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

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.

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

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

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

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

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

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

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.