Intravesical electromotive administration of botulinum toxin type A in improving the bladder and bowel functions: Evidence for novel mechanism of action

Abstract

Objective: To examine the hypothesis that what is the concomitant mechanism of action botulinum toxin type A (BoNTA) administration by intravesical electromotive into the bladder resulting in bladder function improvement. We also tried to confirm the possibility of retrograde trans-axonal transportation of toxin.

Design: Animal study.

Setting: Ten male rabbits were divided into two groups.

Participants: Group 1 (G1) (n = 5) (BoNTA/EMDA), and group 2 (G2) (n = 5) the control group.

Interventions: Animals in G1received 10 IU/Kg of intravesical BoNTA through a specific catheter for electromotive drug administration (BoNTA/EMDA). About 0.1–0.15 ml of toxin was diluted in 1 ml of distilled water. The maximum frequency of the device for drug solution delivery was set at 4 mA for 15 min. In G2 as the control group, the same procedure was performed to deliver normal saline to the bladder.

Outcome measures: Multiple biopsies were taken from bladder’s contiguous structures one month postoperatively. The immunohistochemical (IHC) evaluation was performed with anti-clostridium botulinum toxoid type A mouse IgM monoclonal antibody.

Results: In specimens of G1, BoNTA penetrated through muscular layers of the bladder wall and the staining was uniform in the urothelium, interstitium, and muscular layers. Positive IHC staining showed that BoNTA was traced in the upper and lower spinal cord in addition to pelvic nerve, sacral nerve plexus, intestine wall, and pelvic floor muscle. In G2, all the specimens were intact in IHC staining.

Conclusions: The presence of BoNTA in lower and upper spinal cord suggests the possibility of retrograde trans-axonal transfer of toxin to lower and upper neural pathways which may result in simultaneous improvement in bladder and bowel functions.

Introduction

Botulinum toxin (BTX) is the strongest natural lethal protein and neurotoxin, which is produced by an anaerobic, rod-shaped bacterium called Clostridium botulinum. Botox, as one of its trade names, is used for numerous cosmetic and medical procedures. Among several subtypes of BTX, only types A and B have shown promising widespread results in many neuromuscular disorders. BTX binds rapidly and tightly to the intramuscular nerve terminals and causes a prolonged local effect. Each serotype of the BTX affects one or more proteins, which are involved in the process of acetylcholine release in the synaptic gap. Botulinum toxin type A (BoNTA) is internalized in synaptic vesicles, which are recycled in the terminal axons. BoNTA is currently used to treat lower urinary tract dysfunction, especially in patients with neuropathic overactive bladder (OAB).^1–3

The main goals in managing patients with lower urinary tract dysfunction are to avoid renal failure, increase bladder capacity, and obtain a social urinary continence through a minimally invasive approach.⁴ Numerous studies have successfully demonstrated the effectiveness of intradetrusor BoNTA injection for the treatment of detrusor overactivity (DO) refractory to conservative therapy in adults and children.^1,5–7 A growing unanimity has also confirmed that Botox injection is a well-tolerated and low-risk therapy.⁸ There are also some evidences that BoNTA modulates sensory nerve fibers and afferent signaling mechanisms that are involved in DO, and inhibits nerve-mediated bladder contraction by blocking neurotransmitter release from peripheral afferent nerve endings.^5,9

By the application of electromotive drug administration (EMDA) as an electrical field, any water-soluble drug can be transported into the local tissues in an accelerated manner. Additionally, the efficacy of intravesical EMDA has been confirmed by several previous studies.^10,11 EMDA may be of great value in the treatment of various pathological conditions such as neurogenic or non-neurogenic voiding dysfunctions.

In the previous reports, BoNTA was applied in children with myelomeningocele and neurogenic DO via both intra-detrusor injection¹² and a method of intravesical EMDA,¹¹ the results of which demonstrated a significant improvement in co-existing bowel dysfunction in children. The exact mechanism for this observation is still unclear, but the effect of toxin on common neural pathways for urinary and bowel function, and the diffusion of toxin to bladder’s contiguous structures are existing hypotheses. Our assumption was that this observation could be due to the effect of the toxin on common neural pathways between bladder and the large intestine, or the dissemination of toxin to the contiguous structures including anal sphincter or pelvic diaphragm.

The aim of the current experimental study was to confirm the presence of intravesically administered BoNTA in either bladder’s contiguous structures or upper and lower neural pathways which can culminate in the improvement of bladder and bowel functions.

Materials and methods

Animal preparation

This study was performed at the laboratory of experimental surgery of Pediatric Urology Research Center, Tehran, Iran. The current experiment was carried out following a Bioethics Committee approval and it complied with national and international rules for the protection and care of animals. Precepts of laboratory animal care were administered for treating animals (NIH publication no. 85-23, revised 1985). The animals had access to standard diet and water until the beginning of this experimental study. Animal selection, managements, and surgery protocols were conducted under guidelines approved by the local ethical committee of Tehran University of Medical Sciences. Ten male New Zealand white rabbits at 12–18 months with a body weight of 2–3 kg were selected and divided into two groups (five rabbits in each group). Groups 1 (G1) underwent electromotive intravesical administration of BoNTA (BoNTA/EMDA) while group 2 (G2) received normal saline via EMDA. Rabbits received no food and water around 10–12 h before the operation. Prior to the surgery, the animals were anesthetized by an intramuscular injection of Ketamine (100 mg/ml) (25 mg/kg), Xylazine (20 mg/ml) (5 mg/kg), and Acepromazine (10 mg/ml) (2.5 mg/kg). Animals were positioned in dorsal recumbency with the head held at heart level on the operating table, prepped and draped in the standard sterile manner.

BoNTA/EMDA and electromotive saline administration

Animals in G1 underwent the electromotive intravesical administration of BoNTA (Dysport^®, Ipsen Ltd., UK). In G1, catheterization with an 8 Fr Foley catheter was performed after applying adequate lubricant gel. This Foley catheter contained a thick pure silver wire of 2-mm thickness with corkscrewed distal end as conducting electrode and several tiny perimetrical holes at its tip in order to induce maximum drug distribution and maintain the electrical circuit. Subsequently, the bladder was washed alternately with normal saline until the catheter output became clear. Afterward, the bladder was filled with distilled water containing 10IU/kg BoNTA to its maximal capacity (0.1–0.15 ml of toxin was diluted in 1 ml of distilled water). Two dispersive pads (2 cm × 2 cm) which were covered with contact gel were positioned bilaterally parallel to the midline at the level of distended bladder. Then, the electrodes of the catheter and dispersive pads were connected to a pulsed current generator (Stimulator 733X, NOVIN Ltd., Iran). The device was programed to deliver a maximum of 2–2.4 mA with a frequency of 2.5 kHz for a total duration of 15 min. In the electromotive saline group (G2), animals underwent the same procedure as G1 but the electrical current with similar characteristics was delivered to the saline-filled bladder.

Analysis of the images was carried out using Photoshop 10.0 software (Adobe Systems, Inc., Mountain View, CA, USA), and Image Pro (Image Pro Inc., Boston, MA, USA). Positively stained elements were used for quantification. The mean score obtained from five randomly selected photomicrographs (×100) were used for scoring.

Sampling

One month postoperatively, a midline incision was made from the caudal end of the pubic bone to the mid-abdominal area in each animal of both groups under sterile condition. Then, several samples were taken from intestinal wall laid behind the posterolateral wall of the bladder as well as pelvic nerve, sacral nerve plexus (L4-S5), pelvic floor muscles, lower spinal cord (sacral), and the upper spinal cord (cervical) on both sides. After the surgery, the animals were sacrificed by exsanguinations.

Immunohistochemical (IHC) analysis

Fresh specimens were washed several times with cool phosphate buffered saline (PBS) and embedded in Tissue- Tek OCT compound (Sakura Fine technical, Tokyo, Japan). Then, the tissues were rapidly frozen at −30° C and 3–4 µm thick cryosections were mounted on poly-L lysine (Sigma-Aldrich, USA) coated slides.

Mouse IgM monoclonal to Clostridium botulinum A Toxoid Primary antibody (4:100 dilution, US Biological, Swampscott, MA, USA) was applied for 60 min at room temperature. Antigen retrieval was not performed. After washing the slides in PBS, they were incubated with Dako EnVision⁺ system and diaminobenzidine as chromogen (Dako, Copenhagen, Denmark). Then, the sections were counterstained with hematoxylin and evaluated at a magnification of ×100 under light microscopy. A positive reaction was noted upon the observation of an obvious brown staining compared to the control sections, and 3–5 sections were analyzed per animal.

Results

There were no significant complications such as weight loss, or hind/limb weakness in none of the animals postoperatively. All the animals underwent sonography after the operation in order to evaluate the urinary residue. Four rabbits developed urinary retention postoperatively and the bladder was emptied twice daily for 2 weeks using manual expression.

In G1, the toxin was detected in sacral nerve plexus specimens from both sides (Fig. 1A). There was a trace of the toxin in the intestinal wall laid behind the posterolateral wall of the bladder. The scattered area of the intestine was completely enhanced with Botox staining which confirmed the presence of Botox in the intestine wall (Fig. 1B). Botox was also traced in the upper and lower spinal cord in addition to the pelvic nerve (Fig. 1C–E). IHC analysis also revealed positive BoNTA staining in the pelvic floor muscle samples (Fig. 1F). In electromotive saline administration groups (G2) intestinal wall which was contiguous with the posterolateral wall of the bladder, upper and lower spinal cord, pelvic nerve, pelvic floor muscle, and sacral nerve plexus were not stained (Fig. 1G–L). The results of the negative control samples are also demonstrated in Fig. 2 and Table 1.

Figure 1.

IHC analysis in G1 revealed strong homogeneous BoNTA staining in (A) sacral nerve plexus (B) intestinal wall laid behind the posterolateral wall of the bladder, (C) upper spinal cord, (D) lower spinal cord, (E) pelvic nerve, and (F) pelvic floor muscle. H&E analysis in G2 in (G) sacral nerve plexus (H) intestinal wall laid behind the posterolateral wall of the bladder, (I) upper spinal cord, (J) lower spinal cord, (K) pelvic nerve, and (L) pelvic floor muscle.

Figure 2.

H&E analysis in the negative control group in (A) sacral nerve plexus (B) intestinal wall laid behind the posterolateral wall of the bladder, (C) upper spinal cord, (D) lower spinal cord, (E) pelvic nerve, and (F) pelvic floor muscle.

Table 1. The results of IHC analysis in the negative control and BONTA samples.

Marker	Sacral nerve plexus	Intestinal wall	Upper spinal cord	Lower spinal cord	Pelvic nerve	Pelvic floor muscle
BONTA	98.00	113.25	82.75	97.00	94.25	82.75
Negative control	41.25	32.75	37.00	45.25	51.75	48.00
P value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

Data are means (% of normal tissues). P < 0.05 is considered statistically significant. A significant difference was obtained in all tissues in experimental group regarding Botox spread, in comparison with negative controls after surgery.

Discussion

In the current experimental study, we demonstrated that BoNTA/EMDA has the potential to distribute in either bladder’s contiguous structures or lower and upper neural pathways like sacral nerve plexus. Additionally, we demonstrated that the mechanisms of retrograde axonal transport of BoNTA may play a crucial role in the treatment of patients with bladder and bowel dysfunctions.

BoNTA has recently become one of the most effective treatments in pediatric urology especially in children with non-neurogenic or neurogenic voiding dysfunction.¹³ This toxin has long been thought to induce temporary detrusor muscle paralysis through cleavage of the SNAP-25 and therefore, inhibiting the presynaptic vesicular release of acetylcholine.¹⁴ However, some studies have provided evidences that BoNTA has additional inhibitory effects on neuropeptides, neurotransmitters, and receptors mediating sensory neurotransmission.^{15, 16}

In EMDA, drug transportation is accelerated by multiple electrokinetic forces.¹⁷ In optimal intravesical BoNTA delivery, EMDA, sufficient delivery time, adequate drug concentration with non-harmful intensity are of great value which should be taken into consideration.¹⁸ In the current study, functional density of 0.12 mA/cm² and drug concentration of 1.2 IU/cm² were applied which has been recommended to prevent tissue damage and burning sensation.¹⁹ It has also been demonstrated that drug can be penetrated efficiently within the first 15 min of EMDA.¹⁸ Although it has been reported that the bladder dome and the anterior wall are less susceptible to drug penetration in EMDA as they are far from electrical field,¹⁸ pathological specimens from BoNTA/EMDA group demonstrated that BoNTA has distributed to former areas in an identical pattern. In the current experimental study, a growing body of evidence has supported that the establishment of BoNTA/EMDA may have promising advantages for patients with bladder and bowel dysfunctions. By the application of this method, homogeneous distribution of BoNTA can be feasible in bladder and bowel segments.

The correlation between anti-Dysport antibody titer and repeated Dysport injections was previously evaluated.²⁰ The results demonstrated that increment of antibody titer is impermanent and the immune system may not be boosted by repeated injections, which has also been demonstrated in the IHC staining of the present study. In the study of Gurpinar et al., noticeable submucosal and muscularis methylene blue penetration was confirmed by the application of EMDA in the dog urinary bladder.¹⁸ As in our previous study where we evaluated the penetration of BoNTA in bladder structures via EMDA, we decided to investigate the possibility of more systemically absorption of BoNTA in bladder and bowel structures as well as lower and upper spinal cord, sacral nerve, and pelvic floor muscle. McKenna et al., suggested that abnormal contraction of pelvic musculature stimulates changes in urinary and bowel innervations resulting in concomitant urinary and bowel dysfunctions in children without any obvious neurological defects, a concept termed neuroplasticity.²¹

In one study,²² the effects of sphincteric botulinum toxin A injection has been evaluated in patients with external sphincter dyssynergia. The results demonstrated the efficacy of this method in the treatment of refractory nonneurogenic voiding dysfunction in these selected children. According to the study of Hollingshead et al., functional anal pain as a result of sphincter muscles spams can be also treated by injecting of BoNTA into the internal anal sphincter.²³ The result of another study demonstrated the efficacy and safety of intravesical BoNTA injection for treatment of cystitis/bladder pain syndrome (IC/BPS).²⁴ The retrograde transport of BoNT/A to the CNS after bladder injection has been also verified in a rat model by using the gamma-emitting radionuclide technetium-99m (^99mTc).²⁵ The results of the current study regarding the positive BoNTA staining in the pelvic floor muscle samples are in accordance with another study in which it has been confirmed that the injection of BoNTA can be effective in the treatment of refractory chronic pelvic pain related to pelvic floor muscle spasm.²⁶

The research is interesting from the clinical point of view as EMDA approach is characterized as easier to perform, safer, less painful and with no need for anesthesia in comparison to, currently used, intravesical BoNTA injection. The interesting point of our study was the presence of toxin in the sacral plexus on both sides. Despite the widespread application of BoNTA in several medical fields, little is known about its potential central effects. Electrophysiological studies in human and animal models have demonstrated the central effects of BoNTA on spinal cord circuitry, brainstem, and motor cortex following the intramuscular injection of the toxin in neurological pathologies.²⁷ One of the main proposed mechanisms to justify these observations is the retrograde transport and transcytosis of BoNTA following uptake at the neuromuscular junction. Habermann initially reported retrograde intra-axonal transfer of radio-labeled BoNTA to ventral roots and adjacent spinal cord segments upon intramuscular injection in the cat.²⁸ It has been recently confirmed that intramuscular injection of BoNTA can induce long-distance effects. However, it mostly exerts its effect at the injection site and the neuromuscular junction. Although most of its effects are restricted to the injection site, there are evidences of toxin activity in distant synapses.²⁹ Regarding our findings in the present study, it can be considered that the retrograde trans-axonal transfer of intravesical BoNTA to the sacral parasympathetic plexus, upper and lower spinal cord may be possible. In addition, the possibility of utilizing BoNTA/EMDA in the treatment of patients with constipation should be kept in mind. It seems that synaptic vesicles and fibroblast growth factor receptor 3 (FGF3) play a crucial role in undergoing axonal transport in neurons.²⁷ In our previous study,³⁰ intravesical BoNTA/EMDA demonstrated deep and homogenous penetration of the Dysport throughout the urinary bladder layers but the effect of toxin on common neural pathways, possibility of retrograde intra-axonal transport of toxin, and further probable improvement of bladder and bowel functions were not discussed in the literature.

Developing the modality of BoNTA/EMDA with longer duration of effect, lesser invasive intervention, deeper penetration of toxin to the layers of the bladder wall, without the need for general anesthesia and hospitalization, compared to cystoscopic guidance of BoNTA are among the benefits of the current study. However, in patients with upper motor lesions who have spastic neurogenic bladder and hypertonicity of anal sphincter, electromotive intravesical administration of BoNTA may cause relief in both regions. Although it has been assumed that the effect of BoNTA are confined to the injection region of the toxin, we showed that intravesical administrated BoNTA is retrogradely transported to the upper and lower neural pathways by central neurons. In the present study, we found that BoNTA appears at the site of injection as well as distant regions.

The current study also has some potential limitations which should be underlined. As the electrophysiological responses in sacral parasympathetic plexus were not measured following the toxin administration, it was not possible to determine whether the toxin provoked any significant changes in the neuronal responses in dorsal band, lateral band, and the inter band region. Although there was no trace of toxin in samples taken from bladder’s adjacent structures, we could not rule out the possibility of toxin dissemination to sacral nerves via regional or systemic blood circulation. Another limitation of the current study was the lack of experiments with concomitant use of an axonal transporter blocker for better confirmation of mechanisms of retrograde axonal transport of BoNTA. A high injection volume might increase the risk of systemic effects due to unintended spread of BoNT/A. In the current study, functional density of 0.12 mA/cm² and drug concentration of 1.2 IU/cm² were applied which has been recommended to prevent tissue damage and burning sensation. Although there are evidences of toxin activity in distant synapses in previous studies, we did not detect any deleterious effects in long-term follow ups. However, we should take into consideration the potential deleterious effects of intravesical EMDA to alter colonic and recto-anal function when such changes are not clinically desirable. In addition, more electrophysiological studies are required to evaluate the neural responses of the sacral nerves especially the sacral parasympathetic nucleus following the intravesical BoNTA/EMDA. We will also have the addition of a detrusor-injection group which would strengthen the assertion that EMDA is a better approach in future functional study.

Conclusion

In summary, this experimental study showed that BoNTA can be detected in bladder and bowel structures as well as upper and lower spinal cord following electromotive intravesical administration of BoNTA. It also seems that the toxin reaches the sacral plexus via trans-axonal retrograde transfer mechanism. This mechanism of action can justify our previous reports regarding the beneficiary effects of intravesical BoNTA on both urinary and bowel dysfunctions in children with neural tube defects. As a complementary research, an electrophysiological study on neural responses of sacral nervous system following the intravesical BoNTA administration in an experimental model of neural tube defect might be of great value in drawing a clear scheme in this regard.

Disclaimer statements

Contributors None.

Funding None of the authors has direct or indirect commercial financial incentive associating with publishing the article.

Conflicts of interest The authors report no conflicts of interest.