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Intravesical Botulinum Toxin A Injections Plus Hydrodistension Can Reduce Nerve Growth Factor Production and Control Bladder Pain in Interstitial Cystitis
Adult Urology Intravesical Botulinum Toxin A Injections Plus Hydrodistension Can Reduce Nerve Growth Factor Production and Control Bladder Pain in Interstitial Cystitis Hsin-Tzu Liu and Hann-Chorng Kuo OBJECTIVES METHODS
RESULTS
CONCLUSIONS
To investigate the level of nerve growth factor (NGF) mRNA in bladder tissue and the effect of botulinum toxin A (BTX-A) treatment in patients with interstitial cystitis (IC). A total of 19 patients with IC were treated with 100 U or 200 U of intravesical BTX-A injections followed by cystoscopic hydrodistension 2 weeks later. Bladder mucosa biopsies were performed before BTX-A injection and immediately after hydrodilation and in 12 controls. The NGF mRNA and protein levels in bladder tissues were assessed by real-time polymerase chain reaction and immunohistochemistry studies to determine differences in NGF expression between patients with IC before and after BTX-A treatment and compare with controls. At 3 months, 14 patients had symptomatic improvement (responders) and 5 did not (nonresponders). The NGF mRNA levels at baseline in the overall IC patient group were significantly greater than those in the controls (0.65 ⫾ 0.33 versus 0.42 ⫾ 0.25, P ⫽ 0.046). At 2 weeks after BTX-A treatment, the NGF mRNA levels had decreased to 0.47 ⫾ 0.23 (P ⫽ 0.002, compared with baseline) and were no longer significantly different from those of the controls. The NGF mRNA levels decreased significantly in responders and were significantly decreased after BTX-A in 11 patients with a visual analog pain scale reduction of 2 or more. The immunoreactivity study of bladder tissue from patients with IC showed greater NGF density at baseline compared with controls, but the difference was no longer significant after successful BTX-A treatment. Intravesical BTX-A injections plus hydrodistension reduce bladder pain in patients with IC. The NGF levels in the bladder tissue were significantly increased in patients with IC and decreased to normal level after treatment in responders. UROLOGY 70: 463– 468, 2007. © 2007 Elsevier Inc.
I
nterstitial cystitis (IC) is a debilitating chronic disease of unknown etiology characterized by urgency, frequency, and suprapubic pain. Current treatments are usually unsuccessful in completely eradicating bladder pain and increasing bladder capacity.1 The chronic pain symptoms in IC might be due to central nervous system (CNS) sensitization and persisting abnormality or activation of the afferent sensory system in the urinary bladder.2 In the urinary tract, nerve growth factor (NGF) is produced by bladder smooth muscle and urothelium.3 Previous studies have indicated that NGF is involved in the ongoing regulation of neural function in conditions such as spinal cord injury and denervation, as well as in inflammation and pain.3–5 Increased levels of NGF have From the Department of Urology, Buddhist Tzu Chi General Hospital and Buddhist Tzu Chi University School of Medicine, Hualien, Taiwan Reprint requests: Hann-Chorng Kuo, M.D., Department of Urology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan. E-mail: [email protected] Submitted: January 30, 2007; accepted (with revisions): April 27, 2007
© 2007 Elsevier Inc. All Rights Reserved
also been reported in the bladder tissue and urine of patients with painful inflammatory conditions of the lower urinary tract, such as sensory urgency and IC.6,7 Bladder inflammation caused by intravesical irritants or in patients with IC leads to acute afferent nerve activity and to long-term plasticity that lowers the threshold for nociceptive and mechanoceptive afferent fibers.8 –10 Chronic sensitization of afferent fibers might involve both peripheral and CNS mechanisms. Intravesical irritants cause increased expression of the c-fos protein in the lumbosacral spinal cord.11 An increase in bladder NGF in the muscle or urothelium initiates signals that are transported along the afferent nerves of the bladder to the dorsal root ganglion or spinal cord.12 Recent studies have shown that intravesical botulinum toxin A (BTX-A) reduces bladder levels of NGF in patients with idiopathic and neurogenic detrusor overactivity.13 Although the mechanism responsible for this reduction in bladder NGF has not been elucidated, prevention of neural plasticity by blockade of NGF production has been suggested as a potential treatment to reduce 0090-4295/07/$32.00 doi:10.1016/j.urology.2007.04.038
463
urge incontinence.13 If BTX-A is delivered to the suburothelial space of the bladder wall and can decrease the release of NGF, it might effectively control inflammatory processes such as those in IC. Recent research using rat models showed BTX-A can inhibit not only the release of acetylcholine and norepinephrine, but also that of adenosine triphosphate and calcitonin gene-related peptide from the detrusor muscle and urothelium.14 –16 In clinical experiments, BTX-A has been shown to reduce detrusor overactivity and impaired bladder sensation and decrease visceral pain in chronic inflammatory diseases.17,18 These findings suggest that BTX-A treatment can decrease sensory transmission, as well as detrusor contractility. This study was designed to investigate the NGF mRNA levels in the bladder tissue of patients with IC before and after intravesical BTX-A injection. The association of a change in NGF mRNA levels with the clinical effectiveness of BTX-A treatment was also assessed.
MATERIAL AND METHODS A total of 19 patients with IC in whom conventional treatments had failed were enrolled in this study. A diagnosis of IC was established according to the characteristic symptoms and cystoscopic findings of glomerulation, petechia, or mucosal fissure. All patients underwent a thorough evaluation and were excluded if they did not meet the criteria of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases.19 Twelve patients who underwent ureteroscopic removal of an upper ureteral stone and who were without lower urinary tract symptoms served as the controls. The institutional review board and ethics committee of Buddhist Tzu Chi General Hospital approved this study. Patients were treated with intravesical injections of 100 U or 200 U BTX-A (Botox, Allergan, Irvine, Calif) followed by cystoscopic hydrodistension 2 weeks later under intravenous general anesthesia. The maximal bladder capacity (MBC) during hydrodistension was recorded. All patients underwent bladder mucosa biopsies before BTX-A injection and immediately after cystoscopic hydrodilation. After Botox injection, the patients were followed up in the outpatient clinic. The data from a 3-day voiding diary, functional bladder capacity, pain visual analog scale (VAS, scored from 0 to 10, representing increasing pain levels), and quality of life index using the International Prostate Symptom Score were recorded.20 A videourodynamic study and potassium chloride (KCl) sensitivity test were performed at baseline and 3 months after treatment. A positive KCl test was defined if the patient perceived bladder pain of 2 or more (score of 0 to 5) after instillation of 40 mL 0.4M KCl into the bladder.21 The urodynamic parameters, including cystometric capacity, volume of urge sensation, voiding pressure, maximal flow rate, and postvoid residual urine volume were recorded. The treatment outcome was considered excellent when patients reported improvement in the quality of life index, complete resolution of bladder pain, and the absence of voiding dysfunction. The outcome was considered improved if the patients reported an improvement in the quality of life index, the VAS pain score was reduced by 2 or more, or the daily urinary frequency was reduced by 25% or more. Patients with excellent 464
or improved outcome were classified as responders and those without improvement as nonresponders. The therapeutic outcome was assessed at 3 months after hydrodistension. Three bladder biopsies were performed using small cystoscopic biopsy forceps in all patients just before intravesical BTX-A injection (baseline) and immediately after cystoscopic hydrodilation. The biopsies were performed about 2 cm above the ureteral orifice on the lateral wall, and only bladder mucosa was taken. Two bladder mucosa biopsies were taken before performing intravesical manipulation in the controls. The bladder biopsy specimens were sent to the pathology department to confirm the diagnosis of IC and exclude the possibility of carcinoma in situ. Total RNA was extracted from the bladder tissue using TRIZOL Total RNA Isolation Reagent (Invitrogen Life Technologies, Carlsbad, Calif) combined with the RNeasy Mini Kit (QIAGEN). In brief, the bladder biopsy specimen was homogenized directly in 1 mL TRIZOL denaturing reagent per 3 to 10 mg. The lysate was transferred to a RNeasy column, following the steps in the manufacturer’s instructions. Next, 1 g of total RNA was reverse transcribed into cDNA using the HighCapacity cDNA Archive Kit (Applied Biosystems, Foster City, Calif). NGF, beta polypeptide mRNA expression was analyzed using real-time polymerase chain reaction with the TaqMan protocol. Real-time quantification polymerase chain reaction was performed with an ABI Prism 7300 Sequence Detection System (ABI 7300 SDS) using 96-well microtiter plates. The primers and probes for NGF beta polypeptide, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) were purchased from PE Applied Biosystems (Assays-on-Demand Gene Expression Products). All experiments were performed in duplicate or triplicate. Relative quantification of NGF beta polypeptide gene expression was performed using the comparative threshold cycle method described in User Bulletin 2 provided by the manufacturer (PE Applied Biosystems). For the accurate normalization of realtime quantification, two reference genes, GAPDH and HPRT1, were used as endogenous references. The results confirmed that both GAPDH and HPRT1 reference genes were consistently expressed in the tissues of the case-patient and control groups. Only data for the normalized target amount with GAPDH are shown. The urinary bladder specimens were immersion fixated for 1 hour in an ice-cold solution of 4% formaldehyde in phosphatebuffered saline (pH 7.4) and rinsed with ice-cold phosphatebuffered saline containing 15% sucrose for 12 hours. The specimens were embedded in OCT medium (Miles, Ekhart, Ind) and then stored at ⫺80°C. The tissues were sectioned in 6-m-thick slices and were collected onto poly-L-lysine-coated slides (SuperFrost Plus). The sections were incubated overnight at 4°C with primary antibodies of anti-human beta-NGF (goat polyclonal antibody, R&D Systems, Minneapolis, Minn). After rinsing the sections with Tween-20 phosphate-buffered saline, rabbit anti-goat secondary antibodies (Stressgen, Canada) were applied to the sections, followed by incubation for 1 hour. Subsequently, horseradish peroxidase-conjugated secondary antibodies were added for 30 minutes, and DAB (BioGenix) was added for 3 minutes. Finally, the sections were counterstained with hematoxylin (Sigma Chemical, St. Louis, Mo). Negative controls included omission of the primary antibody. Differences in the normalized baseline expression of NGF mRNA levels between patients with IC and controls were UROLOGY 70 (3), 2007
Figure 1. Scatter plots of relative expression of NGF mRNA levels in (A) patients with IC with symptomatic improvement (responders) and no improvement (nonresponders) after BTX-A treatment at baseline and 2 weeks after BTX-A injection, and (B) patients with pain VAS reduction of 2 or more or pain VAS reduction of less than 2 at baseline and 2 weeks after BTX-A injection. Horizontal lines represent mean values of NGF mRNA.
assessed using an independent Student’s t test. A paired t test was used to analyze the NGF mRNA expression levels at baseline and after BTX-A treatment in patients with IC. The NGF mRNA levels were also compared between the patients with or without (a) an overall satisfactory therapeutic result after treatment, (b) a VAS pain score reduction of 2 or more, and (c) an increase in MBC of 100 mL or greater. All calculations were performed using Statistical Package for Social Sciences for Windows, version 10.0. A P value of less than 0.05 was considered statistically significant.
RESULTS Of the 19 patients with IC, 14 were women (aged 27 to 62 years, mean 37) and 5 were men (aged 35 to 66 years, mean 41). Of the 19 patients, 14 received 100 U and 5 received 200 U of BTX-A. At 3 months after treatment, 14 of the patients had significant symptomatic improvement (responders) and 5 had no improvement (nonresponders). Of the 14 patients in the 100-U BTX-A group and 5 patients in the in 200-U group, 10 (74%) and 4 (80%) were responders, respectively (P ⫽ 0.89). The functional bladder capacity of the overall patients increased by 1.4 times the baseline value. The pain VAS score decreased from 5.16 ⫾ 2.09 to 2.53 ⫾ 1.43 after treatment (P ⬍0.0001), and the daily frequency episodes decreased from 12.6 ⫾ 4.3 to 8.8 ⫾ 2.5 (P ⫽ 0.001). Difficult urination and a large postvoid residual urine volume were problems in 7 patients (50%) who had received 100 U and in 5 (100%) who had received 200 U BTX-A during the initial month after treatment; however, all patients had achieved a balanced bladder at 3 UROLOGY 70 (3), 2007
months after treatment. The overall success rate was 74%. The cystometric bladder capacity (276.2 ⫾ 94.8 versus 372.1 ⫾ 149.3 mL, P ⫽ 0.009) and urge sensation volume (251.3 ⫾ 89.2 versus 321.1 ⫾ 96.9 mL, P ⫽ 0.029) increased significantly after treatment. No significant posttreatment change was noted in voiding pressure, maximal flow rate, or postvoid residual urine volume. All patients had a positive KCl test at baseline, but a negative result was found in 10 patients at 3 months (52.6%, P ⫽ 0.002). The NGF mRNA levels in patients with IC at baseline were significantly greater than those of the controls (0.65 ⫾ 0.33 versus 0.42 ⫾ 0.25, P ⫽ 0.046). At 2 weeks after BTX-A injection, the NGF mRNA had decreased to 0.47 ⫾ 0.23, a 28% reduction compared with baseline (P ⫽ 0.002) and was no longer significantly different from that of the controls. No significant difference was found in the NGF mRNA levels between the responders (0.69 ⫾ 0.37) and nonresponders (0.48 ⫾ 0.16) at baseline (P ⫽ 0.222). The responders had a significantly greater NGF mRNA level at baseline compared with the controls (P ⫽ 0.040), but the nonresponders did not (P ⫽ 0.658). The responders showed a significant reduction in NGF mRNA (0.48 ⫾ 0.26, P ⫽ 0.002), and the nonresponders showed no significant change (0.41 ⫾ 0.15, P ⫽ 0.274) after BTX-A treatment (Fig. 1A). The baseline NGF mRNA levels and changes after treatment showed no significant difference between patients receiving 100 U (0.63 ⫾ 0.38 465
Figure 2. Immunohistochemistry of NGF in bladder tissues of patients with IC and controls. Absence of primary antibody used as negative control. Neither urothelial cells nor nerve fibers in suburothelium were stained (A). NGF immunoreactivity staining was positive on apical urothelium in bladder biopsies of patients with IC (C,D) and controls (B). NGF immunoreactivity of nerve fibers and nerve cells in suburothelium only observed in bladder tissue of patients with IC (C, green arrows) and was not found in suburothelial cells of controls (B). NGF immunoreactivity density in nerve fibers and nerve cells of suburothelium significantly reduced after successful treatment with BTX-A (D). Original magnification ⫻400.
versus 0.45 ⫾ 0.25) and those receiving 200 U (0.71 ⫾ 0.12 versus 0.52 ⫾ 0.20) BTX-A (P ⫽ 0.670). The VAS pain score was reduced by 2 or more in 11 patients and less than 2 in 8. The NGF mRNA levels decreased significantly after treatment in patients with a VAS reduction of 2 or more (from 0.62 ⫾ 0.31 to 0.40 ⫾ 0.19, P ⫽ 0.004) but not in those with a VAS reduction of less than 2 (from 0.65 ⫾ 0.38 to 0.57 ⫾ 0.27, P ⫽ 0.226; Fig. 1B). Using the independent t test to compare the changes in NGF mRNA after BTX-A treatment, responders tended to have a greater reduction in NGF mRNA than nonresponders (P ⫽ 0.098) and patients with a VAS reduction of 2 or more tended to have a greater reduction than those with a VAS reduction of less than 2 (P ⫽ 0.069). At 2 weeks after BTX-A injection, the MBC during hydrodistension was 674.4 ⫾ 231.8 mL, which was significantly increased compared with the level at the diag466
nosis of IC (544.7 ⫾ 136.3 mL, P ⬍0.0001). The MBC increased by more than 100 mL (range 100 to 450, mean 187) in 14 patients. The decrease in NGF mRNA levels in these patients was significant (from 0.71 ⫾ 0.36 to 0.53 ⫾ 0.26, P ⫽ 0.018). In the other 5 patients with an MBC increase of less than 100 mL, the NGF mRNA levels still decreased significantly (from 0.55 ⫾ 0.13 to 0.31 ⫾ 0.09, P ⫽ 0.018). No significant difference was found in the reduction of NGF mRNA levels between these two groups. The NGF immunoreactivity study in the bladder of patients with IC showed a significantly increased density in the urothelium, apical urothelial, and submucosal cells at baseline (Fig. 2C). The density was reduced after successful treatment with BTX-A (Fig. 2D). The density of NGF immunostaining was stronger in patients with stronger NGF mRNA expression. In contrast, controls had NGF immunostaining only in the apical urothelial cells (Fig. 2B). UROLOGY 70 (3), 2007
COMMENT The results of this study have demonstrated that NGF mRNA production in bladder tissue is significantly increased in patients with IC compared with that in controls and that successful intravesical BTX-A injection reduced NGF mRNA expression to normal levels at 2 weeks after BTX-A treatment. NGF mRNA expression preceded NGF protein production in bladder tissue. The decrease in NGF mRNA at 2 weeks after BTX-A treatment was associated with therapeutic effectiveness and pain reduction at 3 months, suggesting that NGF expression plays an important role in the pathogenesis of IC and thus is a reasonable therapeutic target. Intravesical instillation of NGF can induce bladder hyperactivity in rats.10 In the rat chemical cystitis model, detrusor injection of BTX-A was shown to have therapeutic effects on increasing bladder capacity and compliance.22 In human IC, NGF levels have been shown to be increased in both bladder tissue and urine compared with controls.4,7 NGF may act on trkA receptors to mediate biosynthesis and release of substance P and induce neurogenic inflammation.23 Inflammation also induces neuroplastic changes that lead to increased sensory nerve density in the bladder.5 Long-term NGF production in chronic inflammatory processes might induce chronic sensitization of both peripheral nerves and the CNS, resulting in propagation of symptoms in IC that may persist after the inflammatory stimuli subside.24 In this regard, inhibition of the neuroplasticity of the sensory fibers in the suburothelial space by BTX-A injection might have good therapeutic targeting on pain and sensory urgency in patients with IC. The results of this study support this hypothesis and, therefore, the use of this modality in the treatment of IC. In this study, only patients with IC who had a reduction of NGF mRNA levels after treatment responded to BTX-A treatment, suggesting a reduction of NGF might decrease neurogenic inflammation in the IC bladder and, therefore, improve symptoms. Patients with IC who have normal levels of NGF mRNA are unlikely to benefit from BTX-A therapy. A decreased NGF mRNA level after treatment was associated with a decreased pain score, but it was not associated with an increase in the MBC, suggesting that NGF has a direct effect on factors that might contribute to pain perception in the bladder wall such as calcitonin gene-related peptide production, but might not affect the established chronic inflammatory changes in the bladder. Other pathways in the inflammatory process of IC might be responsible for the lack of a response to this treatment. Previous studies of BTX-A treatment on IC did not report consistent results.25–27 The cause of the unsuccessful therapeutic results or short therapeutic duration might have been inadequate CNS desensitization. IC can be an acute or chronic process of inflammation in the bladder. Bladder inflammation increased NGF production and UROLOGY 70 (3), 2007
caused morphologic changes in the sensory and motor neurons.5 Intravesical injection of BTX-A might induce peripheral desensitization. This effect might have an important role in reducing bladder pain in IC, but the CNS desensitization might not be achieved by a single BTX-A treatment. Repeated injections of BTX-A might be necessary to eliminate the neurogenic inflammation and thereby relieve the associated visceral pain. One major concern in this study was that bladder biopsies after BTX-A treatment were obtained after hydrodistension. Changes of other biomarkers such as antiproliferative factor and heparin-binding epidermal growth factor have been demonstrated after bladder hydrodistension in IC, and patients might benefit from hydrodistension alone.28 Therefore, it cannot be excluded that hydrodistension might have an influence on NGF mRNA production. Additional studies are necessary to clarify this point.
CONCLUSIONS The results of our study have indicated that intravesical injection of BTX-A plus hydrodistension can reduce bladder pain in patients with IC. The NGF mRNA levels in the bladder tissue were significantly increased in patients with IC and decreased to normal levels after BTX-A treatment in the responders. References 1. Hanno PM, and Sant GR: Clinical highlights of the national Institute of Diabetes and Digestive and Kidney Diseases/Interstitial Cystitis Association scientific conference on interstitial cystitis. Urology 57(suppl 6A): 2– 6, 2001. 2. Wesselmann U: Interstitial cystitis: a chronic visceral pain syndrome. Urology 57(suppl 6A): 32–39, 2001. 3. Tuttle JB, Steers WD, Albo M, et al: Neural input regulates tissue NGF and growth of the adult urinary bladder. J Auton Nerv Syst 49: 147–158, 1994. 4. Steers WD, Kolbeck S, Creedon D, et al: Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest 88: 1709 –1715, 1991. 5. Dupont MC, Spitsberegen JM, Kim KB, et al: Histological and neurotrophic changes triggered by varying models of bladder inflammation. J Urol 166: 1111–1118, 2001. 6. Lowe EM, Anand P, Terenghi G, et al: Increased nerve growth factor levels in the urinary bladder of women with idiopathic sensory urgency and interstitial cystitis. Br J Urol 79: 572–577, 1997. 7. Okragly AJ, Niles AL, Saban R, et al: Elevated tryptase, nerve growth factor, neurotrophin-3 and glial cell line-derived neurotrophic factor levels in the urine of interstitial cystitis and bladder cancer patients. J Urol 161: 438 – 441, 1999. 8. Murray E, Malley SE, Qiao LY, et al: Cyclophosphamide induced cystitis alters neurotrophin and receptor tyrosine kinase expression in pelvic ganglia and bladder. J Urol 172: 2434 –2439, 2004. 9. Oddiah D, Anand P, McMahon SB, et al: Rapid increase of NGF, BDNF and NT-3 mRNAs in inflamed bladder. Neuroreport 9: 1455–1458, 1998. 10. Chung YC, Fraser MO, Yu Y, et al: The role of bladder afferent pathways in bladder hyperactivity induced by the intravesical administration of nerve growth factor. J Urol 165: 975–979, 2001. 11. Seki S, Sasaki K, Fraser MO, et al: Immunoneutralization of nerve growth factor in lumbosacral spinal cord reduces bladder hyperreflexia in spinal cord injured rats. J Urol 168: 2269 –2274, 2002.
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12. Lamb K, Gebhart GF, and Bielefeldt K: Increased nerve growth factor expression triggers bladder overactivity. J Pain 5: 150 –156, 2004. 13. Giannantoni A, Di Stasi SM, Nardicchi V, et al: Botulinum A toxin injection into the detrusor decrease nerve growth factor bladder tissue levels in patients with neurogenic detrusor overactivity. J Urol 175: 2341–2344, 2006. 14. Smith CP, Boone TB, de Groat WC, et al: Effect of stimulation intensity and botulinum toxin isoform on rat bladder strip contractions. Brain Res Bull 61: 165–171, 2003. 15. Khera M, Somogyi GT, Kiss S, et al: Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury. Neurochem Int 45: 987–993, 2004. 16. Chuang YC, Yoshimura N, Huang CC, et al: Intravesical botulinum toxin A administration produces analgesia against acetic acid induced bladder pain responses in rats. J Urol 172: 1529 –1532, 2004. 17. Kuo HC: Urodynamic evidence of effectiveness of botulinum A toxin injection in treatment of detrusor overactivity refractory to anticholinergic agents. Urology 63: 868 – 872, 2004. 18. Zermann DH, Ishigooka M, and Schubert J: Trigonum and bladder base injection of botulinum toxin A (BTX) in patients with severe urgency-frequency-syndrome refractory to conservative medical treatment and electrical stimulation (abstract). Neurourol Urodyn 20: 412– 413, 2001. 19. Gillenwatewer JY, Wein AJ. Summary of the National Institute of arthritis, diabetes and kidney diseases workshop on interstitial cystitis. National Institute of Health, Bethesda, Maryland, August 28 –29, 1987. J Urol 140:203–206, 1988. 20. Cockett A, Aso Y, Denis L, et al: Recommendation of the International Consensus Committee concerning: 1. Prostate symptom
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score and quality of life assessment, in Cockett ATK, Khoury S, Aso Y, et al (Eds): Proceedings of the 2nd International Consultation on Benign Prostatic Hyperplasia (BPH), Paris, June 27–30, 1993. Jersey, Channel Island, Scientific Communication International, 1994, pp 553–555. Parsons CL, Forrest J, Nickel JC, et al: Effect of pentosan polysulphate therapy on intravesical potassium sensitivity. Urology 59: 329 –333, 2002. Cayan S, Coskun B, Bozlu M, et al: Botulinum toxin type A may improve bladder function in a rat chemical cystitis model. Urol Res 30: 399 – 404, 2003. Dinh QT, Groneberg DA, Peisert C, et al: Nerve growth factorinduced substance P in capsaicin insensitive vagal neurons innervating the lower mouse airway. Clin Exp Allergy 34: 1474 –1479, 2006. Steers WD, and Tuttle JB: Mechanisms of diseases: the role of nerve growth factor in the pathophysiology of bladder disorders. Nature Clin Pract Urol 3: 101–110, 2006. Smith CP, Radziszewski P, Borkowski A, et al: Botulinum toxin A has antinociceptive effects in treating interstitial cystitis. Urology 64: 871– 875, 2004. Kuo HC: Preliminary results of suburothelial injection of botulinum A toxin in the treatment of chronic interstitial cystitis. Urol Int 75: 170 –174, 2005. Giannantoni A, Costantini E, Di Stasi SM, et al: Botulinum A toxin intravesical injections in the treatment of painful bladder syndrome: a pilot study. Eur Urol 49: 704 –709, 2006. Tsai TC, Zhang CO, Shoenfelt JO, et al: Bladder stretch alters urinary heparin-binding epidermal growth factor and antiproliferative factor in patients with interstitial cystitis. J Urol 163: 1440 – 1444, 2000.
UROLOGY 70 (3), 2007
RESULTS
CONCLUSIONS
To investigate the level of nerve growth factor (NGF) mRNA in bladder tissue and the effect of botulinum toxin A (BTX-A) treatment in patients with interstitial cystitis (IC). A total of 19 patients with IC were treated with 100 U or 200 U of intravesical BTX-A injections followed by cystoscopic hydrodistension 2 weeks later. Bladder mucosa biopsies were performed before BTX-A injection and immediately after hydrodilation and in 12 controls. The NGF mRNA and protein levels in bladder tissues were assessed by real-time polymerase chain reaction and immunohistochemistry studies to determine differences in NGF expression between patients with IC before and after BTX-A treatment and compare with controls. At 3 months, 14 patients had symptomatic improvement (responders) and 5 did not (nonresponders). The NGF mRNA levels at baseline in the overall IC patient group were significantly greater than those in the controls (0.65 ⫾ 0.33 versus 0.42 ⫾ 0.25, P ⫽ 0.046). At 2 weeks after BTX-A treatment, the NGF mRNA levels had decreased to 0.47 ⫾ 0.23 (P ⫽ 0.002, compared with baseline) and were no longer significantly different from those of the controls. The NGF mRNA levels decreased significantly in responders and were significantly decreased after BTX-A in 11 patients with a visual analog pain scale reduction of 2 or more. The immunoreactivity study of bladder tissue from patients with IC showed greater NGF density at baseline compared with controls, but the difference was no longer significant after successful BTX-A treatment. Intravesical BTX-A injections plus hydrodistension reduce bladder pain in patients with IC. The NGF levels in the bladder tissue were significantly increased in patients with IC and decreased to normal level after treatment in responders. UROLOGY 70: 463– 468, 2007. © 2007 Elsevier Inc.
I
nterstitial cystitis (IC) is a debilitating chronic disease of unknown etiology characterized by urgency, frequency, and suprapubic pain. Current treatments are usually unsuccessful in completely eradicating bladder pain and increasing bladder capacity.1 The chronic pain symptoms in IC might be due to central nervous system (CNS) sensitization and persisting abnormality or activation of the afferent sensory system in the urinary bladder.2 In the urinary tract, nerve growth factor (NGF) is produced by bladder smooth muscle and urothelium.3 Previous studies have indicated that NGF is involved in the ongoing regulation of neural function in conditions such as spinal cord injury and denervation, as well as in inflammation and pain.3–5 Increased levels of NGF have From the Department of Urology, Buddhist Tzu Chi General Hospital and Buddhist Tzu Chi University School of Medicine, Hualien, Taiwan Reprint requests: Hann-Chorng Kuo, M.D., Department of Urology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan. E-mail: [email protected] Submitted: January 30, 2007; accepted (with revisions): April 27, 2007
© 2007 Elsevier Inc. All Rights Reserved
also been reported in the bladder tissue and urine of patients with painful inflammatory conditions of the lower urinary tract, such as sensory urgency and IC.6,7 Bladder inflammation caused by intravesical irritants or in patients with IC leads to acute afferent nerve activity and to long-term plasticity that lowers the threshold for nociceptive and mechanoceptive afferent fibers.8 –10 Chronic sensitization of afferent fibers might involve both peripheral and CNS mechanisms. Intravesical irritants cause increased expression of the c-fos protein in the lumbosacral spinal cord.11 An increase in bladder NGF in the muscle or urothelium initiates signals that are transported along the afferent nerves of the bladder to the dorsal root ganglion or spinal cord.12 Recent studies have shown that intravesical botulinum toxin A (BTX-A) reduces bladder levels of NGF in patients with idiopathic and neurogenic detrusor overactivity.13 Although the mechanism responsible for this reduction in bladder NGF has not been elucidated, prevention of neural plasticity by blockade of NGF production has been suggested as a potential treatment to reduce 0090-4295/07/$32.00 doi:10.1016/j.urology.2007.04.038
463
urge incontinence.13 If BTX-A is delivered to the suburothelial space of the bladder wall and can decrease the release of NGF, it might effectively control inflammatory processes such as those in IC. Recent research using rat models showed BTX-A can inhibit not only the release of acetylcholine and norepinephrine, but also that of adenosine triphosphate and calcitonin gene-related peptide from the detrusor muscle and urothelium.14 –16 In clinical experiments, BTX-A has been shown to reduce detrusor overactivity and impaired bladder sensation and decrease visceral pain in chronic inflammatory diseases.17,18 These findings suggest that BTX-A treatment can decrease sensory transmission, as well as detrusor contractility. This study was designed to investigate the NGF mRNA levels in the bladder tissue of patients with IC before and after intravesical BTX-A injection. The association of a change in NGF mRNA levels with the clinical effectiveness of BTX-A treatment was also assessed.
MATERIAL AND METHODS A total of 19 patients with IC in whom conventional treatments had failed were enrolled in this study. A diagnosis of IC was established according to the characteristic symptoms and cystoscopic findings of glomerulation, petechia, or mucosal fissure. All patients underwent a thorough evaluation and were excluded if they did not meet the criteria of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases.19 Twelve patients who underwent ureteroscopic removal of an upper ureteral stone and who were without lower urinary tract symptoms served as the controls. The institutional review board and ethics committee of Buddhist Tzu Chi General Hospital approved this study. Patients were treated with intravesical injections of 100 U or 200 U BTX-A (Botox, Allergan, Irvine, Calif) followed by cystoscopic hydrodistension 2 weeks later under intravenous general anesthesia. The maximal bladder capacity (MBC) during hydrodistension was recorded. All patients underwent bladder mucosa biopsies before BTX-A injection and immediately after cystoscopic hydrodilation. After Botox injection, the patients were followed up in the outpatient clinic. The data from a 3-day voiding diary, functional bladder capacity, pain visual analog scale (VAS, scored from 0 to 10, representing increasing pain levels), and quality of life index using the International Prostate Symptom Score were recorded.20 A videourodynamic study and potassium chloride (KCl) sensitivity test were performed at baseline and 3 months after treatment. A positive KCl test was defined if the patient perceived bladder pain of 2 or more (score of 0 to 5) after instillation of 40 mL 0.4M KCl into the bladder.21 The urodynamic parameters, including cystometric capacity, volume of urge sensation, voiding pressure, maximal flow rate, and postvoid residual urine volume were recorded. The treatment outcome was considered excellent when patients reported improvement in the quality of life index, complete resolution of bladder pain, and the absence of voiding dysfunction. The outcome was considered improved if the patients reported an improvement in the quality of life index, the VAS pain score was reduced by 2 or more, or the daily urinary frequency was reduced by 25% or more. Patients with excellent 464
or improved outcome were classified as responders and those without improvement as nonresponders. The therapeutic outcome was assessed at 3 months after hydrodistension. Three bladder biopsies were performed using small cystoscopic biopsy forceps in all patients just before intravesical BTX-A injection (baseline) and immediately after cystoscopic hydrodilation. The biopsies were performed about 2 cm above the ureteral orifice on the lateral wall, and only bladder mucosa was taken. Two bladder mucosa biopsies were taken before performing intravesical manipulation in the controls. The bladder biopsy specimens were sent to the pathology department to confirm the diagnosis of IC and exclude the possibility of carcinoma in situ. Total RNA was extracted from the bladder tissue using TRIZOL Total RNA Isolation Reagent (Invitrogen Life Technologies, Carlsbad, Calif) combined with the RNeasy Mini Kit (QIAGEN). In brief, the bladder biopsy specimen was homogenized directly in 1 mL TRIZOL denaturing reagent per 3 to 10 mg. The lysate was transferred to a RNeasy column, following the steps in the manufacturer’s instructions. Next, 1 g of total RNA was reverse transcribed into cDNA using the HighCapacity cDNA Archive Kit (Applied Biosystems, Foster City, Calif). NGF, beta polypeptide mRNA expression was analyzed using real-time polymerase chain reaction with the TaqMan protocol. Real-time quantification polymerase chain reaction was performed with an ABI Prism 7300 Sequence Detection System (ABI 7300 SDS) using 96-well microtiter plates. The primers and probes for NGF beta polypeptide, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) were purchased from PE Applied Biosystems (Assays-on-Demand Gene Expression Products). All experiments were performed in duplicate or triplicate. Relative quantification of NGF beta polypeptide gene expression was performed using the comparative threshold cycle method described in User Bulletin 2 provided by the manufacturer (PE Applied Biosystems). For the accurate normalization of realtime quantification, two reference genes, GAPDH and HPRT1, were used as endogenous references. The results confirmed that both GAPDH and HPRT1 reference genes were consistently expressed in the tissues of the case-patient and control groups. Only data for the normalized target amount with GAPDH are shown. The urinary bladder specimens were immersion fixated for 1 hour in an ice-cold solution of 4% formaldehyde in phosphatebuffered saline (pH 7.4) and rinsed with ice-cold phosphatebuffered saline containing 15% sucrose for 12 hours. The specimens were embedded in OCT medium (Miles, Ekhart, Ind) and then stored at ⫺80°C. The tissues were sectioned in 6-m-thick slices and were collected onto poly-L-lysine-coated slides (SuperFrost Plus). The sections were incubated overnight at 4°C with primary antibodies of anti-human beta-NGF (goat polyclonal antibody, R&D Systems, Minneapolis, Minn). After rinsing the sections with Tween-20 phosphate-buffered saline, rabbit anti-goat secondary antibodies (Stressgen, Canada) were applied to the sections, followed by incubation for 1 hour. Subsequently, horseradish peroxidase-conjugated secondary antibodies were added for 30 minutes, and DAB (BioGenix) was added for 3 minutes. Finally, the sections were counterstained with hematoxylin (Sigma Chemical, St. Louis, Mo). Negative controls included omission of the primary antibody. Differences in the normalized baseline expression of NGF mRNA levels between patients with IC and controls were UROLOGY 70 (3), 2007
Figure 1. Scatter plots of relative expression of NGF mRNA levels in (A) patients with IC with symptomatic improvement (responders) and no improvement (nonresponders) after BTX-A treatment at baseline and 2 weeks after BTX-A injection, and (B) patients with pain VAS reduction of 2 or more or pain VAS reduction of less than 2 at baseline and 2 weeks after BTX-A injection. Horizontal lines represent mean values of NGF mRNA.
assessed using an independent Student’s t test. A paired t test was used to analyze the NGF mRNA expression levels at baseline and after BTX-A treatment in patients with IC. The NGF mRNA levels were also compared between the patients with or without (a) an overall satisfactory therapeutic result after treatment, (b) a VAS pain score reduction of 2 or more, and (c) an increase in MBC of 100 mL or greater. All calculations were performed using Statistical Package for Social Sciences for Windows, version 10.0. A P value of less than 0.05 was considered statistically significant.
RESULTS Of the 19 patients with IC, 14 were women (aged 27 to 62 years, mean 37) and 5 were men (aged 35 to 66 years, mean 41). Of the 19 patients, 14 received 100 U and 5 received 200 U of BTX-A. At 3 months after treatment, 14 of the patients had significant symptomatic improvement (responders) and 5 had no improvement (nonresponders). Of the 14 patients in the 100-U BTX-A group and 5 patients in the in 200-U group, 10 (74%) and 4 (80%) were responders, respectively (P ⫽ 0.89). The functional bladder capacity of the overall patients increased by 1.4 times the baseline value. The pain VAS score decreased from 5.16 ⫾ 2.09 to 2.53 ⫾ 1.43 after treatment (P ⬍0.0001), and the daily frequency episodes decreased from 12.6 ⫾ 4.3 to 8.8 ⫾ 2.5 (P ⫽ 0.001). Difficult urination and a large postvoid residual urine volume were problems in 7 patients (50%) who had received 100 U and in 5 (100%) who had received 200 U BTX-A during the initial month after treatment; however, all patients had achieved a balanced bladder at 3 UROLOGY 70 (3), 2007
months after treatment. The overall success rate was 74%. The cystometric bladder capacity (276.2 ⫾ 94.8 versus 372.1 ⫾ 149.3 mL, P ⫽ 0.009) and urge sensation volume (251.3 ⫾ 89.2 versus 321.1 ⫾ 96.9 mL, P ⫽ 0.029) increased significantly after treatment. No significant posttreatment change was noted in voiding pressure, maximal flow rate, or postvoid residual urine volume. All patients had a positive KCl test at baseline, but a negative result was found in 10 patients at 3 months (52.6%, P ⫽ 0.002). The NGF mRNA levels in patients with IC at baseline were significantly greater than those of the controls (0.65 ⫾ 0.33 versus 0.42 ⫾ 0.25, P ⫽ 0.046). At 2 weeks after BTX-A injection, the NGF mRNA had decreased to 0.47 ⫾ 0.23, a 28% reduction compared with baseline (P ⫽ 0.002) and was no longer significantly different from that of the controls. No significant difference was found in the NGF mRNA levels between the responders (0.69 ⫾ 0.37) and nonresponders (0.48 ⫾ 0.16) at baseline (P ⫽ 0.222). The responders had a significantly greater NGF mRNA level at baseline compared with the controls (P ⫽ 0.040), but the nonresponders did not (P ⫽ 0.658). The responders showed a significant reduction in NGF mRNA (0.48 ⫾ 0.26, P ⫽ 0.002), and the nonresponders showed no significant change (0.41 ⫾ 0.15, P ⫽ 0.274) after BTX-A treatment (Fig. 1A). The baseline NGF mRNA levels and changes after treatment showed no significant difference between patients receiving 100 U (0.63 ⫾ 0.38 465
Figure 2. Immunohistochemistry of NGF in bladder tissues of patients with IC and controls. Absence of primary antibody used as negative control. Neither urothelial cells nor nerve fibers in suburothelium were stained (A). NGF immunoreactivity staining was positive on apical urothelium in bladder biopsies of patients with IC (C,D) and controls (B). NGF immunoreactivity of nerve fibers and nerve cells in suburothelium only observed in bladder tissue of patients with IC (C, green arrows) and was not found in suburothelial cells of controls (B). NGF immunoreactivity density in nerve fibers and nerve cells of suburothelium significantly reduced after successful treatment with BTX-A (D). Original magnification ⫻400.
versus 0.45 ⫾ 0.25) and those receiving 200 U (0.71 ⫾ 0.12 versus 0.52 ⫾ 0.20) BTX-A (P ⫽ 0.670). The VAS pain score was reduced by 2 or more in 11 patients and less than 2 in 8. The NGF mRNA levels decreased significantly after treatment in patients with a VAS reduction of 2 or more (from 0.62 ⫾ 0.31 to 0.40 ⫾ 0.19, P ⫽ 0.004) but not in those with a VAS reduction of less than 2 (from 0.65 ⫾ 0.38 to 0.57 ⫾ 0.27, P ⫽ 0.226; Fig. 1B). Using the independent t test to compare the changes in NGF mRNA after BTX-A treatment, responders tended to have a greater reduction in NGF mRNA than nonresponders (P ⫽ 0.098) and patients with a VAS reduction of 2 or more tended to have a greater reduction than those with a VAS reduction of less than 2 (P ⫽ 0.069). At 2 weeks after BTX-A injection, the MBC during hydrodistension was 674.4 ⫾ 231.8 mL, which was significantly increased compared with the level at the diag466
nosis of IC (544.7 ⫾ 136.3 mL, P ⬍0.0001). The MBC increased by more than 100 mL (range 100 to 450, mean 187) in 14 patients. The decrease in NGF mRNA levels in these patients was significant (from 0.71 ⫾ 0.36 to 0.53 ⫾ 0.26, P ⫽ 0.018). In the other 5 patients with an MBC increase of less than 100 mL, the NGF mRNA levels still decreased significantly (from 0.55 ⫾ 0.13 to 0.31 ⫾ 0.09, P ⫽ 0.018). No significant difference was found in the reduction of NGF mRNA levels between these two groups. The NGF immunoreactivity study in the bladder of patients with IC showed a significantly increased density in the urothelium, apical urothelial, and submucosal cells at baseline (Fig. 2C). The density was reduced after successful treatment with BTX-A (Fig. 2D). The density of NGF immunostaining was stronger in patients with stronger NGF mRNA expression. In contrast, controls had NGF immunostaining only in the apical urothelial cells (Fig. 2B). UROLOGY 70 (3), 2007
COMMENT The results of this study have demonstrated that NGF mRNA production in bladder tissue is significantly increased in patients with IC compared with that in controls and that successful intravesical BTX-A injection reduced NGF mRNA expression to normal levels at 2 weeks after BTX-A treatment. NGF mRNA expression preceded NGF protein production in bladder tissue. The decrease in NGF mRNA at 2 weeks after BTX-A treatment was associated with therapeutic effectiveness and pain reduction at 3 months, suggesting that NGF expression plays an important role in the pathogenesis of IC and thus is a reasonable therapeutic target. Intravesical instillation of NGF can induce bladder hyperactivity in rats.10 In the rat chemical cystitis model, detrusor injection of BTX-A was shown to have therapeutic effects on increasing bladder capacity and compliance.22 In human IC, NGF levels have been shown to be increased in both bladder tissue and urine compared with controls.4,7 NGF may act on trkA receptors to mediate biosynthesis and release of substance P and induce neurogenic inflammation.23 Inflammation also induces neuroplastic changes that lead to increased sensory nerve density in the bladder.5 Long-term NGF production in chronic inflammatory processes might induce chronic sensitization of both peripheral nerves and the CNS, resulting in propagation of symptoms in IC that may persist after the inflammatory stimuli subside.24 In this regard, inhibition of the neuroplasticity of the sensory fibers in the suburothelial space by BTX-A injection might have good therapeutic targeting on pain and sensory urgency in patients with IC. The results of this study support this hypothesis and, therefore, the use of this modality in the treatment of IC. In this study, only patients with IC who had a reduction of NGF mRNA levels after treatment responded to BTX-A treatment, suggesting a reduction of NGF might decrease neurogenic inflammation in the IC bladder and, therefore, improve symptoms. Patients with IC who have normal levels of NGF mRNA are unlikely to benefit from BTX-A therapy. A decreased NGF mRNA level after treatment was associated with a decreased pain score, but it was not associated with an increase in the MBC, suggesting that NGF has a direct effect on factors that might contribute to pain perception in the bladder wall such as calcitonin gene-related peptide production, but might not affect the established chronic inflammatory changes in the bladder. Other pathways in the inflammatory process of IC might be responsible for the lack of a response to this treatment. Previous studies of BTX-A treatment on IC did not report consistent results.25–27 The cause of the unsuccessful therapeutic results or short therapeutic duration might have been inadequate CNS desensitization. IC can be an acute or chronic process of inflammation in the bladder. Bladder inflammation increased NGF production and UROLOGY 70 (3), 2007
caused morphologic changes in the sensory and motor neurons.5 Intravesical injection of BTX-A might induce peripheral desensitization. This effect might have an important role in reducing bladder pain in IC, but the CNS desensitization might not be achieved by a single BTX-A treatment. Repeated injections of BTX-A might be necessary to eliminate the neurogenic inflammation and thereby relieve the associated visceral pain. One major concern in this study was that bladder biopsies after BTX-A treatment were obtained after hydrodistension. Changes of other biomarkers such as antiproliferative factor and heparin-binding epidermal growth factor have been demonstrated after bladder hydrodistension in IC, and patients might benefit from hydrodistension alone.28 Therefore, it cannot be excluded that hydrodistension might have an influence on NGF mRNA production. Additional studies are necessary to clarify this point.
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