLADDER

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

Efferent Nerve Fibers

In accordance with the robust appearance of cleaved SNAP-25 in parasympathetic fibers after BoNT/A injection in the bladder wall,[13, 16] the toxin decreases acetylcholine (ACh) release in the detrusor smooth muscle, similar to what is known to occur in the skeletal muscle.[5-7] In rat urinary bladders injected with BoNT/A, the release of ACh triggered by electrical field stimulation was significantly inhibited 5 days after injection.[20] Thirty days after toxin injection, release of ACh was restored to the levels recorded in sham-treated animals. Because depression of ACh release occurred during high frequency (20 Hz) but not low frequency (2 Hz) stimulation, one may expect that BoNT/A is more effective in situations of more intense neuronal activity, such as detrusor overactivity. It is unknown whether BoNT/A affects other neurotransmitters that are coreleased with ACh. However, it is highly probable that the corelease of adenosine 5′ triphosphate (ATP)[21] or vasoactive intestinal polypeptide (VIP)[22] from postganglionic parasympathetic terminals is impaired, resulting in as-yet unidentified consequences. The effect of BoNT/A on VIP release may be worthwhile to investigate, since this neuropeptide is known to cause bladder relaxation.[22]

BoNT/A also influences the expression of muscarinic and purinergic receptor subtypes present in the detrusor, urothelium, and suburothelium.[23] The modification of the M3 receptor may be particularly relevant, as it mediates cholinergic-mediated detrusor contractions[7] as well as bladder afferent activity conveyed in both C and Aδ fibers.[24] Changes in purinergic receptors may also be relevant. Although purinergic transmission is not relevant for detrusor contraction in the normal bladder, the picture seems substantially altered in patients who have idiopathic[25] or neurogenic detrusor overactivity.[26] Repeated OnabotA injections in children suffering from NDO were shown to decrease the expression of M2, M3, P2X2, and P2X3 receptors in the detrusor layer. The analysis was performed at the moment children were submitted to bladder augmentation, and controls were children with similar bladder dysfunction who never received toxin injections. The mechanisms accounting for these changes are unclear. No evidence was presented to support either a direct effect of the toxin on the smooth muscle or an indirect effect resulting from the decreased availability of neurotransmitters in the synaptic cleft.[27] Using biopsy samples from patients with NDO or IDO, Datta et al.[23] were able to demonstrate that successful OnabotA treatment largely restored the expression of muscarinic receptors that were found to be decreased in the suburothelium and urothelium. An increased muscarinic receptor immunoreactivity was observed for M1 and M2 subtypes in the suburothelium, whereas an increase in M3 immunoreactivity was observed in the urothelium. The meaning of these changes is still unclear.[23]

BoNT/A-induced impairment of parasympathetic nerves decreases detrusor smooth muscle contractions. A recent study evaluated detrusor activity in mice using a spinal-bladder preparation that allowed L5-S2 spinal nerve electrical stimulation and simultaneous monitoring of detrusor activity using Ca2+ optical mapping and tension measurements.[28] OnabotA was injected only in one side of the bladder. Electric nerve stimulation on the BoNT/A-treated side did not generate Ca2+ transients, and bladder tension was substantially decreased when compared to the same parameters measured after nerve stimulation of the untreated side.[28] These experiments explain why about 60% of NDO patients do not exhibit detrusor contractions during cystometry after bladder injections of OnabotA, while the remaining 40% show a marked decrease in the pressure generated by ongoing contractile detrusor activity.[1]

Intrinsic detrusor contractions are present in normal bladders and are enhanced after chronic spinal cord transection or chronic bladder outlet obstruction.[1, 28, 29] Interestingly, BoNT/A did not show any effect on the spontaneous contractions of guinea-pig isolated detrusor strips.[30] Likewise, bladder injection of BoNT/A did not reduce the amplitude or frequency of spontaneous contractions in spinalized mice.[28] Also, intracellular Ca2+ transients responsible for intrinsic contractions were not affected by BoNT/A administration.[28]

Altogether, these experiments indicate that intrinsic detrusor activity receives little input from parasympathetic nerve activity. As intrinsic detrusor contractions are, at least partially, suppressed by antimuscarinic drugs, it seems reasonable to suppose that ACh release from neuronal or nonneuronal urothelial sites is involved in the process. Whatever the origin of the neurotransmitter, ACh release in these circumstances is likely to be independent of a mechanism of synaptic vesicle fusion facilitated by SNAP-25.

Afferent Nerve Fibers

In the normal human bladder, about 50% of afferents express SV2 and SNAP-25 indicating their sensitivity to the neurotoxin.[13] In the normal guinea-pig bladder, a similar percentage of sensory fibers will express cleaved SNAP-25 within a few hours of injection.[15] Because these are peripheral branches of bladder sensory fibers, it is not a surprise that BoNT/A causes a substantial decrease in baseline and capsaicin-evoked release of calcitonin gene-related peptide (CGRP) in the bladder wall.[31] This may contribute to suppression of the neurogenic component of inflammation.

In cultured dorsal root ganglion cells, BoNT/A cleaves SNAP-25 and decreases the release of glutamate and substance P.[32] It is, therefore, assumed that BoNT/A injection in the bladder wall decreases the release of these pivotal neurotransmitters in the spinal cord. Nevertheless, the appearance of cleaved SNAP-25 in the spinal cord has never been directly correlated with a resultant suppression of glutamate or neuropeptide release in the dorsal horn following injection of the toxin in the bladder wall. Moreover, the effect of BoNT/A on other neurotransmitters coreleased by bladder sensory fibers like VIP, cholecystokinin, and encephalin[33, 34] has not been investigated.

BoNT/A was shown to prevent transient receptor potential vanilloid-1 channel (TRPV1) trafficking to the membrane during bladder inflammation.[35] This process is expected to not only affect the noxious transmission in the bladder but also to prevent activation of micturition reflex during bladder filling. In accordance with these experimental findings, TRPV1 immunoreactivity in the urinary bladder of patients with DO was shown to decrease after BoNT/A administration.[6, 36] An identical decrease in immunoreactivity was observed for another pivotal receptor in bladder function, the purinergic receptor subtype P2X3.[6, 36] The decrease of this receptor combined with the decrease of urothelial ATP release in the bladder wall is expected to markedly suppress bladder sensory activity of small diameter bladder afferents.

BoNT/A injection in the bladder wall also decreases bladder sensory activity by preventing neurotrophin release in the bladder wall. A marked decrease of the concentration of nerve growth factor in the bladder wall or in the urine of patients with NDO and IDO was documented after BoNT/A injections.[37, 38] More recently, a decrease in urinary concentration of brain-derived nerve factor was also found after BoNT/A injection in the bladder wall.[39]

These mechanisms are expected to impair nociceptive input from the urinary bladder. In rats, BoNT/A administration to the bladder resulted in a decrease by half in the amount of spinal cord C-fos expression caused by bladder inflammation.[40] Detrusor activity induced by capsaicin administration to the mouse bladder was suppressed after BoNT/A injection.[28] In patients with BPS/IC, BoNT/A injection either in the whole bladder[41, 42] or restricted to the trigone, where most nociceptive fibers are located,[39, 43] produced a consistent analgesic effect (Fig. 5). Effectiveness of BoNT/A in reducing forms of somatic pain, including postherpetic pain,[44] underscores its potential analgesic effect.

image

Figure 5. Bladder pain intensity in 14 patients before and after (3 months) OnabotA injection (100 U) in the trigone. Pain was quantified by a visual analogue scale where 0 is no pain and 10 is the maximal tolerable pain. OnabotA consistently induced analgesia. The differences to baseline after each injection are statistically significant (P < 0.05). OnabotA, onabotulinum toxin A. Courtesy of Rui Pinto.

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In addition to the analgesic effect of BoNT/A in the bladder, one might expect the toxin to reduce the afferent noise arising from sensory fibers apposed to smooth muscle cells. These sensory fibers are in an ideal location to detect spontaneous detrusor activity which is enhanced in experimental animals after spinal cord injury (SCI)[28] or bladder obstruction,[29] and they can constitute the basis for localized detrusor activity that increases in patients with IDO.[45] Excess sensory input can, in theory, facilitate activation of the micturition reflex.

Sympathetic Nerve Fibers

The effects of BoNT/A administration on the sympathetic nervous system have not been well studied. Sympathetic nerve endings, which are predominant in the trigone and bladder neck, release norepinephrine, which by binding beta3- and alpha1A-adrenoreceptors, induces detrusor relaxation and bladder neck contraction, respectively.[46] In addition to norepinephrine, sympathetic nerve endings also contain somatostatin, neuropeptide Y, and ATP, whose functions are yet unclear in the context of sympathetic activity.[21, 47]

Following BoNT/A injections in the ventral, left, and right lateral rat bladder walls, the expected diffusion to the trigone and urethra does not affect the release of norepinephrine from bladder strips during electrical field stimulation, possibly due to the sparse sympathetic innervation of the bladder walls and dome. On the contrary, the release of norepinephrine from urethral strips during electrical field stimulation was found to decrease by 42% when measured 30 days after BoNT/A injection.[20] In accordance with these experimental study results, trigone injections of OnabotA in patients with BPS/IC markedly decreased the 24-hr urine content of norepinephrine.[48]

The way in which BoNT/A-induced impairment of norepinephrine release affects bladder function is unclear. One could speculate that a lack of norepinephrine would reduce detrusor relaxation and, in its maximum effect, facilitate detrusor contraction. However, in NDO patients, bladder injection of OnabotA above the trigone undoubtedly increased bladder capacity.[1] The trigonal injection of the toxin in BPS/IC patients, which is expected to impair a larger number of sympathetic fibers, also did not prevent the increment of maximal cystometric capacity.[39, 43]

Urothelium

SV2 and SNAP-25 immunoreactivity has not been detected in urothelial cells from human or guinea-pig bladders.[13, 15] Nevertheless, these results should be interpreted with some caution since other investigators, using reverse transcriptase polymerase chain reaction and Western blot techniques, revealed SNAP-25 expression in human and mouse urothelium.[49]

Although the mechanism of action of BoNT/A in urothelial function has not been elucidated, from what is understood about urinary bladder urothelial cells,[50] there is no doubt that urothelial release of ATP and nitric oxide (NO) is modified after BoNT/A administration. This effect has profound influences on bladder function. ATP released from the urothelium is thought to enhance bladder activity by activating suburothelial P2X3-expressing sensory nerve fibers, thereby increasing sensory transmission.[51] On the contrary, NO reduces the frequency of bladder contractions.[52, 53]

Urothelial ATP release is increased in both SCI and inflamed bladder preparations. Interestingly, however, ATP and NO have a basal and evoked form of release. While BoNT/A instillation in the bladder has been shown to inhibit ATP release in bladder urothelium in chronic SCI and chronic bladder inflammation models,[39, 54-56] this effect only occurs in evoked ATP and NO release, evoked by hypo-osmatic stimulation.[54-56] In addition to bladder preparations, BoNT/A has also been shown to inhibit stretch-evoked ATP from cultured urothelial cells.[57] These data seem to consistently indicate that only evoked release of ATP and NO is carried out by exocytotic, SNARE-dependent mechanisms.

SNAP-23 is a homologue of the neuronal SNAP-25. SNAP-23 regulates, in part, the exocytotic insertion or endocytotic retrieval of H+-ATPase-loaded vesicles in inner medullary collecting duct cells of the kidney.[58] Experiments carried out in cultured rat inner medullary-collecting duct cells document that BoNT/A (and the BoNT/E subtype) cleaves rat SNAP-23 and reduces immune-detectable and 35S-labeled SNAP-23 by up to 60%.[58] A strong immunoreaction for SNAP-23 has been observed in the human and rodent urothelium (Antonio Avelino, PhD, unpublished data; Fig. 6). It is therefore conceivable that SNAP-23 may have an important role in urothelial ATP release.

image

Figure 6. Immunolabeling for SNAP-23 in the mouse urothelium. Labeling appears throughout the urothelium although with more intensity in umbrella cells. Green: Rabbit anti-human SNAP-23 (synaptic Systems, 1:2,000, 88% homology with mouse). Blue: DAPI nuclear staining. SNAP-23, synaptosome-associated protein of 23 kDa. Magnification bar indicates 50 µm. Courtesy of Antonio Avelino.

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Myofibroblast Cells

Myofibroblast cells form a syncytium through extensive coupling via the gap-junction protein connexin 43 and have close contacts with sensory nerves.[59] These observations led to the hypothesis that myofibroblasts act as modulators of bladder behavior.[60, 61] However, there is no evidence that myofibroblasts express SV2 or SNAP-25. Moreover, BoNT/A does not alter the expression of connexin 43.[62] Hence, further studies evaluating the effects of BoNT/A on bladder myofibroblast cells are difficult to support at this moment.

OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

Repeated injections of OnabotA into the detrusor muscle have not been found to cause inflammatory infiltration or fibrotic activity in the bladder wall.[6, 63] One study demonstrated that NDO patients treated with BoNT/A exhibited less fibrosis than untreated patients.[63] However, the presence of eosinophilic infiltrate was shown to increase in specimens from patients receiving multiple treatments, a finding that could not be fully explained.[6] Injection of BoNT/A in the bladder does not cause apoptosis. Kessler et al.[64] performed terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reactions in human bladder samples after OnabotA administration, and the staining was consistently negative.

Recent investigations of OnabotA injection in the rat, dog, and human prostate produced a decrease in prostate volume.[65-67] This was associated with an increase in TUNEL staining in acinar and stromal cells[67-69] and in immunolabeling for pro-apoptotic proteins, like Caspase 3 and BAX (Fig. 7).[70] Prostate apoptosis was suggested to be dependent on sympathetic nerve impairment and consequent decrease of the adrenergic stimulation of the gland.[67] In addition to prostate volume changes, BoNT/A induced a dose-dependent decrease on the contractile function of the dog prostate,[71] further reinforcing the importance of sympathetic nerve signaling.

image

Figure 7. Increased immunostaining for the pro-apoptotic protein BAX in the rat prostate epithelium after OnabotA administration. Left panel: Control animal; Right panel: OnabotA-treated animal. OnabotA, onabotulinum toxin A. Magnification bar indicates 100 µm. Adapted from Gorgal et al.[70]

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These findings suggest that BoNT/A exerts an intraprostatic mechanism of action that does not occur in other tissues. Nevertheless, the human prostate is also unique in that it increases in size with age. These experimental data must be interpreted with caution when extrapolated to humans. Studies have reported a decrease in prostate volume within a few months after OnabotA injection,[72, 73] but another study failed to demonstrate a variation in the prostate volumes of OnabotA-injected patients.[74]

CONCLUDING REMARKS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

There is now robust information to support that the mechanism of action of BoNT/A in the bladder involves nerve fibers and the urothelium. Parasympathetic and sensory fibers seem to be particularly sensitive to BoNT/A. However, there are some points that still require intense research. The impairment of release of neurotransmitters other than ACh, neuropeptides, or glutamate has not been investigated yet. The mechanism through which BoNT/A interferes with urothelial activity remains unclear. An explanation for the large duration of the effect of BoNT/A in the bladder compared with in the skeletal muscle has not yet been advanced. The importance of the volume of the injection to the final effect of the toxin has yet to be investigated in clinical trials. Finally, considering that parasympathetic and sensory fibers have characteristic distributions in the human bladder, the consequences of individualizing the local of the injections may be valuable.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES

I would like to acknowledge Prof. Ana Charrua for her invaluable contribution toward drafting this manuscript and Prof. Antonio Avelino for the images kindly supplied. Additional editorial support was provided by Christina Sarabhai and Gregory Bezkorovainy of Adelphi Communications NY and was funded by Astellas.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DISTRIBUTION OF BINDING SITES AND TARGET PROTEIN
  5. PARASYMPATHETIC GANGLIA IMPAIRMENT AFTER BoNT/A INJECTION IN THE BLADDER
  6. DISTRIBUTION OF OnabotA IN THE BLADDER
  7. SITES OF ACTION OF BoNT/A IN THE BLADDER
  8. OTHER CHANGES IN THE LOWER URINARY TRACT AFTER BoNT/A ADMINISTRATION
  9. CONCLUDING REMARKS
  10. ACKNOWLEDGMENTS
  11. REFERENCES
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