Exogenous overexpression of nerve growth factor in the urinary bladder produces bladder overactivity and altered micturition circuitry in the lumbosacral spinal cord

Peter Zvara, Margaret A Vizzard, Peter Zvara, Margaret A Vizzard

Abstract

Background: Exogenous NGF or saline was delivered to the detrusor smooth muscle of female rats for a two-week period using osmotic mini-pumps. We then determined: (1) bladder function using conscious cystometry; (2) organization of micturition reflexes using Fos protein expression in lumbosacral (L5-S1) spinal cord neurons; (3) calcitonin gene-related peptide (CGRP)-immunoreactivity (IR) in lumbosacral spinal cord segments.

Methods: An osmotic pump infused 0.9% NaCl (n = 6) or NGF (n = 6)(2.5 microg/microl solution; 0.5 microl/hr) for two weeks into the bladder wall. NGF bladder content was determined by enzyme-linked immunoassays. Bladder function was assessed with conscious cystometry. Immunohistochemical and imaging techniques were used to determine the distribution of Fos-IR cells and CGRP expression in the L5-S1 spinal cord in saline and NGF-treated rats two hours after intravesical saline distention. Fos expression and CGRP-IR in NGF-treated rats with bladder distention was compared to that observed in cyclophosphamide (CYP; 75 mg/kg; i.p.) treated rats with bladder distention.

Results: Two-week infusion of NGF into the bladder wall increased bladder weight, reduced bladder capacity (60%), reduced the intercontraction interval (60%) and increased the amplitude of non-voiding contractions. NGF treatment and intravesical saline distention (2 hr) increased expression of Fos protein in L6-S1 spinal cord and altered the distribution pattern of Fos-IR cells. CGRP-IR in the lumbosacral spinal cord was also increased after NGF treatment.

Conclusion: These data suggest that NGF infusion into the bladder wall induces bladder overactivity, can reveal a "nociceptive" Fos expression pattern in the spinal cord in response to a non-noxious bladder stimulus and increases CGRP-IR in the lumbosacral spinal cord.

Figures

Figure 1
Figure 1
Intraoperative procedure for tubing implantation. Intraoperative picture depictingthe insertion of PE10 tubing into the urinary bladder wall. Bundles of detrusor muscle are dissected from the urothelium (A). A 4–5 mm length of tubing is inserted into the bladder wall (B). Tubing is secured in place by two 10-0 nylon sutures (C). Significant (p ≤ 0.001) increase in total urinary bladder nerve growth factor (NGF; D) as detected with ELISA after two-week exogenous delivery of NGF to bladder wall. *, p ≤ 0.001. 'n' = 6 for each group in D.
Figure 2
Figure 2
Effects of NGF on cystometry variables. Summary bar graphs depict the significant (*, p ≤ 0.01) increase in bladder weight (A), decrease in bladder capacity (B), and decrease in intercontraction interval (C) in NGF-treated rats. 'n' = 6 for each group.
Figure 3
Figure 3
Cystometrogram recordings. Exogenous delivery of NGF (2.5 μg/μl) decreased bladder capacity (increased voiding frequency). Continuous cystometrogram recordings in saline (A) and NGF-treated rats (B). Arrows point to some non-voiding bladder contractions. The x-axis represents the time (minutes, min) and the y-axis represents the intravesical pressure (cm H2O). The amount of saline voided (ml) is also illustrated.
Figure 4
Figure 4
Effects of NGF on cystometry variables. Summary bar graphs depict the significant (*, p ≤ 0.05) increase in peak micturition pressure (B) and significant (*, p ≤ 0.01) increase in amplitude of non-voiding contractions (NVCs)(C) in NGF-treated rats. No changes in filling pressure (FP) or threshold pressure (TP) were observed (B). 'n' = 6 for each group.
Figure 5
Figure 5
Fos induction in NGF-treated rats. Brightfield photographs from sections (40 μm) of the L6 spinal cord showing the distribution of Fos-IR cells after intravesical saline distention (2 hr) in: saline treated rats (A), NGF-treated rats (B), or cyclophosphamide (CYP) treated rats (C). MDH, medial dorsal horn; LDH, lateral dorsal horn; DCM, dorsal commissure; CC, central canal; SPN, sacral parasympathetic nucleus. Calibration bar represents 100 μm.
Figure 6
Figure 6
Magnitude and Distribution of Fos in NGF-treated rats. Histogram (A) showing the segmental distribution of Fos-immunoreactive (IR) cells/section (s) in the rat spinal cord (L5-S1) after intravesical saline distention in saline or NGF-treated rats. *, p ≤ 0.005. Histogram (B) showing the distribution of Fos-IR cells in four regions of the L6 spinal cord after intravesical saline distention in saline or NGF-treated rats. Values represent the percentage of the total population of Fos-IR cells induced in each experimental paradigm. The four regions analyzed include: SPN, sacral parasympathetic nucleus; DCM, dorsal commissure; MDH, medial dorsal horn; LDH, lateral dorsal horn. #, p ≤ 0.01; *, p ≤ 0.005. 'n' = 6 for the saline and NGF groups.
Figure 7
Figure 7
CGRP Spinal Cord Expression in NGF-treated rats. CGRP-IR increases in lumbosacral spinal cord with exogenous NGF treatment. CGRP-IR in the L6 (A-B) and S1 (D-E) spinal segment in control (A, D) and NGF-treated (B, E) rats. A, D. Fluorescence photographs showing CGRP-IR in the L6 (A) and S1 (D) spinal segment of control (saline) + bladder distention. B, E. Fluorescence photographs showing CGRP-IR in the L6 (B) and S1 (E) spinal segment with NGF treatment + bladder distention. Increased density of CGRP-IR was observed in the medial (MDH) to lateral (LDH) extent of the superficial laminae (I-II) of the dorsal horn (DH) with NGF treatment in L6 (C) and S1 (F) segments. Changes in CGRP-IR in other spinal cord regions were more dramatic in the S1 spinal segment. Increased CGRP-IR was present in a fiber bundle extending from Lissauer's tract in lamina I along the lateral edge of the DH to the region of the sacral parasympathetic nucleus (SPN) (lateral collateral pathway of Lissauer, LCP) in the S1 segment (F). Although this fiber bundle was present in control tissue sections, the staining was less intense (D) and was less frequently observed in transverse sections compared to NGF treatment (E). Faint CGRP-IR was present in the region of the SPN in control sections (A, D). With NGF treatment, CGRP-IR in the SPN region also increased in the S1 segment. Increased CGRP-IR was also present in the dorsal commissure (DCM) with NGF treatment (D, E, F). Summary bar graphs of CGRP-IR optical density (O.D.) as measured in specific regions of the L6-S1 spinal cord (C, F). Calibration bar represents 125 μm. *, p ≤ 0.01.

References

    1. Clemow DB, Spitsbergen JM, McCarty R, Steers WD, Tuttle JB. Altered NGF regulation may link a genetic predisposition for hypertension with hyperactive voiding. J Urol. 1999;161:1372–1377. doi: 10.1016/S0022-5347(01)61686-0.
    1. Steers WD, Kolbeck S, Creedon D, Tuttle JB. Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest. 1991;88:1709–1715.
    1. Zvara P, Kliment J, Jr., DeRoss AL, Irwin BH, Malley SE, Plante MK, Vizzard MA. Differential expression of bladder neurotrophic factor mRNA in male and female rats after bladder outflow obstruction. J Urol. 2002;168:2682–2688. doi: 10.1016/S0022-5347(05)64244-9.
    1. Steers WD, Tuttle JB. Mechanisms of Disease: the role of nerve growth factor in the pathophysiology of bladder disorders. Nat Clin Pract Urol. 2006;3:101–110. doi: 10.1038/ncpuro0408.
    1. Yoshimura N, Bennett NE, Hayashi Y, Ogawa T, Nishizawa O, Chancellor MB, de Groat WC, Seki S. Bladder overactivity and hyperexcitability of bladder afferent neurons after intrathecal delivery of nerve growth factor in rats. J Neurosci. 2006;26:10847–10855. doi: 10.1523/JNEUROSCI.3023-06.2006.
    1. Dmitrieva N, Shelton D, Rice AS, McMahon SB. The role of nerve growth factor in a model of visceral inflammation. Neuroscience. 1997;78:449–459. doi: 10.1016/S0306-4522(96)00575-1.
    1. Lamb K, Gebhart GF, Bielefeldt K. Increased nerve growth factor expression triggers bladder overactivity. J Pain. 2004;5:150–156. doi: 10.1016/j.jpain.2004.01.001.
    1. Yoshimura N, Seki S, Chancellor MB, de Groat WC, Ueda T. Targeting afferent hyperexcitability for therapy of the painful bladder syndrome. Urology. 2002;59:61–67. doi: 10.1016/S0090-4295(01)01639-9.
    1. Hu VY, Zvara P, Dattilio A, Redman TL, Allen SJ, Dawbarn D, Stroemer RP, Vizzard MA. Decrease in bladder overactivity with REN1820 in rats with cyclophosphamide induced cystitis. J Urol. 2005;173:1016–1021. doi: 10.1097/01.ju.0000155170.15023.e5.
    1. Okragly AJ, Niles AL, Saban R, Schmidt D, Hoffman RL, Warner TF, Moon TD, Uehling DT, Haak-Frendscho M. 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. 1999;161:438–41; discussion 441-2. doi: 10.1016/S0022-5347(01)61915-3.
    1. Lowe EM, Anand P, Terenghi G, Williams-Chestnut RE, Sinicropi DV, Osborne JL. Increased nerve growth factor levels in the urinary bladder of women with idiopathic sensory urgency and interstitial cystitis. Br J Urol. 1997;79:572–577.
    1. Birder LA, Wolf-Johnston A, Griffiths D, Resnick NM. Role of urothelial nerve growth factor in human bladder function. Neurourol Urodyn. 2007
    1. Vizzard MA. Up-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways after chronic cystitis. J Comp Neurol. 2000;420:335–348. doi: 10.1002/(SICI)1096-9861(20000508)420:3<335::AID-CNE5>;2-#.
    1. Vizzard MA. Alterations in neuropeptide expression in lumbosacral bladder pathways following chronic cystitis. J Chem Neuroanat. 2001;21:125–138. doi: 10.1016/S0891-0618(00)00115-0.
    1. Vizzard MA. Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am J Physiol Regul Integr Comp Physiol. 2000;278:R1027–39.
    1. Vizzard MA. Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction. Exp Neurol. 2000;161:273–284. doi: 10.1006/exnr.1999.7254.
    1. LaBerge J, Malley SE, Zvarova K, Vizzard MA. Expression of corticotropin-releasing factor and CRF receptors in micturition pathways after cyclophosphamide-induced cystitis. Am J Physiol Regul Integr Comp Physiol. 2006;291:R692–703.
    1. Yuridullah R, Corrow KA, Malley SE, Vizzard MA. Expression of fractalkine and fractalkine receptor in urinary bladder after cyclophosphamide (CYP)-induced cystitis. Auton Neurosci. 2006;126-127:380–389. doi: 10.1016/j.autneu.2006.02.030.
    1. Birder LA, de Groat WC. Induction of c-fos expression in spinal neurons by nociceptive and nonnociceptive stimulation of LUT. Am J Physiol. 1993;265:R326–33.
    1. Keast JR, de Groat WC. Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J Comp Neurol. 1992;319:615–623. doi: 10.1002/cne.903190411.
    1. Driscoll A, Teichman JM. How do patients with interstitial cystitis present? J Urol. 2001;166:2118–2120. doi: 10.1016/S0022-5347(05)65517-6.
    1. Sant GR, Hanno PM. Interstitial cystitis: current issues and controversies in diagnosis. Urology. 2001;57:82–88. doi: 10.1016/S0090-4295(01)01131-1.
    1. Dray A. Inflammatory mediators of pain. Br J Anaesth. 1995;75:125–131.
    1. Pang X, Marchand J, Sant GR, Kream RM, Theoharides TC. Increased number of substance P positive nerve fibres in interstitial cystitis. Br J Urol. 1995;75:744–750.
    1. Marchand JE, Sant GR, Kream RM. Increased expression of substance P receptor-encoding mRNA in bladder biopsies from patients with interstitial cystitis. Br J Urol. 1998;81:224–228.
    1. Callsen-Cencic P, Mense S. Expression of neuropeptides and nitric oxide synthase in neurones innervating the inflamed rat urinary bladder. J Auton Nerv Syst. 1997;65:33–44. doi: 10.1016/S0165-1838(97)00032-5.
    1. Maggi CA. The role of neuropeptides in the regulation of the micturition reflex: An update. Gen Pharmacol. 1991;22:1–24.
    1. Ahluwalia A, Maggi CA, Santicioli P, Lecci A, Giuliani S. Characterization of the capsaicin-sensitive component of cyclophosphamide-induced inflammation in the rat urinary bladder. Br J Pharmacol. 1994;111:1017–1022.
    1. Ahluwalia A, Giuliani S, Scotland R, Maggi CA. Ovalbumin-induced neurogenic inflammation in the bladder of sensitized rats. Br J Pharmacol. 1998;124:190–196. doi: 10.1038/sj.bjp.0701793.
    1. Rapp DE, Turk KW, Bales GT, Cook SP. Botulinum toxin type a inhibits calcitonin gene-related peptide release from isolated rat bladder. J Urol. 2006;175:1138–1142. doi: 10.1016/S0022-5347(05)00322-8.
    1. Chuang YC, Yoshimura N, Huang CC, Chiang PH, Chancellor MB. Intravesical botulinum toxin a administration produces analgesia against acetic acid induced bladder pain responses in rats. J Urol. 2004;172:1529–1532. doi: 10.1097/01.ju.0000137844.77524.97.
    1. Lecci A, Giulani S, Santiciolo P, Maggi CA. Involvement of spinal tachykinin NK1 and NK2 receptors in detrusor hyperreflexia during chemical cystitis in anaesthetized rats. Eur J Pharmacol. 1994;259:129–135. doi: 10.1016/0014-2999(94)90501-0.

Source: PubMed

Sponsorit ja yhteistyökumppanit

Sairaudet

Huumeiden interventiot

3
Tilaa