INTRAVESICAL BOTULINUM TOXIN A ADMINISTRATION PRODUCES ANALGESIA AGAINST ACETIC ACID INDUCED BLADDER PAIN RESPONSES IN RATS

0022-5347/04/1724-1529/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 172, 1529 –1532, October 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000137844.77524.97

INTRAVESICAL BOTULINUM TOXIN A ADMINISTRATION PRODUCES ANALGESIA AGAINST ACETIC ACID INDUCED BLADDER PAIN RESPONSES IN RATS YAO-CHI CHUANG,* NAOKI YOSHIMURA, CHAO-CHENG HUANG, PO-HUI CHIANG , AND MICHAEL B. CHANCELLOR† ‡ From the Departments of Urology (Y-CC, P-HC) and Pathology (C-CH), Chang Gung Memorial Hospital, Kaohsiung, Taiwan, and Department of Urology, University of Pittsburgh School of Medicine (NY, MBC), Pittsburgh, Pennsylvania

ABSTRACT

Purpose: There is evidence that botulinum toxin A (BTX-A) might have analgesic properties. However, the mechanisms by which BTX-A alters pain remain largely unexplored. In the bladder afferent nerve fibers contain calcitonin gene-related peptide (CGRP). In this study we investigated the effect of intravesical BTX-A administration on CGRP immunoreactivity and bladder hyperactivity in an acetic acid induced bladder pain model in rats. Materials and Methods: Experimental and control animals were catheterized and intravesically exposed to protamine sulfate (1 ml, 10 mg/ml), followed by BTX-A (1 ml, 25 U/ml) or saline, respectively. Three or 7 days after intravesical therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemistry was used to detect CGRP. Results: The intercontraction interval (ICI) was decreased after acetic acid instillation (50.2% and 65.0%) in the control group at days 3 and 7, respectively. However, rats that received BTX-A showed a significantly decreased response (28.6% ICI decrease) to acetic acid instillation at day 7. This effect was not observed at day 3 (62.2% ICI decrease). Increased CGRP immunoreactivity was detected in the BTX treated group at day 7, which was not detected at day 3. Conclusions: Intravesical BTX administration blocked acetic acid induced bladder pain responses and inhibited CGRP release from afferent nerve terminals. These results support the clinical application of BTX-A for the treatment of interstitial cystitis and other types of visceral pain. KEY WORDS: bladder; pain; urodynamics; cystitis, interstitial; botulinum toxin type A

Interstitial cystitis is a clinical syndrome characterized by bladder pain, frequency and urgency in the absence of any identifiable pathological factors.1, 2 Although the etiology and pathogenesis of interstitial cystitis are still undetermined, there is currently increasing support for the hypothesis of neurogenic inflammation, which activates bladder afferent nerves and provokes bladder pain and a hypersensitive bladder.3, 4 Botulinum toxin type A (BTX-A) has been used effectively for years for different conditions with muscular hypercontractions.5, 6 The therapeutic value of BTX-A stems from its ability to inhibit acetylcholine release and correct focal dystonia when injected into a muscle.5, 6 During recent years there has been increasing evidence that BTX-A might also have analgesic properties but the mechanisms of pain relief are not completely understood.7–9 Calcitonin gene-related peptide (CGRP) is one of the capsaicin sensitive nerve fibers containing neuropeptide, which has a role in the regulation of micturition and mediation of painful sensation from the bladder.10 –12 In the current study we combined the intravesical instillation of protamine sulfate (PS), thought to break down urothelial barrier function,

and BTX-A in an acetic acid induced bladder pain model. We hypothesized that intravesical BTX-A administration could inhibit the release of CGRP and suppress bladder pain. Our current results show that BTX-A pretreatment can retain CGRP and decrease the bladder pain response.

MATERIALS AND METHODS

Animal preparation. All experimental procedures were performed in female Sprague-Dawley rats weighing 270 to 320 gm. Using halothane anesthesia PE-50 tubing (Clay-Adams, Parsippany, New Jersey) was inserted into the bladder through the urethra and tied in place by a ligature around the urethral orifice. The bladder was emptied of urine and slowly filled with 1 ml sterile saline solution containing 10 mg PS (Sigma Chemical Co., St. Louis, Missouri) through the catheter. One hour later the solutions were removed from the bladder and replaced with BTX-A (1 ml, 25 U/ml in saline) (Allergan, Irvine, California) or sterile saline for 1 hour. Cystometrogram (CMG). Three or 7 days after intravesical instillation of PS/BTX-A (6 rats per group) or PS/saline (6 rats per group) the animals were anesthetized by subcutaneous injection of urethane (1.2 gm/kg). PE-50 tubing was inserted into the bladder through the urethra and connected via a 3-way stopcock to a pressure transducer, and to a syringe pump for recording intravesical pressure and for infusing solutions into the bladder. Control CMG was performed by filling the bladder with saline (0.08 ml per minute) to elicit repetitive voiding. The amplitude, pressure threshold (PT), pressure baseline (PB) and intercontraction interval (ICI) of reflex bladder contractions were recorded after 2

Accepted for publication April 16, 2004. Study received institutional animal care and use committee approval. Supported by Fishbein Family CURE-IC HD39768, DK066138, DK06695 and DK068556, PVA Spinal Cord Research Foundation and National Science Council Taiwan NSC92-2314-B-182A-195. * Financial interest and/or other relationship with Allergan. † Correspondence: Suite 700, Kaufmann Building, 3471 Fifth Ave., Pittsburgh, Pennsylvania 15213 (telephone: 412-692-4096; FAX: 412-692-4101; e-mail: [email protected]). ‡ Financial interest and/or other relationship with OrthoMcNeil, Pfizer, Lilly, Watson, Cook and Yamanouchi. 1529

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BOTULINUM TOXIN A PRODUCES ANALGESIA

hours of infusion. Measurements in each animal represented the average of 3 to 5 bladder contractions. After a baseline measurement was established during saline infusion we infused acetic acid (0.3%) into the bladder at 0.08 ml per minute to promote acutely bladder hyperactivity. We measured 3 to 5 bladder contractions after 30 minutes of infusion. Transcardiac perfusion. At the end of CMG the animals were deeply anesthetized and sacrificed via transcardiac perfusion with Krebs buffer, followed by 4% paraformaldehyde fixative. The animals were then dissected to harvest the bladder. Histology and immunohistochemistry. Bladder tissues for histology were fixed in 4% paraformaldehyde in phosphate buffered saline for 4 hours and then in 30% sucrose in phosphate buffered saline overnight. Samples for histology were embedded in paraffin, cut in 10 ␮m pieces and stained with hematoxylin and eosin. Alternatively samples were cryosectioned to 10 ␮m and blocked in 5% normal bovine serum (Santa Cruz Biotechnology, Santa Cruz, California) and stored in a moist chamber overnight at 4C. Slides were then stained for sensory nerve endings using CGRP (goat polyclonal IgG, N-20, Santa Cruz Biotechnology) at 1:100 dilution in 5% normal bovine serum and 0.2% Triton-X 100 octylphenoxypolyethoxyethanol (Sigma Chemical Co.), and stored in a moist chamber for 20 hours at 4C. The following day bovine antigoat IgG-fluorescein isothiocyanate and containing DAPI (4,6-diamidino-2-phenylindol) was added at 1:1,250 dilution in 5% normal bovine serum and 0.2% Triton-X 100 octylphenoxypolyethoxyethanol for 1 hour at room temperature. Slides were read by 2 independent observers using fluorescence/light microscopy. Sections were stained with a fluorescent solution and cytoplasma was counterstained with hematoxylin. A set of section was stained in a similar way without CGRP antibody (primary antibody) to confirm specific binding of the antibody to the protein. Statistical analysis. Quantitative data are expressed as the mean ⫾ SD. Statistical analyses were performed using Student’s t test for paired or unpaired data as applicable with p ⬍0.05 considered significant. RESULTS

Bladder activity in the control group (PS/saline) and after PS/BTX-A treatment at day 3. CMG parameters in the control group during intravesical saline and acetic acid instillation were not significantly different from those in the PS/BTX-A pretreated group (table 1, fig. 1, A and B). These results indicate that bladder function under normal or hyperactive condition was not affected by BTX-A treatment at day 3. Bladder activity in the control group (PS/saline) and after PS/BTX-A treatment at day 7. CMG parameters in the control group during intravesical saline instillation were not

significantly different from those in the PS/BTX-A pretreated group (table 2, fig. 1, C and D). These results indicate that normal bladder function was not affected by BTX-A pretreatment at day 7. The irritative effect of acetic acid was evident 20 to 30 minutes after the start of infusion. ICI was significantly decreased by 65.0% (20.6 ⫾ 2.1 to 7.2 ⫾ 1.5 minutes) in the control group. However, the decrease in ICI was significantly smaller in the BTX-A pretreated group (ICI decrease 28.6%, 26.9 ⫾ 2.4 to 18.2 ⫾ 3.1 minutes, fig. 2). These results indicate that the therapeutic effect of BTX-A on bladder hyperactivity could be observed at day 7. Histology and immunohistochemistry. The control and PS/ BTX-A treated groups at day 3 had mucosal sloughing, as determined by the histopathological evaluation of tissue section stained with hematoxylin and eosin (fig. 2, A and C). However, the mucosa recovered in the control and BTX-A treated groups at day 7 (fig. 2, B and D). These results indicate that PS caused urothelial sloughing that was repaired at day 7. When comparing the control and BTX-A treated groups, the former demonstrated a more severe edematous change in the bladder mucosa associated with a moderate amount of inflammatory cells, while the latter showed a mild edematous change associated with few inflammatory cells. These results suggest that BTX-A pretreatment might have anti-inflammatory effects. CGRP immunostaining was confirmed at the bladder mucosal layer in the BTX-A treated group at day 7, which was not observed at day 3 or in the control groups (fig. 3). These results indicate that BTX-A treatment inhibited CGRP release at day 7. DISCUSSION

The major findings of the current study are that intravesical instillation of PS/BTX-A suppresses acetic acid induces bladder hyperactivity, inhibits CGRP release and decreases the bladder inflammatory reaction. Although the mechanisms of visceral pain, including that of bladder pain, are poorly understood, the activation of C and A-J afferent fibers from the peripheral organs has been suggested.11, 12 Previous experiments infusing acetic acid into the bladder revealed the stimulation of nociceptive afferent fibers, release of inflammatory neuropeptides and evocation of a hyperactive bladder.13, 14 The effect of intravesical acetic acid was decreased but not completely eliminated by capsaicin pretreatment. These results prove that sensitization of A-J and C afferent fibers has a role in the bladder pain model induced by acetic acid.13 BTX-A has been effectively used for years for different conditions with somatic and autonomic motor disorders.5, 6, 15

TABLE 1. Effects of saline and 0.3% acetic acid on CMG parameters on day 3 in rats pretreated with PS/saline as control or PS/BTX-A Mean ICI ⫾ SE (mins)

Mean PB ⫾ SE (cm H2O)

Saline control (6 rats) 22.1 ⫾ 1.8 6.2 ⫾ 1.2 Acetic acid 11.3 ⫾ 1.8* 5.2 ⫾ 1.0 Saline ⫹ BTX-A (6 rats) 26.2 ⫾ 0.9 6.7 ⫾ 0.6 Acetic acid 9.9 ⫾ 1.2* 6.9 ⫾ 0.6 No differences between saline and BTX-A groups after acetic acid instillation. * p ⬍0.05.

Mean PT ⫾ SE (cm H2O)

Mean Amplitude ⫾ SE (cm H2O)

10.0 ⫾ 1.5 6.5 ⫾ 0.9* 10.4 ⫾ 0.7 8.2 ⫾ 0.6*

32.9 ⫾ 3.4 36.7 ⫾ 3.9 29.7 ⫾ 2.0 34.4 ⫾ 2.0

TABLE 2. Effects of saline and 0.3% acetic acid on CMG parameters on day 7 in rats pretreated with PS/saline as control or PS/BTX-A

Saline control (6 rats) Acetic acid Saline ⫹ BTX-A (6 rats) Acetic acid * p ⬍0.05.

Mean ICI ⫾ SE (mins)

Mean PB ⫾ SE (cm H2O)

Mean PT ⫾ SE (cm H2O)

Mean Amplitude ⫾ SE (cm H2O)

20.6 ⫾ 2.1 7.2 ⫾ 1.5 26.9 ⫾ 2.4 18.2 ⫾ 3.1*

5.7 ⫾ 0.9 4.2 ⫾ 1.1 6.3 ⫾ 0.3 6.9 ⫾ 0.5

9.2 ⫾ 1.2 5.4 ⫾ 1.1 9.9 ⫾ 0.6 8.2 ⫾ 0.3

31.5 ⫾ 3.5 31.3 ⫾ 2.5 30.0 ⫾ 1.9 33.9 ⫾ 2.8

BOTULINUM TOXIN A PRODUCES ANALGESIA

FIG. 1. Representative traces of in vivo continuous CMGs in urethane anesthetized rats. CMG was performed in control rat at days 3 (A) and 7 (C) after PS/saline instillation, and in PS/BTX-A treated rat at days 3 (B) and 7 (D). After acetic acid (AA.) infusion control rat and BTX-A pretreated rat showed decreased ICI but decrease was significantly smaller (antinociceptive effect) in BTX-A pretreated rat at day 7.

Apart from its therapeutic effects on muscular hypercontraction there has been increasing evidence that BTX-A might also have analgesic properties in animals and clinically in humans. Welch et al observed that the release of neuropep-

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tide from rat dorsal root ganglion was inhibited by various botulinum toxins (BTX-A, B, C1 and F),7 while Cui et al noted that BTX-A pretreatment significantly decreased formalin induced pain in a rat model.16 Jabbari et al reported that segmental burning pain was relieved by multiple subcutaneous injection of BTX-A.17 In another pilot study Freund and Schwata successfully treated patients with posttherapeutic neuralgia with multiple subcutaneous injection of BTX-A.18 They hypothesized that there may be a direct effect of BTX-A on noncholinergic neurons, resulting in decreased release of substance P, CGRP, glutamate and other substances.9 The molecular weight of BTX-A is 150 kD, which is hard to get through the intact urothelium. Previous experiments showed that exposing the bladder to protamine sulfate was necessary to increase epithelial permeability and induce an inflammatory effect or activate bladder afferents by lipopolysaccharide or potassium chloride, respectively.3, 19 The method used in the current study revealed urothelial sloughing at day 3, which resolved at day 7. Thus, PS, which temporarily increases epithelial permeability, was efficient for increasing the penetration of BTX-A without permanent damage. Many C-fibers that can be seen in the mucosa of the bladder contain neuropeptides, such as substance P, neurokinin A and CGRP, which upon release could modulate the micturition reflex and perhaps cause bladder hyperactivity.20 Furthermore, these neuropeptides have direct vasoactive effects with subsequent leukocyte adhesion and tissue edema.20 CGRP-like immunoreactivity has been demonstrated in sensory ganglion cells and capsaicin sensitive visceral nerve endings.12 The current study reveals that pretreatment with BTX-A inhibited CGRP release and decreased acetic acid induced hyperactive bladder at day 7 but not at day 3. This result implicates a delayed onset of BTX-A effects on bladder afferents. Although the mechanisms by which BTX-A inhibits CGRP release are not clear, the effects might relate to decrease edematous change and inflammatory reaction to acetic acid instillation. The appearance of CGRP staining 7 days after BTX-A treatment is consistent with the inhibition of CGRP release but it was not directly proved in the current study. There are a number of alternative processes that could lead to these

FIG. 2. Photomicrographs of bladder sections after PS/saline treatment at days 3 (A) and 7 (B), and PS/BTX-A treatment at days 3 (C) and day 7 (D). Sloughing of urothelial cells (arrows) in response to intravesical PS (10 mg/ml) was detected at day 3 (A and C), which resolved at day 7 (B and D). Edematous change and polymorphonuclear neutrophil leukocyte accumulation (triangles) were detected in control group (A and B), which were less significant in BTX-A pretreated group (C and D). Reduced from ⫻100.

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BOTULINUM TOXIN A PRODUCES ANALGESIA CONCLUSIONS

Intravesical BTX-A administration blocked acetic acid induced bladder pain responses and inhibited CGRP release from afferent nerve terminals. These results support the clinical application of BTX-A for the treatment of interstitial cystitis and other types of visceral pain.21 REFERENCES

FIG. 3. CGRP staining (arrow) in representative section of experimental rat bladder 7 days after PS/saline instillation (A), and 3 (B) and 7 (C) days after PS/BTX-A instillation. CGRP was detected at mucosal layer of bladder 7 days after PS/BTX-A instillation, which was not detected in other groups.

results, such as increased CGRP synthesis and decreased CGRP breakdown. Additional experiments for future reports using radioimmunoassay to quantify the tissue concentration of CGRP are underway.10 In our current study BTX-A pretreatment did not change bladder contraction amplitude. We assume that a weakened urothelium enhances the attachment of BTX-A in the urothelium but not deep enough for the muscular layer. This result proves that intravesical PS/BTX-A might be a safe and effective method for the treatment of bladder pain syndrome without compromise bladder contractility.

1. Parsons, C. L., Lilly, J. D. and Stein, P.: Epithelial dysfunction in nonbacterial cystitis (interstitial cystitis). J Urol, 145: 732, 1991 2. Parsons, C. L., Housley, T., Schmidt, J. D. and Lebow, D.: Treatment of interstitial cystitis with intravesical heparin. Br J Urol, 73: 504, 1994 3. Chuang, Y.-C., Fraser, M. O., Yu, Y., Chancellor, M. B., de Groat, W. C. and Yoshimura, N.: The role of bladder afferent pathways in bladder hyperactivity induced by the intravesical administration of nerve growth factor. J Urol, 165: 975, 2001 4. Hohenfellner, M., Nunes, L., Schmidt, R. A., Lampel, A., Thuroff, J. W. and Tanagho, E.: Interstitial cystitis: increased sympathetic innervation and related neuropeptide synthesis. J Urol, 147: 587, 1992 5. Grazko, M. A., Polo, K. B. and Jabbari, B.: Botulinum toxin A for spasticity, muscle spasms, and rigidity. Neurology, 45: 712, 1995 6. Jankovic, J., Schwartz, K. and Donovan, D. T.: Botulinum toxin treatment of cranial-cervical dystonia, spasmodic dysphonia, other focal dystonias and hemifacial spasm. J Neurol Neurosurg Psychiatry, 53: 633, 1990 7. Welch, M. J., Purkiss, J. R. and Foster, K. A.: Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins. Toxicon, 38: 245, 2000 8. Purkiss, J. R., Welch, M. J., Doward, S., Foster, K. A. and Quinn, C. P.: A method for the measurement of [3H]-glutamate release from cultured dorsal root ganglion neurons. Biochem Soc Trans, 26: S108, 1998 9. Argoff, C. E.: A focused review on the use of botulinum toxins for neuropathic pain. Clinic J Pain, 18: S177, 2002 10. Su, H. C., Wharton, J., Polak, J. M., Mulderry, P. K., Ghatei, M. A., Gibson, S. J. et al: Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry. Neuroscience, 18: 727, 1986 11. Morrison, J.: The activation of bladder wall afferent nerves. Exp Physiol, 84: 131, 1999 12. Gabella, G. and Davis, C.: Distribution of afferent axons in the bladder of rats. J Neurocytol, 27: 141, 1998 13. Fraser, M. O., Chuang, Y. C., Tyagi, P., Yokoyama, T., Yoshimura, N., Huang, L. et al: Intravesical liposome administration—a novel treatment for hyperactive bladder in the rat. Urology, 61: 656, 2003 14. Birder, L. A. and deGroat, W. C.: Increased c-fos expression in spinal neurons after irritation of the lower urinary tract in the rat. J Neurosci, 12: 4878, 1992 15. Chuang, Y. C., Smith, C. P., Somogyi, G. T. and Chancellor, M. B.: Botulinum toxin treatment of urethral and bladder dysfunction. J Formos Med Assoc, 102: 5, 2003 16. Cui, M., Khanijou, S., Rubino, J. and Aoki, K. R.: Subcutaneous administration of botulinum toxin A reduces formalin-induced pain. Pain, 107: 125, 2004 17. Jabbari, B., Maher, N. and Difazio, M. P.: Botulinum toxin A improved burning pain and allodynia in two patients with spinal cord pathology. Pain Med, 4: 206, 2003 18. Freund, B. and Schwata, M.: Subcutaneous BTX-A in the treatment of neurogenic pain: a pilot study. Presented at 38th Integragency Botulinum Research Coordinating Committee meeting, Easton, Maryland, October 17–19, 2001 19. Stein, P. C., Pham, H., Ito, T. and Parsons, C. L.: Bladder injury model induced in rats by exposure to protamine sulfate followed by bacterial endotoxin. J Urol, 155: 1133, 1996 20. Doggweiler, R., Jasmin, L. and Schmidt, R. A.: Neurogenically mediated cystitis in rats: an animal model. J Urol, 160: 1551, 1998 21. Smith, C. P. and Chancellor, M. B.: Emerging role of botulinum toxin in the management of voiding dysfunction. J Urol, 171: 2128, 2004