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Immunoneutralization of Nerve Growth Factor in Lumbosacral Spinal Cord Reduces Bladder Hyperreflexia in Spinal Cord Injured Rats.
0022-5347/02/1684-2269/0 THE JOURNAL OF UROLOGY® Copyright © 2002 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 168, 2269 –2274, November 2002 Printed in U.S.A.
DOI: 10.1097/01.ju.0000025338.65642.09
IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR IN THE LUMBOSACRAL SPINAL CORD REDUCES BLADDER HYPERREFLEXIA IN SPINAL CORD INJURED RATS SATOSHI SEKI, KATSUMI SASAKI, MATTHEW O. FRASER,* YASUHIKO IGAWA, OSAMU NISHIZAWA, MICHAEL B. CHANCELLOR, WILLIAM C. DE GROAT AND NAOKI YOSHIMURA From the Departments of Urology and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, and Department of Urology, Shinshu University, Matsumoto, Japan
ABSTRACT
Purpose: We investigated the effects of intrathecal application of nerve growth factor (NGF) antibodies (Ab) on bladder hyperreflexia in chronic spinalized rats. Materials and Methods: In adult female rats an intrathecal catheter was implanted at the level of the L6 to S1 spinal cord, followed by complete transection of the Th8 to 9 spinal cord. At 10 days after spinalization the intrathecal catheter was connected to an osmotic pump for continuous delivery of vehicle or NGF Ab (10 g. daily) for 2 weeks. Awake cystometry was then performed. NGF levels in the L5 to S1 dorsal root ganglia, L6 spinal cord and bladder were also measured using enzyme-linked immunosorbent assay. Results: The number of uninhibited bladder contractions per voiding cycle, maximal pressure of uninhibited bladder contraction and maximal voiding pressure were significantly decreased in NGF Ab treated versus vehicle treated spinalized rats. Intercontraction interval, baseline intravesical pressure, pressure threshold for voiding and voiding efficiency were not significantly changed by NGF Ab treatment. NGF levels in the bladder, L6 spinal cord and L5 to S1 dorsal root ganglia of vehicle treated spinalized rats was 1.6 to 4.8 times higher than in spinal cord intact rats. After intrathecal NGF Ab treatment NGF levels were significantly lower in the L6 to S1 dorsal root ganglia (30% to 35%) and L6 spinal cord (53%) but not in the bladder or L5 dorsal root ganglia compared with levels in vehicle treated spinalized rats. Conclusions: Increased levels of NGF in the bladder, spinal cord and dorsal root ganglia were associated with bladder hyperreflexia after spinal cord injury. Immuno-neutralization of NGF in the spinal cord suppressed NGF levels in the L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, and also suppressed bladder hyperreflexia. Thus, suppression of NGF levels in afferent pathways could be useful for treating bladder hyperreflexia associated with spinal cord injury. KEY WORDS: bladder; spinal cord injuries; rats, Sprague-Dawley; nerve growth factor; ganglia, spinal
Spinal cord injury rostral to the lumbosacral level impairs voluntary and supraspinal control of voiding, leading to reorganization of the neural pathways regulating bladder and urethral sphincter function. Spinal cord injury initially induces an areflexic bladder and urinary retention, followed by the emergence of automatic voiding mediated by spinal reflex mechanisms.1, 2 The bladder becomes hyperreflexic and bladder sphincter coordination is impaired, leading to detrusorsphincter dyssynergia. These lower urinary tract dysfunctions then produce various problems, such as urinary incontinence, recurrent urinary tract infection and vesicoureteral reflux with or without upper urinary tract deterioration.1 Clinical and experimental studies indicate that bladder hyperreflexia after spinal cord injury is induced at least in part by phenotypic changes in unmyelinated C-fiber bladder afferent pathways.1, 2 Multiple mechanisms may be involved in the plasticity of bladder afferent neurons in pathological conditions. For example, it has been suggested that target
organ-neural interactions in bladder afferent pathways after spinal cord injury may be mediated by an increase in neurotrophic factors produced in target organ tissues and transported retrograde back to the neuronal cell bodies. It is known that trophic factors such as nerve growth factor (NGF) increase in hypertrophied bladders in rats with spinal cord injury3 or partial urethral ligation.4 It has also been shown that the hypertrophy in afferent and efferent neurons innervating the hypertrophic bladder in rats with partial urethral obstruction was antagonized in part by systemic autoimmunization against NGF.4 In addition, recent studies revealed that acute or chronic local administration of NGF can sensitize bladder afferent pathways and induce bladder hyperactivity.5, 6 Overall these data suggest that NGF may be involved in inducing the neuroplasticity that underlies the emergence of bladder hyperreflexia after spinal cord injury. However, many aspects of this hypothesis remain unexplored. For example, it is not known whether NGF levels in bladder afferent pathways are actually increased after spinal cord injury, nor is it certain whether a peripheral or central nervous system source of NGF is most important for the development of bladder hyperreflexia. It has been reported that NGF protein and NGF receptor mRNA increase
Accepted for publication May 17, 2002. Supported by Grants DK 49430, DK 57267 and P01 HD39768 from the National Institutes of Health and Grant 1861– 01/02 from Spinal Cord Research Foundation, PVA. * Financial interest and/or other relationship with Pfizer UK, Roche, Aventis and Cook Myocyte. 2269
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throughout the spinal cord after spinal cord injury in rats, while little NGF is found in the spinal cord under normal conditions.7, 8 A recent study also demonstrated that intrathecal administration of NGF Ab in spinalized rats suppressed the hypertensive response (autonomic dysreflexia) induced by colon distention, which is mediated by C-fiber afferents.9 Thus, it seems reasonable to assume that NGF produced not only by the bladder, but also by the injured spinal cord could be transported to bladder afferent pathways to induce bladder hyperreflexia. We tested this hypothesis by administrating NGF Ab intrathecally in spinal cord injured rats. We determined if immuno-neutralization of NGF in the spinal cord can reduce NGF levels in L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, and if this treatment can suppress bladder hyperreflexia after spinal cord injury. MATERIALS AND METHODS
Surgical preparation. Experiments were performed in adult female Sprague-Dawley rats weighing 182 to 255 gm. Care and handling of animals were done in accordance with institutional guidelines and approved by the University of Pittsburgh Institutional Animal Care and Use Committee. An PE-10 intrathecal catheter (Clay-Adams, Parsippany, New Jersey) was implanted at the level of the L6 to S1 spinal cord after laminectomy at the Th11 vertebra with the rat under halothane anesthesia. The end of the catheter was heat sealed and placed subcutaneously. At 3 to 4 days after intrathecal catheter implantation spinal cord transection was performed between Th8 to Th9 with the rat under halothane anesthesia. After T8 laminectomy the dura and spinal cord were cut with scissors and a sterile Gelform sponge (The Upjohn Co., Kalamazoo, Michigan) was placed between the cut ends of the spinal cord. The overlying muscle and skin were sutured. Postoperatively the animals were treated with antibiotics (ampicillin, 150 mg./kg. intramuscularly) for 7 days. The bladder of spinalized rats was manually emptied twice daily until reflex voiding recovered, usually 10 to 14 days after spinalization. At 10 days after spinalization the intrathecal catheter was connected to an Model 2002 osmotic mini pump (Alza Co., Palo Alto, California) placed subcutaneously in the back for continuous delivery of vehicle (artificial cerebrospinal fluid) in 8 rats or NGF Ab (200 l. 700 g/ml., 10 g. daily) in 11 for 14 days. Monoclonal NGF Ab (clone 911) (Genentech, South San Francisco, California) was used. It has been demonstrated that this antibody specifically binds to NGF but not to other neurotrophic factors, such as brain derived neurotrophic factor or neurotrophin-3.10 Cystometry. At 14 days after intrathecal injection of vehicle or NGF Ab cystometry in conscious rats was performed according to previously published methods. With the rat under halothane anesthesia a PE-50 catheter was inserted via a midline abdominal incision into the bladder through the bladder dome. After surgery the rat was placed in a restraining cage and allowed to recover from anesthesia for 1 to 2 hours. The intravesical catheter was connected via a 3-way stopcock to a pressure transducer and a syringe pump for recording intravesical bladder pressure and infusing saline into the bladder, respectively. Saline at room temperature was infused at a rate of 0.04 ml. per minute to elicit repetitive bladder contractions. Saline infusion was continued at least 3 hours before measuring voiding parameters. Saline voided from the urethral meatus was collected and measured to determine voided volume (VV). After constant voided volume measurements were collected the infusion was stopped and post-void residual volume (RV) was measured. Residual saline was withdrawn through the intravesical catheter by gravity and then the bladder was completely emptied by manual compression through the abdominal wall.
FIG. 1. Representative cystometrogram traces in awake spinalized (SCI) rats. A, vehicle treated. Arrows indicate voiding contractions. B, NGF Ab treated.
Bladder capacity (BC) was calculated using the formula, BC ⫽ VV ⫹ RV. Based on these values voiding efficiency (VE) expressed as a percent could be estimated by the formula, VE ⫽ [(VV/BC) ⫻ 100]. Intercontraction interval, maximal voiding pressure, pressure threshold for voiding and baseline intravesical pressure were also measured. The number of uninhibited bladder contractions per voiding cycle and maximal uninhibited bladder contraction pressure were measured. Uninhibited bladder contractions were defined as rhythmic intravesical pressure increases greater than 7 cm. water from baseline pressure without a release of fluid from the urethra. Enzyme-linked immunosorbent assay (ELISA). After cystometry the bladder, L5 to 6 and S1 dorsal root ganglia, and L6 spinal cord were removed from vehicle or NGF Ab treated spinalized rats under urethane anesthesia (1.2 mg./kg. intraperitoneally). Bladder, dorsal root ganglia and spinal cord tissues were also obtained from 8 untreated spinal cord intact rats. In the L5, L6 and S1 dorsal root ganglia ELISA measurements was performed in each animal using the 2 sides of the dorsal root ganglia tissues. The tissues were rapidly frozen on dry ice and stored at – 80C until NGF extraction. The samples were homogenized in buffer (2.66% Trisma HCl, 0.985% Trisma Base, 0.5 mM. phenylmethylsulfonyl fluoride, 1M. leupeptin, 1 M. pepstatin A and 0.3 M. aprotinin in 400 and 100 l. for the bladder and dorsal root ganglia, respectively). The homogenate was centrifuged at 10,000 ⫻ gravity for 4 minutes. The supernatant was diluted with 4 volumes Dulbecco’s phosphate buffered saline (0.02% KCl, 0.8% NaCl, 0.02% KH2PO4, 0.115% Na2HPO4, 0.0133% CaCl2䡠2H2O and 0.01% MgCl2䡠6H2O, pH 7.35). Samples were stored in a deep freezer at – 80C until assayed. The samples were assayed in triplicate in an antigen capture ELISA Emax ImmunoAssay System (Promega, Madison, Wisconsin) according to manufacturer instructions. ELISA plates were read at 450 nm. on an Elx800 microplate reader (Bio-Tek Instruments, Winooski, Vermont). Total protein concentration in the same samples was also determined with a BCA Protein Assay Kit (Pierce, Rockford, Illinois). All tissue NGF values were standardized by tissue protein levels and are expressed in pg./g. total protein. Evaluation and statistical analysis. All values are expressed as the mean plus or minus SE. The Mann-Whitney U test was used to determine significance. For all statistical tests p ⬍0.05 was considered significant. RESULTS
Cystometry. During awake cystometry all 7 spinalized rats that received vehicle treatment showed uninhibited bladder
IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR REDUCES BLADDER HYPERREFLEXIA
contraction before large amplitude voiding bladder contractions occurred (fig. 1). Uninhibited bladder contraction amplitude increased as the bladder was filled, reaching a mean maximum of 39.6 ⫾ 5.49 cm. water. There was an average of 6.7 ⫾ 0.81 uninhibited bladder contractions per voiding cycle (fig. 2). Of the 10 spinalized rats that received intrathecal NGF Ab 8 (80%) showed uninhibited bladder contractions before voiding bladder contractions. However, in 10 NGF Ab treated animals the mean number of uninhibited bladder contraction per voiding (2.5 ⫾ 0.61 contractions, p ⬍0.01) and mean maximal uninhibited bladder contraction pressure (26.8 ⫾ 2.82 cm. water, p ⬍0.05) were significantly less than in vehicle treated spinalized animals (figs. 1 and 2). Mean maximal voiding pressure was also less in NGF Ab treated than in vehicle treated rats (42.7 ⫾ 3.20 versus 57.1 ⫾ 6.30 cm. water, p ⬍0.05). However, NGF Ab treatment did not affect other cystometric parameters, such as intercontraction interval, baseline intravesical pressure, pressure threshold for voiding efficiency or bladder capacity (see table). NGF levels in the bladder, L6 spinal cord and dorsal root ganglia. In vehicle treated spinalized rats NGF levels in the bladder, L6 spinal cord and dorsal root ganglia (L5 to 6 and S1) were significantly higher than in spinal cord intact rats (figs. 3 and 4). In 8 spinalized rats injected with NGF Ab at the L6 to S1 intrathecal space for 2 weeks mean NGF levels in the L6 and S1 dorsal root ganglia were significantly lower than in 8 vehicle treated spinalized rats (2.53 ⫾ 0.20 and 3.05 ⫾ 0.30 versus 3.87 ⫾ 0.48 and 4.41 ⫾ 0.48 pg./g. protein, respectively). Mean NGF levels in the L6 spinal cord of 8 NGF Ab treated spinalized animals were also significantly lower than in 8 vehicle treated spinalized rats (0.49 ⫾ 0.08 versus 1.06 ⫾ 0.21 pg./g. protein). However, NGF levels in the L6 spinal cord and L6 to S1 dorsal root ganglia were still significantly higher in NGF Ab treated than in spinal cord intact rats (figs. 3 and 4). In 8 vehicle treated and 8 NGF Ab treated spinalized rats
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mean NGF levels in the bladder (4.16 ⫾ 0.56 versus 4.34 ⫾ 0.18 pg./g. protein) and L5 dorsal root ganglia (3.01 ⫾ 0.40 versus 2.40 ⫾ 0.20 pg./g. protein, respectively) were not significantly different. Mean bladder weight in 11 vehicle treated and 11 NGF Ab treated spinalized rats (425 ⫾ 58 versus 420 ⫾ 43 mg., respectively) was not different but each value was significantly (4 to 5 times) greater than mean bladder weight in 6 spinal cord intact rats (80.6 ⫾ 12 mg., see table). DISCUSSION
The results of the current study indicate that NGF levels in the bladder, L6 spinal cord and dorsal root ganglia were increased in chronic spinalized rats with bladder hyperreflexia and immuno-neutralization of NGF in the spinal cord (which in turn decreased NGF in the L6 spinal cord and L6 to S1 dorsal root ganglia, where bladder afferent fibers originate) reduced uninhibited bladder contractions and maximal voiding pressure in chronic spinalized rats. Since intrathecal NGF Ab treatment decreased NGF levels in the spinal cord but not in the bladder, it is likely that NGF Ab administered intrathecally localized and reduced NGF levels in the spinal cord without effects at the periphery. It has been proposed that elevated expression of neurotrophic factors such as NGF in the hypertrophied bladder after spinal cord injury is induced by increased bladder work due to uncoordinated bladder and urethral sphincter activity (detrusor-sphincter dyssynergia), which increases urethral resistance.3, 11 Similar mechanisms are also suggested to be involved in another rat model of bladder hypertrophy induced by partial urethral obstruction that involves bladder hyperactivity and increased NGF levels in the bladder.4 Autoimmunization against NGF antagonized at least in part bladder hyperactivity as well as afferent neuron hypertrophy in rats with partial urethral obstruction.4 Thus, target organ-
FIG. 2. Spinalized vehicle and NGF Ab treated rats. A, number of uninhibited bladder contractions (UIC) per voiding cycle. Double asterisks indicate p ⬍0.01. B, uninhibited bladder contraction amplitude. Single asterisk indicates p ⬍0.05. C, maximal voiding pressure.
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IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR REDUCES BLADDER HYPERREFLEXIA Spinalized Vehicle Treated
Intercontraction interval (secs.) Bladder capacity (ml.) Baseline intravesical pressure (cm. H2O) Pressure threshold for voiding (cm. H2O) % Voiding efficiency Bladder wt. (mg.)
Mean ⫾ SE 628 ⫾ 88 0.67 ⫾ 0.09 9.8 ⫾ 2.6 11.3 ⫾ 2.4 0.90 ⫾ 0.05 424 ⫾ 58
neural interactions mediated by increased NGF in the hypertrophied bladder and increased transport of NGF to neuronal cell bodies in the afferent pathways may contribute to the emergence of bladder hyperactivity in cases of spinal cord injury or partial urethral obstruction.4, 11 The current study seems to be consistent with this hypothesis because NGF levels in the lumbosacral dorsal root ganglia were in fact increased as bladder NGF expression was elevated after spinal cord injury. However, our study also shows that the NGF level in the L6 spinal cord was increased in spinalized rats and immuno-neutralization of NGF at the L6 and S1 spinal cord segment levels significantly suppressed increased NGF levels in the L6 to S1 dorsal root ganglia as well as in the L6 spinal cord without affecting bladder NGF levels. Thus, it is assumed that increased NGF levels in the dorsal root ganglia were at least in part attributable to NGF production in the spinal cord after spinal cord injury. This finding is also supported by earlier studies showing that NGF proteins7 and NGF receptor mRNA8 were increased throughout the spinal cord almost immediately after spinal cord injury in rats. In addition, in our experiments NGF levels were increased not only in the L6 to S1
Spinalized NGF Ab Treated
No. Rats
Mean ⫾ SE
No. Rats
7 7 7 7 7 11
761 ⫾ 187 0.82 ⫾ 0.12 7.5 ⫾ 1.7 10.2 ⫾ 1.8 0.88 ⫾ 0.03 419 ⫾ 43
10 10 10 10 10 10
dorsal root ganglia, but also in the L5 dorsal root ganglia, which do not contain bladder afferent neurons.12 Therefore, NGF in the L5 dorsal root ganglia cannot be attributable to changes in bladder NGF expression. Overall it seems likely that NGF produced in the spinal cord has an important role in inducing bladder hyperreflexia after spinal cord transection, although retrograde transport of NGF from the bladder can also contribute to the NGF increase in the dorsal root ganglia after spinal cord injury since NGF levels in the dorsal root ganglia after intrathecal NGF Ab treatment were still significantly higher in spinalized than in spinal cord intact rats. Because immuno-neutralization of NGF in the spinal cord reduced NGF levels in the L6 to S1 dorsal root ganglia, bladder hyperreflexia in spinalized rats could be suppressed by modulating neural activity in the spinal cord or in bladder afferent pathways. Various indirect evidence suggests that a change in bladder afferent activity may be important for the effect of NGF Ab treatment. It is well known that 2 types of afferent fibers, namely A␦ and C-fibers, carry sensory information from bladder to spinal cord.12, 13 Previous studies in cats and rats showed that A␦-fiber bladder afferents trigger
FIG. 3. ELISA measurements of NGF in normal spinal cord intact, vehicle treated spinalized and NGF Ab treated spinalized rats. NGF values are standardized to tissue protein and expressed in pg./g. total protein. A, bladder. Single asterisk indicates p ⬍0.05. Double asterisks indicate p ⬍0.01. B, L5 dorsal root ganglia (DRG). C, L6 dorsal root ganglia. D, S1 dorsal root ganglia.
IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR REDUCES BLADDER HYPERREFLEXIA
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2, 17
FIG. 4. ELISA measurements of NGF in L6 spinal cord in normal spinal cord intact, vehicle treated spinalized and NGF Ab treated spinalized rats. NGF values are standardized to tissue protein and expressed in pg./g. total protein. Single asterisk indicates p ⬍0.05. Double asterisks indicate p⬍0.01.
the normal micturition reflex via a long latency supraspinal pathway passing through the pons.1, 13 In chronic spinal injured rats A␦ bladder afferents trigger the voiding reflex but C-fiber afferents appear to initiate bladder hyperactivity during filling. Desensitizing C-fiber afferents by systemic capsaicin administration suppressed the uninhibited bladder contractions that occurred before voiding.14 This effect is similar to that in the current experiments after NGF-Ab experiments. In addition, because chronic administration of exogenous NGF increases the excitability of C-fiber bladder afferents and induces bladder hyperactivity in spinal cord intact rats,15 it seems reasonable to propose that NGF Ab treatment in the spinal cord could reduce uninhibited bladder contractions by eliminating NGF induced sensitization of C-fiber afferents in chronic spinalized rats. The current study also shows that NGF Ab treatment reduced maximal voiding pressure. Since a previous study indicated that hyperexcitability of C-fiber bladder afferents is also involved in dyssynergic urethral sphincter contractions during voiding after spinal cord injury,16 the reduction in maximal voiding pressure may also be elicited by a reduction in detrusor-sphincter dyssynergia as a result of suppressing C-fiber activity after NGF Ab treatment. Further studies are necessary to evaluate the effect of NGF Ab treatment on urethral function. NGF modulation of C-fiber afferents may also be important for the initiation of bladder hyperactivity in other species after spinal cord injury. Electrophysiological recording in spinalized cats revealed that the afferent limb of the spinal micturition reflex consists of unmyelinated C-fiber afferents.1 This finding was confirmed in pharmacological studies of the C-fiber neurotoxin capsaicin. In chronic spinal cord injured cats 3 to 6 weeks after spinal cord injury subcutaneously administered capsaicin (20 to 30 mg./kg.) completely blocked bladder distention induced by bladder contractions, whereas capsaicin had only a facilitatory effect on reflex bladder contractions in spinal cord intact cats.1 Clinical studies also showed that intravesical instillation of capsaicin suppressed bladder hyperreflexia in patients with spinal cord injury or multiple sclerosis. When administered intravesically, capsaicin increased bladder capacity and reduced the number of incontinence episodes in those
patients. Resiniferatoxin, an agent with similar C-fiber desensitizing properties, has been recently reported to have similar effects for neurogenic bladder hyperreflexia.18 Together it is clear that the functional properties of C-fiber afferents in the bladder are altered after spinal cord injury, thereby, inducing hyperreflexic bladder activity in humans as well as animals. The contribution of NGF to visceral C-fiber afferent hyperexcitability is also further supported by findings in another laboratory that intrathecal application of NGF Ab in spinalized rats reduced autonomic dysreflexia (that is reflex hypertension) induced by colon distention.9 Autonomic dysreflexia induced by bladder or colon distention is another example of a reorganization of C-fiber mediated reflex pathways after spinal cord injury because capsaicin treatment suppresses autonomic dysreflexia in humans with spinal cord injury19 and similar arterial pressure responses induced by bladder distention in rats.20 Thus, it is plausible that an interaction between the spinal cord and bladder afferent pathways, leading to the reorganization of bladder reflex activities after spinal cord injury, could be mediated at least in part by increased expression of NGF in the spinal cord, which is then transported into bladder afferent pathways. CONCLUSIONS
This study provides direct evidence that increased levels of NGF in the bladder, L6 spinal cord and L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, are involved in bladder hyperreflexia induced by C-fiber bladder afferents. It is also suggested that NGF protein produced in the injured spinal cord could be an important source of increased levels of NGF in the afferent pathways after spinal cord injury. Therefore, manipulating NGF levels in bladder afferent pathways could be an effective modality for treating bladder hyperreflexia in spinal cord injury cases. REFERENCES
1. de Groat, W. C.: Mechanisms underlying the recovery of lower urinary tract function following spinal cord injury. Paraplegia, 33: 493, 1995 2. Fowler, C. J., Beck, R. O., Gerrard, S., Betts, C. D. and Fowler, C. G.: Intravesical capsaicin for treatment of detrusor hyperreflexia. J Neurol Neurosurg Psychiatry, 57: 169, 1994 3. Vizzard, M. A.: Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction. Exp Neurol, 161: 273, 2000 4. Steers, W. D., Creedon, D. J. and Tuttle, J. B.: Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. J Urol, 155: 379, 1996 5. Dmitrieva, N. and McMahon, S. B.: Sensitisation of visceral afferents by nerve growth factor in the adult rat. Pain, 66: 87, 1996 6. Chuang, Y. C., Fraser, M. O., Yu, Y., Chancellor, M. B., de Groat, W. C. and Yoshimura, L.: The role of bladder afferent pathways in bladder hyperactivity induced by the intravesical administration of nerve growth factor. J Urol, 165: 975, 2001 7. Bakhit, C., Armanini, M., Wong, W. L., Bennett, G. L. and Wrathall, J. R.: Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase following contusive spinal cord injury. Brain Res, 554: 264, 1991 8. Brunello, N., Reynolds, M., Wrathall, J. R. and Mocchetti, I.: Increased nerve growth factor receptor mRNA in contused rat spinal cord. Neurosci Lett, 118: 238, 1990 9. Krenz, N. R., Meakin, S. O., Krassioukov, A. V. and Weaver, L. C.: Neutralizing intraspinal nerve growth factor blocks autonomic dysreflexia caused by spinal cord injury. J Neurosci, 19: 7045, 1999 10. Hongo, J. S., Laramee, G. R., Urfer, R., Shelton, D. L., Restivo, T, Sadick, M. et al: Antibody binding regions on human nerve growth factor identified by homolog-and alanine-scanning mutagenesis. Hybridoma, 19: 215, 2000 11. Yoshimura, N.: Bladder afferent pathway and spinal cord injury: possible mechanisms inducing hyperreflexia of the urinary
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bladder. Prog Neurobiol, 57: 583, 1999 12. Keast, J. R. and de Groat, W. C.: Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J Comp Neurol, 319: 615, 1992 13. Mallory, B., Steers, W. D. and de Groat, W. C.: Electrophysiological study of micturition reflexes in rats. Am J Physiol, 257: R410, 1989 14. Cheng, C. L., Ma, C. P. and de Groat, W. C.: Effect of capsaicin on micturition and associated reflexes in chronic spinal rats. Brain Res, 678: 40, 1995 15. Yoshimura, N., Bennet, N. E., Phelan, M. W. and de Groat, W. C.: Effects of chronic nerve growth factor treatment on rat urinary bladder and bladder afferent neurons. Soc Neurosci Abstr, 25: 1946, 1999 16. Cheng, C. L, Chai, C. Y. and de Groat, W. C.: Detrusor-sphincter
17.
18. 19. 20.
dyssynergia induced by cold stimulation of the urinary bladder of rats. Am J Physiol, 272: R1271, 1997 Cruz, F., Guimara˜ es, M., Silva, C., Rio, M. E., Gimbra, A. and Reis, M.: Desensitization of bladder sensory fibers by intravesical capsaicin has long lasting clinical and urodynamic effects in patients with hyperactive or hypersensitive bladder dysfunction. J Urol, 157: 585, 1997 Cruz, F., Guimaraes, M., Silva, C. and Reis, M.: Suppression of bladder hyperreflexia by intravesical resiniferatoxin. Lancet, 350: 640, 1997 Igawa, Y., Komiyama, I., Nishizawa, S. et al: Intravesical capsaicin inhibits autonomic dysreflexia in patients with spinal cord injury. Neurourol Urodyn, 15: 374, 1996 Cheng, C. L., Ma, C. P. and de Groat, W. C.: Effects of capsaicin on micturition and associated reflexes in rat. Am J Physiol, 265: R132, 1993
Vol. 168, 2269 –2274, November 2002 Printed in U.S.A.
DOI: 10.1097/01.ju.0000025338.65642.09
IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR IN THE LUMBOSACRAL SPINAL CORD REDUCES BLADDER HYPERREFLEXIA IN SPINAL CORD INJURED RATS SATOSHI SEKI, KATSUMI SASAKI, MATTHEW O. FRASER,* YASUHIKO IGAWA, OSAMU NISHIZAWA, MICHAEL B. CHANCELLOR, WILLIAM C. DE GROAT AND NAOKI YOSHIMURA From the Departments of Urology and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, and Department of Urology, Shinshu University, Matsumoto, Japan
ABSTRACT
Purpose: We investigated the effects of intrathecal application of nerve growth factor (NGF) antibodies (Ab) on bladder hyperreflexia in chronic spinalized rats. Materials and Methods: In adult female rats an intrathecal catheter was implanted at the level of the L6 to S1 spinal cord, followed by complete transection of the Th8 to 9 spinal cord. At 10 days after spinalization the intrathecal catheter was connected to an osmotic pump for continuous delivery of vehicle or NGF Ab (10 g. daily) for 2 weeks. Awake cystometry was then performed. NGF levels in the L5 to S1 dorsal root ganglia, L6 spinal cord and bladder were also measured using enzyme-linked immunosorbent assay. Results: The number of uninhibited bladder contractions per voiding cycle, maximal pressure of uninhibited bladder contraction and maximal voiding pressure were significantly decreased in NGF Ab treated versus vehicle treated spinalized rats. Intercontraction interval, baseline intravesical pressure, pressure threshold for voiding and voiding efficiency were not significantly changed by NGF Ab treatment. NGF levels in the bladder, L6 spinal cord and L5 to S1 dorsal root ganglia of vehicle treated spinalized rats was 1.6 to 4.8 times higher than in spinal cord intact rats. After intrathecal NGF Ab treatment NGF levels were significantly lower in the L6 to S1 dorsal root ganglia (30% to 35%) and L6 spinal cord (53%) but not in the bladder or L5 dorsal root ganglia compared with levels in vehicle treated spinalized rats. Conclusions: Increased levels of NGF in the bladder, spinal cord and dorsal root ganglia were associated with bladder hyperreflexia after spinal cord injury. Immuno-neutralization of NGF in the spinal cord suppressed NGF levels in the L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, and also suppressed bladder hyperreflexia. Thus, suppression of NGF levels in afferent pathways could be useful for treating bladder hyperreflexia associated with spinal cord injury. KEY WORDS: bladder; spinal cord injuries; rats, Sprague-Dawley; nerve growth factor; ganglia, spinal
Spinal cord injury rostral to the lumbosacral level impairs voluntary and supraspinal control of voiding, leading to reorganization of the neural pathways regulating bladder and urethral sphincter function. Spinal cord injury initially induces an areflexic bladder and urinary retention, followed by the emergence of automatic voiding mediated by spinal reflex mechanisms.1, 2 The bladder becomes hyperreflexic and bladder sphincter coordination is impaired, leading to detrusorsphincter dyssynergia. These lower urinary tract dysfunctions then produce various problems, such as urinary incontinence, recurrent urinary tract infection and vesicoureteral reflux with or without upper urinary tract deterioration.1 Clinical and experimental studies indicate that bladder hyperreflexia after spinal cord injury is induced at least in part by phenotypic changes in unmyelinated C-fiber bladder afferent pathways.1, 2 Multiple mechanisms may be involved in the plasticity of bladder afferent neurons in pathological conditions. For example, it has been suggested that target
organ-neural interactions in bladder afferent pathways after spinal cord injury may be mediated by an increase in neurotrophic factors produced in target organ tissues and transported retrograde back to the neuronal cell bodies. It is known that trophic factors such as nerve growth factor (NGF) increase in hypertrophied bladders in rats with spinal cord injury3 or partial urethral ligation.4 It has also been shown that the hypertrophy in afferent and efferent neurons innervating the hypertrophic bladder in rats with partial urethral obstruction was antagonized in part by systemic autoimmunization against NGF.4 In addition, recent studies revealed that acute or chronic local administration of NGF can sensitize bladder afferent pathways and induce bladder hyperactivity.5, 6 Overall these data suggest that NGF may be involved in inducing the neuroplasticity that underlies the emergence of bladder hyperreflexia after spinal cord injury. However, many aspects of this hypothesis remain unexplored. For example, it is not known whether NGF levels in bladder afferent pathways are actually increased after spinal cord injury, nor is it certain whether a peripheral or central nervous system source of NGF is most important for the development of bladder hyperreflexia. It has been reported that NGF protein and NGF receptor mRNA increase
Accepted for publication May 17, 2002. Supported by Grants DK 49430, DK 57267 and P01 HD39768 from the National Institutes of Health and Grant 1861– 01/02 from Spinal Cord Research Foundation, PVA. * Financial interest and/or other relationship with Pfizer UK, Roche, Aventis and Cook Myocyte. 2269
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throughout the spinal cord after spinal cord injury in rats, while little NGF is found in the spinal cord under normal conditions.7, 8 A recent study also demonstrated that intrathecal administration of NGF Ab in spinalized rats suppressed the hypertensive response (autonomic dysreflexia) induced by colon distention, which is mediated by C-fiber afferents.9 Thus, it seems reasonable to assume that NGF produced not only by the bladder, but also by the injured spinal cord could be transported to bladder afferent pathways to induce bladder hyperreflexia. We tested this hypothesis by administrating NGF Ab intrathecally in spinal cord injured rats. We determined if immuno-neutralization of NGF in the spinal cord can reduce NGF levels in L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, and if this treatment can suppress bladder hyperreflexia after spinal cord injury. MATERIALS AND METHODS
Surgical preparation. Experiments were performed in adult female Sprague-Dawley rats weighing 182 to 255 gm. Care and handling of animals were done in accordance with institutional guidelines and approved by the University of Pittsburgh Institutional Animal Care and Use Committee. An PE-10 intrathecal catheter (Clay-Adams, Parsippany, New Jersey) was implanted at the level of the L6 to S1 spinal cord after laminectomy at the Th11 vertebra with the rat under halothane anesthesia. The end of the catheter was heat sealed and placed subcutaneously. At 3 to 4 days after intrathecal catheter implantation spinal cord transection was performed between Th8 to Th9 with the rat under halothane anesthesia. After T8 laminectomy the dura and spinal cord were cut with scissors and a sterile Gelform sponge (The Upjohn Co., Kalamazoo, Michigan) was placed between the cut ends of the spinal cord. The overlying muscle and skin were sutured. Postoperatively the animals were treated with antibiotics (ampicillin, 150 mg./kg. intramuscularly) for 7 days. The bladder of spinalized rats was manually emptied twice daily until reflex voiding recovered, usually 10 to 14 days after spinalization. At 10 days after spinalization the intrathecal catheter was connected to an Model 2002 osmotic mini pump (Alza Co., Palo Alto, California) placed subcutaneously in the back for continuous delivery of vehicle (artificial cerebrospinal fluid) in 8 rats or NGF Ab (200 l. 700 g/ml., 10 g. daily) in 11 for 14 days. Monoclonal NGF Ab (clone 911) (Genentech, South San Francisco, California) was used. It has been demonstrated that this antibody specifically binds to NGF but not to other neurotrophic factors, such as brain derived neurotrophic factor or neurotrophin-3.10 Cystometry. At 14 days after intrathecal injection of vehicle or NGF Ab cystometry in conscious rats was performed according to previously published methods. With the rat under halothane anesthesia a PE-50 catheter was inserted via a midline abdominal incision into the bladder through the bladder dome. After surgery the rat was placed in a restraining cage and allowed to recover from anesthesia for 1 to 2 hours. The intravesical catheter was connected via a 3-way stopcock to a pressure transducer and a syringe pump for recording intravesical bladder pressure and infusing saline into the bladder, respectively. Saline at room temperature was infused at a rate of 0.04 ml. per minute to elicit repetitive bladder contractions. Saline infusion was continued at least 3 hours before measuring voiding parameters. Saline voided from the urethral meatus was collected and measured to determine voided volume (VV). After constant voided volume measurements were collected the infusion was stopped and post-void residual volume (RV) was measured. Residual saline was withdrawn through the intravesical catheter by gravity and then the bladder was completely emptied by manual compression through the abdominal wall.
FIG. 1. Representative cystometrogram traces in awake spinalized (SCI) rats. A, vehicle treated. Arrows indicate voiding contractions. B, NGF Ab treated.
Bladder capacity (BC) was calculated using the formula, BC ⫽ VV ⫹ RV. Based on these values voiding efficiency (VE) expressed as a percent could be estimated by the formula, VE ⫽ [(VV/BC) ⫻ 100]. Intercontraction interval, maximal voiding pressure, pressure threshold for voiding and baseline intravesical pressure were also measured. The number of uninhibited bladder contractions per voiding cycle and maximal uninhibited bladder contraction pressure were measured. Uninhibited bladder contractions were defined as rhythmic intravesical pressure increases greater than 7 cm. water from baseline pressure without a release of fluid from the urethra. Enzyme-linked immunosorbent assay (ELISA). After cystometry the bladder, L5 to 6 and S1 dorsal root ganglia, and L6 spinal cord were removed from vehicle or NGF Ab treated spinalized rats under urethane anesthesia (1.2 mg./kg. intraperitoneally). Bladder, dorsal root ganglia and spinal cord tissues were also obtained from 8 untreated spinal cord intact rats. In the L5, L6 and S1 dorsal root ganglia ELISA measurements was performed in each animal using the 2 sides of the dorsal root ganglia tissues. The tissues were rapidly frozen on dry ice and stored at – 80C until NGF extraction. The samples were homogenized in buffer (2.66% Trisma HCl, 0.985% Trisma Base, 0.5 mM. phenylmethylsulfonyl fluoride, 1M. leupeptin, 1 M. pepstatin A and 0.3 M. aprotinin in 400 and 100 l. for the bladder and dorsal root ganglia, respectively). The homogenate was centrifuged at 10,000 ⫻ gravity for 4 minutes. The supernatant was diluted with 4 volumes Dulbecco’s phosphate buffered saline (0.02% KCl, 0.8% NaCl, 0.02% KH2PO4, 0.115% Na2HPO4, 0.0133% CaCl2䡠2H2O and 0.01% MgCl2䡠6H2O, pH 7.35). Samples were stored in a deep freezer at – 80C until assayed. The samples were assayed in triplicate in an antigen capture ELISA Emax ImmunoAssay System (Promega, Madison, Wisconsin) according to manufacturer instructions. ELISA plates were read at 450 nm. on an Elx800 microplate reader (Bio-Tek Instruments, Winooski, Vermont). Total protein concentration in the same samples was also determined with a BCA Protein Assay Kit (Pierce, Rockford, Illinois). All tissue NGF values were standardized by tissue protein levels and are expressed in pg./g. total protein. Evaluation and statistical analysis. All values are expressed as the mean plus or minus SE. The Mann-Whitney U test was used to determine significance. For all statistical tests p ⬍0.05 was considered significant. RESULTS
Cystometry. During awake cystometry all 7 spinalized rats that received vehicle treatment showed uninhibited bladder
IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR REDUCES BLADDER HYPERREFLEXIA
contraction before large amplitude voiding bladder contractions occurred (fig. 1). Uninhibited bladder contraction amplitude increased as the bladder was filled, reaching a mean maximum of 39.6 ⫾ 5.49 cm. water. There was an average of 6.7 ⫾ 0.81 uninhibited bladder contractions per voiding cycle (fig. 2). Of the 10 spinalized rats that received intrathecal NGF Ab 8 (80%) showed uninhibited bladder contractions before voiding bladder contractions. However, in 10 NGF Ab treated animals the mean number of uninhibited bladder contraction per voiding (2.5 ⫾ 0.61 contractions, p ⬍0.01) and mean maximal uninhibited bladder contraction pressure (26.8 ⫾ 2.82 cm. water, p ⬍0.05) were significantly less than in vehicle treated spinalized animals (figs. 1 and 2). Mean maximal voiding pressure was also less in NGF Ab treated than in vehicle treated rats (42.7 ⫾ 3.20 versus 57.1 ⫾ 6.30 cm. water, p ⬍0.05). However, NGF Ab treatment did not affect other cystometric parameters, such as intercontraction interval, baseline intravesical pressure, pressure threshold for voiding efficiency or bladder capacity (see table). NGF levels in the bladder, L6 spinal cord and dorsal root ganglia. In vehicle treated spinalized rats NGF levels in the bladder, L6 spinal cord and dorsal root ganglia (L5 to 6 and S1) were significantly higher than in spinal cord intact rats (figs. 3 and 4). In 8 spinalized rats injected with NGF Ab at the L6 to S1 intrathecal space for 2 weeks mean NGF levels in the L6 and S1 dorsal root ganglia were significantly lower than in 8 vehicle treated spinalized rats (2.53 ⫾ 0.20 and 3.05 ⫾ 0.30 versus 3.87 ⫾ 0.48 and 4.41 ⫾ 0.48 pg./g. protein, respectively). Mean NGF levels in the L6 spinal cord of 8 NGF Ab treated spinalized animals were also significantly lower than in 8 vehicle treated spinalized rats (0.49 ⫾ 0.08 versus 1.06 ⫾ 0.21 pg./g. protein). However, NGF levels in the L6 spinal cord and L6 to S1 dorsal root ganglia were still significantly higher in NGF Ab treated than in spinal cord intact rats (figs. 3 and 4). In 8 vehicle treated and 8 NGF Ab treated spinalized rats
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mean NGF levels in the bladder (4.16 ⫾ 0.56 versus 4.34 ⫾ 0.18 pg./g. protein) and L5 dorsal root ganglia (3.01 ⫾ 0.40 versus 2.40 ⫾ 0.20 pg./g. protein, respectively) were not significantly different. Mean bladder weight in 11 vehicle treated and 11 NGF Ab treated spinalized rats (425 ⫾ 58 versus 420 ⫾ 43 mg., respectively) was not different but each value was significantly (4 to 5 times) greater than mean bladder weight in 6 spinal cord intact rats (80.6 ⫾ 12 mg., see table). DISCUSSION
The results of the current study indicate that NGF levels in the bladder, L6 spinal cord and dorsal root ganglia were increased in chronic spinalized rats with bladder hyperreflexia and immuno-neutralization of NGF in the spinal cord (which in turn decreased NGF in the L6 spinal cord and L6 to S1 dorsal root ganglia, where bladder afferent fibers originate) reduced uninhibited bladder contractions and maximal voiding pressure in chronic spinalized rats. Since intrathecal NGF Ab treatment decreased NGF levels in the spinal cord but not in the bladder, it is likely that NGF Ab administered intrathecally localized and reduced NGF levels in the spinal cord without effects at the periphery. It has been proposed that elevated expression of neurotrophic factors such as NGF in the hypertrophied bladder after spinal cord injury is induced by increased bladder work due to uncoordinated bladder and urethral sphincter activity (detrusor-sphincter dyssynergia), which increases urethral resistance.3, 11 Similar mechanisms are also suggested to be involved in another rat model of bladder hypertrophy induced by partial urethral obstruction that involves bladder hyperactivity and increased NGF levels in the bladder.4 Autoimmunization against NGF antagonized at least in part bladder hyperactivity as well as afferent neuron hypertrophy in rats with partial urethral obstruction.4 Thus, target organ-
FIG. 2. Spinalized vehicle and NGF Ab treated rats. A, number of uninhibited bladder contractions (UIC) per voiding cycle. Double asterisks indicate p ⬍0.01. B, uninhibited bladder contraction amplitude. Single asterisk indicates p ⬍0.05. C, maximal voiding pressure.
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IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR REDUCES BLADDER HYPERREFLEXIA Spinalized Vehicle Treated
Intercontraction interval (secs.) Bladder capacity (ml.) Baseline intravesical pressure (cm. H2O) Pressure threshold for voiding (cm. H2O) % Voiding efficiency Bladder wt. (mg.)
Mean ⫾ SE 628 ⫾ 88 0.67 ⫾ 0.09 9.8 ⫾ 2.6 11.3 ⫾ 2.4 0.90 ⫾ 0.05 424 ⫾ 58
neural interactions mediated by increased NGF in the hypertrophied bladder and increased transport of NGF to neuronal cell bodies in the afferent pathways may contribute to the emergence of bladder hyperactivity in cases of spinal cord injury or partial urethral obstruction.4, 11 The current study seems to be consistent with this hypothesis because NGF levels in the lumbosacral dorsal root ganglia were in fact increased as bladder NGF expression was elevated after spinal cord injury. However, our study also shows that the NGF level in the L6 spinal cord was increased in spinalized rats and immuno-neutralization of NGF at the L6 and S1 spinal cord segment levels significantly suppressed increased NGF levels in the L6 to S1 dorsal root ganglia as well as in the L6 spinal cord without affecting bladder NGF levels. Thus, it is assumed that increased NGF levels in the dorsal root ganglia were at least in part attributable to NGF production in the spinal cord after spinal cord injury. This finding is also supported by earlier studies showing that NGF proteins7 and NGF receptor mRNA8 were increased throughout the spinal cord almost immediately after spinal cord injury in rats. In addition, in our experiments NGF levels were increased not only in the L6 to S1
Spinalized NGF Ab Treated
No. Rats
Mean ⫾ SE
No. Rats
7 7 7 7 7 11
761 ⫾ 187 0.82 ⫾ 0.12 7.5 ⫾ 1.7 10.2 ⫾ 1.8 0.88 ⫾ 0.03 419 ⫾ 43
10 10 10 10 10 10
dorsal root ganglia, but also in the L5 dorsal root ganglia, which do not contain bladder afferent neurons.12 Therefore, NGF in the L5 dorsal root ganglia cannot be attributable to changes in bladder NGF expression. Overall it seems likely that NGF produced in the spinal cord has an important role in inducing bladder hyperreflexia after spinal cord transection, although retrograde transport of NGF from the bladder can also contribute to the NGF increase in the dorsal root ganglia after spinal cord injury since NGF levels in the dorsal root ganglia after intrathecal NGF Ab treatment were still significantly higher in spinalized than in spinal cord intact rats. Because immuno-neutralization of NGF in the spinal cord reduced NGF levels in the L6 to S1 dorsal root ganglia, bladder hyperreflexia in spinalized rats could be suppressed by modulating neural activity in the spinal cord or in bladder afferent pathways. Various indirect evidence suggests that a change in bladder afferent activity may be important for the effect of NGF Ab treatment. It is well known that 2 types of afferent fibers, namely A␦ and C-fibers, carry sensory information from bladder to spinal cord.12, 13 Previous studies in cats and rats showed that A␦-fiber bladder afferents trigger
FIG. 3. ELISA measurements of NGF in normal spinal cord intact, vehicle treated spinalized and NGF Ab treated spinalized rats. NGF values are standardized to tissue protein and expressed in pg./g. total protein. A, bladder. Single asterisk indicates p ⬍0.05. Double asterisks indicate p ⬍0.01. B, L5 dorsal root ganglia (DRG). C, L6 dorsal root ganglia. D, S1 dorsal root ganglia.
IMMUNONEUTRALIZATION OF NERVE GROWTH FACTOR REDUCES BLADDER HYPERREFLEXIA
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2, 17
FIG. 4. ELISA measurements of NGF in L6 spinal cord in normal spinal cord intact, vehicle treated spinalized and NGF Ab treated spinalized rats. NGF values are standardized to tissue protein and expressed in pg./g. total protein. Single asterisk indicates p ⬍0.05. Double asterisks indicate p⬍0.01.
the normal micturition reflex via a long latency supraspinal pathway passing through the pons.1, 13 In chronic spinal injured rats A␦ bladder afferents trigger the voiding reflex but C-fiber afferents appear to initiate bladder hyperactivity during filling. Desensitizing C-fiber afferents by systemic capsaicin administration suppressed the uninhibited bladder contractions that occurred before voiding.14 This effect is similar to that in the current experiments after NGF-Ab experiments. In addition, because chronic administration of exogenous NGF increases the excitability of C-fiber bladder afferents and induces bladder hyperactivity in spinal cord intact rats,15 it seems reasonable to propose that NGF Ab treatment in the spinal cord could reduce uninhibited bladder contractions by eliminating NGF induced sensitization of C-fiber afferents in chronic spinalized rats. The current study also shows that NGF Ab treatment reduced maximal voiding pressure. Since a previous study indicated that hyperexcitability of C-fiber bladder afferents is also involved in dyssynergic urethral sphincter contractions during voiding after spinal cord injury,16 the reduction in maximal voiding pressure may also be elicited by a reduction in detrusor-sphincter dyssynergia as a result of suppressing C-fiber activity after NGF Ab treatment. Further studies are necessary to evaluate the effect of NGF Ab treatment on urethral function. NGF modulation of C-fiber afferents may also be important for the initiation of bladder hyperactivity in other species after spinal cord injury. Electrophysiological recording in spinalized cats revealed that the afferent limb of the spinal micturition reflex consists of unmyelinated C-fiber afferents.1 This finding was confirmed in pharmacological studies of the C-fiber neurotoxin capsaicin. In chronic spinal cord injured cats 3 to 6 weeks after spinal cord injury subcutaneously administered capsaicin (20 to 30 mg./kg.) completely blocked bladder distention induced by bladder contractions, whereas capsaicin had only a facilitatory effect on reflex bladder contractions in spinal cord intact cats.1 Clinical studies also showed that intravesical instillation of capsaicin suppressed bladder hyperreflexia in patients with spinal cord injury or multiple sclerosis. When administered intravesically, capsaicin increased bladder capacity and reduced the number of incontinence episodes in those
patients. Resiniferatoxin, an agent with similar C-fiber desensitizing properties, has been recently reported to have similar effects for neurogenic bladder hyperreflexia.18 Together it is clear that the functional properties of C-fiber afferents in the bladder are altered after spinal cord injury, thereby, inducing hyperreflexic bladder activity in humans as well as animals. The contribution of NGF to visceral C-fiber afferent hyperexcitability is also further supported by findings in another laboratory that intrathecal application of NGF Ab in spinalized rats reduced autonomic dysreflexia (that is reflex hypertension) induced by colon distention.9 Autonomic dysreflexia induced by bladder or colon distention is another example of a reorganization of C-fiber mediated reflex pathways after spinal cord injury because capsaicin treatment suppresses autonomic dysreflexia in humans with spinal cord injury19 and similar arterial pressure responses induced by bladder distention in rats.20 Thus, it is plausible that an interaction between the spinal cord and bladder afferent pathways, leading to the reorganization of bladder reflex activities after spinal cord injury, could be mediated at least in part by increased expression of NGF in the spinal cord, which is then transported into bladder afferent pathways. CONCLUSIONS
This study provides direct evidence that increased levels of NGF in the bladder, L6 spinal cord and L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, are involved in bladder hyperreflexia induced by C-fiber bladder afferents. It is also suggested that NGF protein produced in the injured spinal cord could be an important source of increased levels of NGF in the afferent pathways after spinal cord injury. Therefore, manipulating NGF levels in bladder afferent pathways could be an effective modality for treating bladder hyperreflexia in spinal cord injury cases. REFERENCES
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bladder. Prog Neurobiol, 57: 583, 1999 12. Keast, J. R. and de Groat, W. C.: Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J Comp Neurol, 319: 615, 1992 13. Mallory, B., Steers, W. D. and de Groat, W. C.: Electrophysiological study of micturition reflexes in rats. Am J Physiol, 257: R410, 1989 14. Cheng, C. L., Ma, C. P. and de Groat, W. C.: Effect of capsaicin on micturition and associated reflexes in chronic spinal rats. Brain Res, 678: 40, 1995 15. Yoshimura, N., Bennet, N. E., Phelan, M. W. and de Groat, W. C.: Effects of chronic nerve growth factor treatment on rat urinary bladder and bladder afferent neurons. Soc Neurosci Abstr, 25: 1946, 1999 16. Cheng, C. L, Chai, C. Y. and de Groat, W. C.: Detrusor-sphincter
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