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MICTURITION IN THORACIC SPINAL CORD INJURED CATS WITH AUTOGRAFTING OF THE ADRENAL MEDULLA TO THE SACRAL SPINAL CORD
0022-5347/01/1666-2525/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 166, 2525–2529, December 2001 Printed in U.S.A.
MICTURITION IN THORACIC SPINAL CORD INJURED CATS WITH AUTOGRAFTING OF THE ADRENAL MEDULLA TO THE SACRAL SPINAL CORD KIMIO SUGAYA, YOSHIHIDE OGAWA, TADASHI HATANO, SAORI NISHIJIMA OSAMU NISHIZAWA
AND
From the Departments of Urology, Faculty of Medicine, University of the Ryukyus, Okinawa and Shinshu University School of Medicine, Matsumoto, Japan
ABSTRACT
Purpose: The role of noradrenergic projection from the pontine micturition center to the sacral spinal cord during micturition was examined in thoracic spinal cord injured cats after autografting the adrenal medulla to the sacral spinal cord. Materials and Methods: In 13 female cats the lower thoracic cord was transected and the right adrenal gland was removed under halothane anesthesia. The resected adrenal medulla was divided into several small pieces, which were subsequently autografted to the sacral spinal cord in 7 cats. Another 6 cats underwent sham operation and served as controls. Continuous cystometry and electromyography of the external urethral sphincter were performed every 2 weeks postoperatively without anesthesia. At week 8 the sacral spinal cord was removed and immunohistochemical testing was done to assess tyrosine hydroxylase immunoreactivity. Results: At week 6 the relative mean duration of detrusor-external sphincter coordination plus or minus standard error during bladder contraction was 62.4% ⫾ 4.9% in adrenal grafted cats, which was significantly (p ⫽ 0.0485) longer than in controls (34.2% ⫾ 12.6%). However, maximum bladder contraction pressure, bladder contraction duration and post-void residual urine volume were not significantly different in the 2 groups. Tyrosine hydroxylase immunoreactive cells were observed in and on the sacral spinal cord in adrenal grafted animals but not in controls. Conclusions: Autografting the adrenal medulla to the sacral spinal cord prolonged detrusorexternal sphincter coordination during bladder contraction in thoracic spinal cord injured cats, although other urodynamic parameters did not change. Therefore, noradrenergic projections to the sacral spinal cord may relax the external urethral sphincter during bladder contraction. KEY WORDS: bladder; spinal cord injuries; cats; transplantation, autologous; adrenal glands
Electrical stimulation of the pontine micturition center causes micturition. This center is reportedly present in the nucleus locus coeruleus ␣ in cats1 and dogs.2 The majority of the neurons in this nucleus contain noradrenaline as a transmitter and project to the spinal cord, including the sacral spinal cord.3, 4 Intrathecal injection of an ␣1-adrenoceptor antagonist was reported to inhibit bladder contraction induced by electrical stimulation of the pontine micturition center in cats under ␣-chloralose anesthesia.5 However, Espey et al reported that intrathecal injection of an ␣1adrenoceptor antagonist did not affect bladder contraction in alert cats.6 Shimoda also noted that intrathecal injection of an ␣1-adrenoceptor antagonist did not affect bladder or urethral contraction, while intrathecal injection of an ␣1adrenoceptor agonist increased bladder volume and urethral pressure in decerebrate dogs.7 ␣2-Adrenoceptors in the spinal cord have been reported to relax the urethra in decerebrate dogs7 and female rats8 but contract it in male rats.9 Thus, controversy remains regarding whether descending noradrenergic projections from the pontine micturition center to the sacral spinal cord can evoke bladder contraction and relax the urethra. We examined the effect of autografting the adrenal medulla, which contains catecholamine secreting cells to the sacral spinal cord on bladder and urethral function in thoracic spinal cord injured cats to determine the role
of noradrenergic projections from the pontine micturition center to the sacral spinal cord during micturition. MATERIALS AND METHODS
A total of 13 female cats weighing 1.7 to 3.2 kg. were anesthetized with halothane and underwent laminectomy to expose the lower thoracic spinal cord at T13 and the sacral spinal cord at S1 to S2. After the dura was opened the spinal cord was completely transected at T13. In 7 of the 13 spinal cord injured cats the right adrenal gland was removed via the retroperitoneal approach and the adrenal medulla was cut into 1 to 1.5 mm.3 pieces. Using the tip of a 21 gauge needle the adrenal pieces were inserted into the center of S1 to S2 through the dorsal funiculus. Three pieces were autografted into the gray matter of S1 to S2, where the cell bodies of the neurons are localized in the pelvic and pudendal nerves,10, 11 while another 3 were placed between the dura and S1 to S2. The remaining 6 spinal cord injured cats underwent sham operative grafting of the sacral spinal cord by inserting a needle 3 times. Postoperatively each group received 200 mg. ampicillin intramuscularly twice daily for 5 days. Urine was voided using a urethral catheter or manual bladder compression twice daily for 10 days. At weeks 2, 4 and 6 a 1.2 mm. catheter was inserted into the bladder and the urine was emptied. The cat was placed in a narrow dark box with a hole for the tail and urethral catheter. The tail was fixed to the edge of the hole to prevent catheter dislodgment by movement related to the spinal micturition reflex. Without anesthesia the bladder was continuously per-
Accepted for publication June 29, 2001. Supported by Grant-in-Aid 11671564 for Scientific Research (C) from the Japanese Ministry of Education, Science, Sports and Culture. 2525
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fused with 2.5 to 5 ml. saline per minute through the catheter and continuous cystometry was performed with a 3-way stopcock. At the same time electromyography of the external urethral sphincter was also done with 2 wire electrodes 50 m. in diameter inserted into the muscle from the perineum. Initial bladder volume at which micturition occurred was measured. Mean maximum bladder contraction pressure, mean bladder contraction duration and mean relative detrusor-sphincter coordination duration per bladder contraction were determined from relatively stable continuous cystometry and electromyography data obtained for 20 minutes. Before the catheter was removed post-void residual urine volume after bladder contraction was measured. At week 8 all animals were transcardinally perfused with 0.1 M. phosphate buffered saline (PBS) (1.5 to 2 l.) at 4C for 10 minutes under deep anesthesia with 40 mg./kg. sodium pentobarbital intraperitoneally. After the blood was washed out perfusion was continued with 1.5 l. of ice-cold fixative, composed of 4% paraformaldehyde, 0.1% glutaraldehyde and 0.2% picric acid in 0.1 M. PBS, pH 7.4. The brainstem, lumbosacral spinal cord and adrenal gland were removed and post-fixed for 2 days at 4C in the same fixative minus glutaraldehyde. They were then immersed in 0.1 M. PBS containing 15% sucrose for at least 2 days. Horizontal serial sections (50 m.) of the specimens were cut on a cryotome. Freely floating sections were washed for 4 days in PBS containing 0.3% Triton X-100, incubated for 3 days at 4C with rabbit antiserum for tyrosine hydroxylase,12 diluted 1:1,000 and incubated for 1 hour with biotinylated goat anti-rabbit IgG, diluted 1:1,000 (Vector Laboratories, Burlingame, California). Tyrosine hydroxylase is the first enzyme involved in the process of catecholamine biosynthesis from tyrosine. After each incubation the sections were washed with 3 changes of 0.3% Triton X-100 in PBS. The sections were then reacted for 4 minutes with a mixture of 0.02% 3,3⬘-diaminobenzidine, 0.3% nickel ammonium sulfate and 0.005% H2O2 in 0.05 M. tris-HCl buffer, mounted on gelatin coated glass slides and counterstained with neutral red. Microscopy was performed to examine the location and the number of tyrosine hydroxylase immunoreactive cells. Results are reported as the mean plus or minus standard error. Student’s t-test for paired or unpaired data was used for statistical analysis with p ⬍0.05 considered statistically significant. RESULTS
In control cats mean initial bladder volume plus or minus standard error (SE) inducing micturition was 73.7 ⫾ 27.8 ml. at week 2. This volume showed a significant (p ⫽ 0.0264) decrease to 15.5 ⫾ 8.2 ml. at week 6 (see table). Single voiding volume was small and variable at about 0.5 to 5 ml. after each bladder contraction between weeks 2 and 6. Postvoid residual urine volume also decreased significantly (p ⫽ 0.0200) from 98.0 ⫾ 28.6 ml. at week 2 to 13.5 ⫾ 7.4 at week 6. Mean maximum bladder contraction pressure was 30 to 41 cm. water and mean bladder contraction duration was 16 to 18 seconds between weeks 2 and 6. Persistent detrusorexternal sphincter dyssynergia was detected in 4, 2 and 2 of
the 6 control cats at weeks 2, 4 and 6, respectively (fig. 1, A). The relative duration of detrusor-external sphincter coordination per bladder contraction ranged widely from 0% to 70%. The mean relative duration of detrusor-external sphincter coordination increased from 20.0% ⫾ 12.9% at week 2 to 39.5% ⫾ 12.8% at week 4 and 34.2% ⫾ 12.6% at week 6 but there were no significant change with time (p ⫽ 0.1749 and 0.3839, respectively). In adrenal grafted cats initial mean bladder volume inducing micturition decreased significantly (p ⫽ 0.0102) from 48.9 ⫾ 12.3 ml. at week 2 to 12.6 ⫾ 3.3 at week 6 (see table). Single voiding volume was small and varied after each bladder contraction between weeks 2 and 6, as in controls. Postvoid residual urine volume also decreased significantly (p ⫽ 0.0093) from 50.0 ⫾ 13.3 ml. at week 2 to 10.1 ⫾ 4.3 at week 6. Maximum bladder contraction pressure increased significantly (p ⫽ 0.0033) from 26.6 ⫾ 3.1 cm. water at week 2 to 40.0 ⫾ 3.7 at week 6, while mean bladder contraction duration was 16 to 19 seconds between weeks 2 and 6. Persistent detrusor-external sphincter dyssynergia was noted in 3 of 7 adrenal grafted cats at week 2 only (fig. 1, B). The relative duration of detrusor-external sphincter coordination per bladder contraction was 0% to 66% at week 2, similar to that in control cats, but it became longer (50% to 88%) by week 6. Although the mean relative duration of detrusor-external sphincter coordination increased from 34.4% ⫾ 12.3% at week 2 to 62.4% ⫾ 4.9% at week 6, there was no significant change with time (p ⫽ 0.0876). Electromyography activity in adrenal grafted cats was similar to that in controls during the urine collection period. When urodynamic parameters were compared in the 2 groups, mean threshold bladder volume was smaller in adrenal grafted than in control cats during weeks 2 and 4 but there were no significant differences (p ⫽ 0.3943 and 0.1797, respectively) in the 2 groups because of the large SE. Postvoid residual urine volume was also smaller in adrenal grafted than in control cats during weeks 2 and 4 but again there were no significant differences (p ⫽ 0.1377 and 0.0632, respectively) in the 2 groups. The relative duration of detrusor-external sphincter coordination per bladder contraction showed a significant difference in the 2 groups at week 6 (p ⫽ 0.0485) but it was the only difference in all parameters tested (see table). In adrenal grafted cats tyrosine hydroxylase immunoreactive cells were detected in the adrenal medulla, nucleus locus coeruleus, nucleus locus coeruleus ␣ in the rostral pons and sacral spinal cord. Small nodules on the surface of the sacral cord were easily removed. Immunohistochemical testing revealed that these nodules contained many tyrosine hydroxylase immunoreactive cells. In the adrenal medulla all cells showed strong tyrosine hydroxylase immunoreactivity and were cuboidal without dendrites or axons. Many neurons in the nucleus locus coeruleus ␣ also showed strong tyrosine hydroxylase immunoreactivity and there were numerous immunoreactive dendrites or axons (fig. 2, A). There were 5 to 14 tyrosine hydroxylase immunoreactive cells (mean 9 ⫾ 1) in the sacral spinal cord. These spindle-shaped cells were scattered in the intermediate gray matter and showed weak
Urodynamic parameters in 6 control and 7 adrenal autografted cats 2, 4 and 6 weeks after operation Urodynamic Parameters Threshold bladder vol. inducing micturition (ml.) Max. bladder contraction pressure (cm. water) Bladder contraction duration (sec.) % Relative detrusor-sphincter coordination duration Post-void residual urine vol. (ml.) * Versus 2 weeks p ⬍0.05. † Versus 2 weeks p ⬍0.01. ‡ Versus control p ⫽ 0.0485.
Mean 2 Wks. ⫾ SE
Mean 4 Wks. ⫾ SE
Mean 6 Wks. ⫾ SE
Control
Autografted
Control
Autografted
Control
Autografted
73.7 ⫾ 27.8 30.3 ⫾ 7.2 17.5 ⫾ 1.9 20.0 ⫾ 12.9 98.0 ⫾ 28.6
48.9 ⫾ 12.3 26.6 ⫾ 3.1 16.3 ⫾ 2.4 34.4 ⫾ 12.3 50.0 ⫾ 13.3
51.7 ⫾ 18.3 37.0 ⫾ 3.0 16.0 ⫾ 1.4 39.5 ⫾ 12.8 46.5 ⫾ 17.0
24.9 ⫾ 7.6 38.0 ⫾ 2.4* 17.6 ⫾ 2.2 50.4 ⫾ 5.6 12.6 ⫾ 4.7*
15.5 ⫾ 8.2* 40.7 ⫾ 2.0 17.3 ⫾ 1.1 34.2 ⫾ 12.6 13.5 ⫾ 7.4*
12.6 ⫾ 3.3* 40.0 ⫾ 3.7† 19.0 ⫾ 2.0 62.4 ⫾ 4.9‡ 10.1 ⫾ 4.3†
MICTURITION IN SPINAL CORD INJURED CATS WITH AUTOGRAFT
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FIG. 1. Continuous cystometrography (CMG) and electromyography (EMG) of external urethral sphincter. In control cat detrusorsphincter dyssynergia persisted from weeks 2 to 6. In adrenal grafted cat detrusor-sphincter coordination was present from weeks 2 to 6. FIG. 3. In adrenal grafted cat 6 and 3 tyrosine hydroxylase immunoreactive cells (filled circles) were evident in S1 and S2, respectively. Many tyrosine hydroxylase immunoreactive cells were located in intermediate gray matter. PSN, parasympathetic nucleus. N, nucleus.
FIG. 2. Photomicrographs of pons (A) and sacral spinal cord (B) of adrenal grafted cat. In dorsolateral pontine tegmentum (A) almost all cells in nucleus locus coeruleus (LC) and many in nucleus locus coeruleus ␣ (LCa) were tyrosine hydroxylase immunoreactive cells (dark brown areas). BC, brachium conjunctivum. MTV, trigeminal nerve mesencephalic tract. 4V, fourth ventricle. Bar represents 500 m. In intermediate gray matter of S1 (B) there are 2 spindle-shaped tyrosine hydroxylase immunoreactive cells are visible (arrows). Bar represents 100 m.
immunoreactivity of a few dendrite or axon-like fibers, indicating that they were neurons (figs. 2, B and 3). Tyrosine hydroxylase immunoreactive cells in the small nodules on the surface of the sacral spinal cord were cuboidal. There were no tyrosine hydroxylase immunoreactive cells in the sacral spinal cord in control cats. DISCUSSION
Detrusor-external sphincter dyssynergia was identified during micturition in our thoracic spinal cord injured cats. Our study also demonstrated that transplantation of the adrenal medulla to the sacral cord increased detrusorexternal sphincter coordination during bladder contraction. Therefore, noradrenergic projections from the pontine micturition center to the sacral spinal cord may inhibit external urethral sphincter activity during bladder contraction. The pontine micturition center in cats and dogs is the nucleus locus coeruleus ␣1, 2 but the pontine micturition center in rats is Barrington’s nucleus, which is located just inside the locus coeruleus.13 In rats the neurons of Barrington’s nucleus do not use catecholamines as a neurotrans-
mitter.3, 14 Therefore, there may be species difference in the location of the pontine micturition center and the neurotransmitter of this center. Regarding the role of noradrenergic projections from the upper central nervous system to the sacral cord there have been various conflicting reports.5–9 It has been reported that systemic or intrathecal injection of glutamate receptor antagonists can strongly inhibit the bladder contraction elicited by filling the bladder or by electrical stimulation of the pontine micturition center and can markedly reduce external urethral sphincter activity in rats with and without spinal cord injury.15–17 Therefore, glutaminergic transmission to the sacral spinal cord may mainly induce bladder and urethral contraction, while noradrenergic transmission to the sacral spinal cord may be involved in bladder and urethral contraction or relaxation. After spinal cord injury the spinal micturition reflex appears and gradually develops as a result of neuronal plasticity.18 Neuronal plasticity may have gradually induced a decrease in threshold bladder volume and post-void residual urine as well as an increase in maximum bladder contraction pressure in our spinal cord injured cats. Detrusor-external sphincter dyssynergia also improved gradually but it was persistent, especially in control spinal cats. Detrusorsphincter dyssynergia is a characteristic of patients with suprasacral spinal cord injury.19 Galeano et al reported that the main reasons for such detrusor-sphincter dyssynergia were an increase in the urethro-urethral contraction reflex and weakness of the vesicourethral relaxation reflex.20 In addition, we have shown that blocking the urethro-urethral contraction reflex by urethral anesthesia can improve detrusor-external sphincter dyssynergia and decrease postvoid residual urine in spinal cord injured cats as well as in patients with spinal cord disorders.21 Therefore, it seems possible that projections from the pontine micturition center to the sacral cord may inhibit the urethro-urethral contraction reflex pathway. In our autografted spinal cord injured cats it seems possible that catecholamines from the adrenal medulla grafts acted on the inhibitory ␣2 receptors of motoneurons in the pudendal nerve or interneurons activating these motoneurons and, thus, inhibited this pathway.7 However, it is also possible that motoneuron inhibition developed as a result of neuronal plasticity18 caused by an influence of the implant.
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MICTURITION IN SPINAL CORD INJURED CATS WITH AUTOGRAFT
This study showed a large differences in mean values of threshold bladder volume and post-void residual urine in the 2 groups during weeks 2 and 4 but the differences were not significant because of the large SE. Despite this finding it seems possible that catecholamines from the autografted adrenal medulla acted to decrease threshold bladder volume or post-void residual urine volume. However, these parameters became almost equal in the 2 groups during week 6, and so the effect of neuronal plasticity after spinal cord injury may be greater than that of autografting the adrenal medulla. Also, maximum bladder contraction pressure did not differ in the 2 groups and a reason may have been the existence of detrusor-internal sphincter dyssynergia.22, 23 Electrical stimulation of the pontine micturition center has been reported to evoke relaxation of the internal urethral sphincter in decerebrate dogs with the urethra separated from the bladder.24 The internal urethral sphincter is innervated by the hypogastric nerves, which are sympathetic nerves originating from the upper and middle lumbar cord segments in cats.25 Therefore, autografting adrenal medulla pieces to the lumbar and sacral spinal cord may improve urodynamic parameters by abolishing detrusor-internal sphincter and detrusorexternal sphincter dyssynergia. We found tyrosine hydroxylase immunoreactive cells in the intermediate gray matter of the sacral spinal cord. Although cell bodies for motoneurons of the pudendal nerves innervating the external urethral sphincter are located in the ventrolateral part (Onuf’s nucleus) of the ventral horn of the sacral cord, the neurons in this nucleus project their dendrites into the intermediate gray matter.11 Therefore, it may be possible that the tyrosine hydroxylase immunoreactive cells detected in our cats were connected to the dendrites of neurons in Onuf’s nucleus. However, small nodules of tyrosine hydroxylase immunoreactive cells were also located on the surface of the sacral cord in each autografted cat. These nodules were thought to arise from the 3 pieces of adrenal medulla that were placed between the dura and sacral spinal cord. Since the number of tyrosine hydroxylase immunoreactive cells in the sacral spinal cord was small, most catecholamines acting on the sacral cord may have been produced by these tyrosine hydroxylase immunoreactive cells on the surface of the spinal cord. However, the concentration of catecholamines produced by these cells on the sacral spinal cord may have been low in the upper and mid segments of the lumbar spinal cord, where the sympathetic nucleus innevating the internal urethral sphincter is located, because catecholamine metabolism is rapid in the central nervous system.26 Transplanting sympathetic ganglionic neurons, cells of the adrenal medulla or fetal brain tissue into the brain has been performed in animals27 and in patients with Parkinson’s disease.28 The grafted cells have been reported to change shape,27 as did the adrenal cells grafted to the sacral spinal cord in our subjects. Our study suggests that autografting sympathetic ganglionic neurons or adrenal medulla cells to the spinal cord may eventually be done to treat micturition disorders in patients with spinal cord injury and severe detrusor-sphincter dyssynergia.
CONCLUSIONS
Autografting adrenal medulla pieces to the sacral spinal cord can increase the duration of detrusor-external sphincter coordination during bladder contraction in thoracic spinal cord cats. Noradrenergic projections to the sacral spinal cord may relax the external urethral sphincter during bladder contraction. Although more detailed experiments are necessary, autografting catecholamine secreting cells to the lumbosacral spinal cord may represent possible treatment for severe detrusor-sphincter dyssynergia in patients with spinal cord injury.
REFERENCES
1. Sugaya, K., Matsuyama, K., Takakusaki, K. et al: Electrical and chemical stimulations of the pontine micturition center. Neurosci Lett, 80: 197, 1987 2. Nishizawa, O., Sugaya, K., Noto, H. et al: Pontine micturition center in the dog. J Urol, 140: 872, 1988 3. Brodal, A.: The reticular formation and some related nuclei. In: Neurological Anatomy in Relation to Clinical Medicine, 3rd ed. Edited by A. Brodal. New York: Oxford University Press, p. 394, 1981 4. Sugaya, K., Mori, S. and Tsuchida, S.: Input and output neuronal structures of the pontine micturition center. Part 2. Mainly output neuronal structures. Nippon Hinyokika Gakkai Zasshi, 79: 1219, 1988 5. Yoshimura, N., Sasa, M., Ohno, Y. et al: Contraction of urinary bladder by central norepinephrine originating in the locus coeruleus. J Urol, 139: 423, 1988 6. Espey, M. J., Downie, J. W. and Fine, A.: Effect of 5-HT receptor and adrenoceptor antagonists on micturition in conscious cats. Eur J Pharmacol, 221: 167, 1992 7. Shimoda, N.: Roles of sacral spinal alpha-adrenoceptive mechanism in micturition reflex in the decerebrate dog. Nippon Hinyokika Gakkai Zasshi, 85: 1747, 1994 8. Ishizuka, O., Mattiasson, A. and Andersson, K.-E.: Role of spinal and peripheral alpha2 adrenoceptors in micturition in normal conscious rats. J Urol, 156: 1853, 1996 9. Durant, P. A., Lucas, P. C. and Yaksh, T. L.: Micturition in the unanesthetized rat: spinal vs. peripheral pharmacology of the adrenergic system. J Pharmacol Exp Ther, 245: 426, 1988 10. Nadelhaft, I., de Groat, W. C. and Morgan, C.: Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat revealed by retrograde axonal transport of horseradish peroxidase. J Comp Neurol, 193: 265, 1980 11. Ueyama, T., Mizuno, N., Nomura, S. et al: Central distribution of afferent and efferent components of the pudendal nerve in cat. J Comp Neurol, 222: 38, 1984 12. Nagatsu, I., Komori, K., Takeuchi, T. et al: Transient tyrosine hydroxylase-immunoreactive neurons in the region of the anterior olfactory nucleus of pre- and postnatal mice do not contain dopamine. Brain Res, 511: 55, 1990 13. Sugaya, K., Ogawa, Y., Hatano, T. et al: Evidence for involvement of the subcoeruleus nucleus and nucleus raphe magnus in urine storage and penile erection in decerebrate rats. J Urol, 159: 2172, 1998 14. Nieuwenhuys, R.: Survey of chemical defined cell groups and pathways. In: Chemoarchitecture of the Brain. Berlin: Springer-Verlag, p. 7, 1985 15. Yoshiyama, M., Roppolo, J. R. and de Groat, W. C.: Effects of LY215490, a competitive alpha-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA) receptor antagonist, on the micturition reflex in the rat. J Pharmacol Exp Ther, 280: 894, 1997 16. Matsumoto, G., Hisamitsu, T. and de Groat, W. C.: Non-NMDA glutamatergic excitatory transmission in the descending limb of the spinobulbospinal micturition reflex pathway of the rat. Brain Res, 693: 246, 1995 17. Nishizawa, O., Igawa, Y., Satoh, T. et al: Effects of glutamate receptor antagonists on lower urinary tract function in conscious unanesthetized rats. Adv Exp Med Biol, 462: 275, 1999 18. Villablanca, J. R. and Hovda, D. A.: Developmental neuroplasticity in a model of cerebral hemispherectomy and stroke. Neuroscience, 95: 625, 2000 19. Blaivas, J. G.: The neurophysiology of micturition: a clinical study of 550 patients. J Urol, 127: 958, 1982 20. Galeano, C., Jubelin, B., Germain, L. et al: Micturitional reflexes in chronic spinalized cats: the underactive detrusor and detrusor-sphincter dyssynergia. Neurourol Urodyn, 5: 45, 1986 21. Sugaya, K., Nishizawa, O. et al: Experimental and clinical studies of urethral anesthesia on etiologies and treatments of detrusor-sphincter dyssynergia. Nippon Hinyokika Gakkai Zasshi, 82: 1133, 1991 22. Al-Ali, M., Salman, G., Rasheed, A. et al: Phenoxybenzamine in the management of neuropathic bladder following spinal cord injury. Aust NZJ Surg, 69: 660, 1999 23. Sakakibara, R., Fowler, C. J., Hattori, T. et al: Pressure-flow study as an evaluating method of neurogenic urethral relax-
MICTURITION IN SPINAL CORD INJURED CATS WITH AUTOGRAFT ation failure. J Auto Nerv Syst, 80: 85, 2000 24. Satoh, K.: Pontine control of the internal urethral sphincter activity on decerebrate dogs. Nippon Hinyokika Gakkai Zasshi, 84: 497, 1993 25. Sugaya, K., Mori, S. and Tsuchida, S.: Efferent and primary afferent neural pathways innervating the lower urinary tract of the cat. Part I. Efferent neural pathways. Nippon Hinyokika Gakkai Zasshi, 79: 868, 1988 26. Sugaya, K.: The effects of chemical stimulations of the pontine
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micturition center in decerebrate cats. Nippon Hinyokika Gakkai Zasshi, 79: 1364, 1988 27. Nishino, H., Ono, T., Shibata, R. et al: Adrenal medullary cells transmute into dopaminergic neurons in dopamine-depleted rat caudate and ameliorate motor disturbances. Brain Res, 445: 325, 1988 28. Clarkson, E. D. and Freed, C. R.: Development of fetal neural transplantation as a treatment for Parkinson’s disease. Life Sci, 65: 2427, 1999
Vol. 166, 2525–2529, December 2001 Printed in U.S.A.
MICTURITION IN THORACIC SPINAL CORD INJURED CATS WITH AUTOGRAFTING OF THE ADRENAL MEDULLA TO THE SACRAL SPINAL CORD KIMIO SUGAYA, YOSHIHIDE OGAWA, TADASHI HATANO, SAORI NISHIJIMA OSAMU NISHIZAWA
AND
From the Departments of Urology, Faculty of Medicine, University of the Ryukyus, Okinawa and Shinshu University School of Medicine, Matsumoto, Japan
ABSTRACT
Purpose: The role of noradrenergic projection from the pontine micturition center to the sacral spinal cord during micturition was examined in thoracic spinal cord injured cats after autografting the adrenal medulla to the sacral spinal cord. Materials and Methods: In 13 female cats the lower thoracic cord was transected and the right adrenal gland was removed under halothane anesthesia. The resected adrenal medulla was divided into several small pieces, which were subsequently autografted to the sacral spinal cord in 7 cats. Another 6 cats underwent sham operation and served as controls. Continuous cystometry and electromyography of the external urethral sphincter were performed every 2 weeks postoperatively without anesthesia. At week 8 the sacral spinal cord was removed and immunohistochemical testing was done to assess tyrosine hydroxylase immunoreactivity. Results: At week 6 the relative mean duration of detrusor-external sphincter coordination plus or minus standard error during bladder contraction was 62.4% ⫾ 4.9% in adrenal grafted cats, which was significantly (p ⫽ 0.0485) longer than in controls (34.2% ⫾ 12.6%). However, maximum bladder contraction pressure, bladder contraction duration and post-void residual urine volume were not significantly different in the 2 groups. Tyrosine hydroxylase immunoreactive cells were observed in and on the sacral spinal cord in adrenal grafted animals but not in controls. Conclusions: Autografting the adrenal medulla to the sacral spinal cord prolonged detrusorexternal sphincter coordination during bladder contraction in thoracic spinal cord injured cats, although other urodynamic parameters did not change. Therefore, noradrenergic projections to the sacral spinal cord may relax the external urethral sphincter during bladder contraction. KEY WORDS: bladder; spinal cord injuries; cats; transplantation, autologous; adrenal glands
Electrical stimulation of the pontine micturition center causes micturition. This center is reportedly present in the nucleus locus coeruleus ␣ in cats1 and dogs.2 The majority of the neurons in this nucleus contain noradrenaline as a transmitter and project to the spinal cord, including the sacral spinal cord.3, 4 Intrathecal injection of an ␣1-adrenoceptor antagonist was reported to inhibit bladder contraction induced by electrical stimulation of the pontine micturition center in cats under ␣-chloralose anesthesia.5 However, Espey et al reported that intrathecal injection of an ␣1adrenoceptor antagonist did not affect bladder contraction in alert cats.6 Shimoda also noted that intrathecal injection of an ␣1-adrenoceptor antagonist did not affect bladder or urethral contraction, while intrathecal injection of an ␣1adrenoceptor agonist increased bladder volume and urethral pressure in decerebrate dogs.7 ␣2-Adrenoceptors in the spinal cord have been reported to relax the urethra in decerebrate dogs7 and female rats8 but contract it in male rats.9 Thus, controversy remains regarding whether descending noradrenergic projections from the pontine micturition center to the sacral spinal cord can evoke bladder contraction and relax the urethra. We examined the effect of autografting the adrenal medulla, which contains catecholamine secreting cells to the sacral spinal cord on bladder and urethral function in thoracic spinal cord injured cats to determine the role
of noradrenergic projections from the pontine micturition center to the sacral spinal cord during micturition. MATERIALS AND METHODS
A total of 13 female cats weighing 1.7 to 3.2 kg. were anesthetized with halothane and underwent laminectomy to expose the lower thoracic spinal cord at T13 and the sacral spinal cord at S1 to S2. After the dura was opened the spinal cord was completely transected at T13. In 7 of the 13 spinal cord injured cats the right adrenal gland was removed via the retroperitoneal approach and the adrenal medulla was cut into 1 to 1.5 mm.3 pieces. Using the tip of a 21 gauge needle the adrenal pieces were inserted into the center of S1 to S2 through the dorsal funiculus. Three pieces were autografted into the gray matter of S1 to S2, where the cell bodies of the neurons are localized in the pelvic and pudendal nerves,10, 11 while another 3 were placed between the dura and S1 to S2. The remaining 6 spinal cord injured cats underwent sham operative grafting of the sacral spinal cord by inserting a needle 3 times. Postoperatively each group received 200 mg. ampicillin intramuscularly twice daily for 5 days. Urine was voided using a urethral catheter or manual bladder compression twice daily for 10 days. At weeks 2, 4 and 6 a 1.2 mm. catheter was inserted into the bladder and the urine was emptied. The cat was placed in a narrow dark box with a hole for the tail and urethral catheter. The tail was fixed to the edge of the hole to prevent catheter dislodgment by movement related to the spinal micturition reflex. Without anesthesia the bladder was continuously per-
Accepted for publication June 29, 2001. Supported by Grant-in-Aid 11671564 for Scientific Research (C) from the Japanese Ministry of Education, Science, Sports and Culture. 2525
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MICTURITION IN SPINAL CORD INJURED CATS WITH AUTOGRAFT
fused with 2.5 to 5 ml. saline per minute through the catheter and continuous cystometry was performed with a 3-way stopcock. At the same time electromyography of the external urethral sphincter was also done with 2 wire electrodes 50 m. in diameter inserted into the muscle from the perineum. Initial bladder volume at which micturition occurred was measured. Mean maximum bladder contraction pressure, mean bladder contraction duration and mean relative detrusor-sphincter coordination duration per bladder contraction were determined from relatively stable continuous cystometry and electromyography data obtained for 20 minutes. Before the catheter was removed post-void residual urine volume after bladder contraction was measured. At week 8 all animals were transcardinally perfused with 0.1 M. phosphate buffered saline (PBS) (1.5 to 2 l.) at 4C for 10 minutes under deep anesthesia with 40 mg./kg. sodium pentobarbital intraperitoneally. After the blood was washed out perfusion was continued with 1.5 l. of ice-cold fixative, composed of 4% paraformaldehyde, 0.1% glutaraldehyde and 0.2% picric acid in 0.1 M. PBS, pH 7.4. The brainstem, lumbosacral spinal cord and adrenal gland were removed and post-fixed for 2 days at 4C in the same fixative minus glutaraldehyde. They were then immersed in 0.1 M. PBS containing 15% sucrose for at least 2 days. Horizontal serial sections (50 m.) of the specimens were cut on a cryotome. Freely floating sections were washed for 4 days in PBS containing 0.3% Triton X-100, incubated for 3 days at 4C with rabbit antiserum for tyrosine hydroxylase,12 diluted 1:1,000 and incubated for 1 hour with biotinylated goat anti-rabbit IgG, diluted 1:1,000 (Vector Laboratories, Burlingame, California). Tyrosine hydroxylase is the first enzyme involved in the process of catecholamine biosynthesis from tyrosine. After each incubation the sections were washed with 3 changes of 0.3% Triton X-100 in PBS. The sections were then reacted for 4 minutes with a mixture of 0.02% 3,3⬘-diaminobenzidine, 0.3% nickel ammonium sulfate and 0.005% H2O2 in 0.05 M. tris-HCl buffer, mounted on gelatin coated glass slides and counterstained with neutral red. Microscopy was performed to examine the location and the number of tyrosine hydroxylase immunoreactive cells. Results are reported as the mean plus or minus standard error. Student’s t-test for paired or unpaired data was used for statistical analysis with p ⬍0.05 considered statistically significant. RESULTS
In control cats mean initial bladder volume plus or minus standard error (SE) inducing micturition was 73.7 ⫾ 27.8 ml. at week 2. This volume showed a significant (p ⫽ 0.0264) decrease to 15.5 ⫾ 8.2 ml. at week 6 (see table). Single voiding volume was small and variable at about 0.5 to 5 ml. after each bladder contraction between weeks 2 and 6. Postvoid residual urine volume also decreased significantly (p ⫽ 0.0200) from 98.0 ⫾ 28.6 ml. at week 2 to 13.5 ⫾ 7.4 at week 6. Mean maximum bladder contraction pressure was 30 to 41 cm. water and mean bladder contraction duration was 16 to 18 seconds between weeks 2 and 6. Persistent detrusorexternal sphincter dyssynergia was detected in 4, 2 and 2 of
the 6 control cats at weeks 2, 4 and 6, respectively (fig. 1, A). The relative duration of detrusor-external sphincter coordination per bladder contraction ranged widely from 0% to 70%. The mean relative duration of detrusor-external sphincter coordination increased from 20.0% ⫾ 12.9% at week 2 to 39.5% ⫾ 12.8% at week 4 and 34.2% ⫾ 12.6% at week 6 but there were no significant change with time (p ⫽ 0.1749 and 0.3839, respectively). In adrenal grafted cats initial mean bladder volume inducing micturition decreased significantly (p ⫽ 0.0102) from 48.9 ⫾ 12.3 ml. at week 2 to 12.6 ⫾ 3.3 at week 6 (see table). Single voiding volume was small and varied after each bladder contraction between weeks 2 and 6, as in controls. Postvoid residual urine volume also decreased significantly (p ⫽ 0.0093) from 50.0 ⫾ 13.3 ml. at week 2 to 10.1 ⫾ 4.3 at week 6. Maximum bladder contraction pressure increased significantly (p ⫽ 0.0033) from 26.6 ⫾ 3.1 cm. water at week 2 to 40.0 ⫾ 3.7 at week 6, while mean bladder contraction duration was 16 to 19 seconds between weeks 2 and 6. Persistent detrusor-external sphincter dyssynergia was noted in 3 of 7 adrenal grafted cats at week 2 only (fig. 1, B). The relative duration of detrusor-external sphincter coordination per bladder contraction was 0% to 66% at week 2, similar to that in control cats, but it became longer (50% to 88%) by week 6. Although the mean relative duration of detrusor-external sphincter coordination increased from 34.4% ⫾ 12.3% at week 2 to 62.4% ⫾ 4.9% at week 6, there was no significant change with time (p ⫽ 0.0876). Electromyography activity in adrenal grafted cats was similar to that in controls during the urine collection period. When urodynamic parameters were compared in the 2 groups, mean threshold bladder volume was smaller in adrenal grafted than in control cats during weeks 2 and 4 but there were no significant differences (p ⫽ 0.3943 and 0.1797, respectively) in the 2 groups because of the large SE. Postvoid residual urine volume was also smaller in adrenal grafted than in control cats during weeks 2 and 4 but again there were no significant differences (p ⫽ 0.1377 and 0.0632, respectively) in the 2 groups. The relative duration of detrusor-external sphincter coordination per bladder contraction showed a significant difference in the 2 groups at week 6 (p ⫽ 0.0485) but it was the only difference in all parameters tested (see table). In adrenal grafted cats tyrosine hydroxylase immunoreactive cells were detected in the adrenal medulla, nucleus locus coeruleus, nucleus locus coeruleus ␣ in the rostral pons and sacral spinal cord. Small nodules on the surface of the sacral cord were easily removed. Immunohistochemical testing revealed that these nodules contained many tyrosine hydroxylase immunoreactive cells. In the adrenal medulla all cells showed strong tyrosine hydroxylase immunoreactivity and were cuboidal without dendrites or axons. Many neurons in the nucleus locus coeruleus ␣ also showed strong tyrosine hydroxylase immunoreactivity and there were numerous immunoreactive dendrites or axons (fig. 2, A). There were 5 to 14 tyrosine hydroxylase immunoreactive cells (mean 9 ⫾ 1) in the sacral spinal cord. These spindle-shaped cells were scattered in the intermediate gray matter and showed weak
Urodynamic parameters in 6 control and 7 adrenal autografted cats 2, 4 and 6 weeks after operation Urodynamic Parameters Threshold bladder vol. inducing micturition (ml.) Max. bladder contraction pressure (cm. water) Bladder contraction duration (sec.) % Relative detrusor-sphincter coordination duration Post-void residual urine vol. (ml.) * Versus 2 weeks p ⬍0.05. † Versus 2 weeks p ⬍0.01. ‡ Versus control p ⫽ 0.0485.
Mean 2 Wks. ⫾ SE
Mean 4 Wks. ⫾ SE
Mean 6 Wks. ⫾ SE
Control
Autografted
Control
Autografted
Control
Autografted
73.7 ⫾ 27.8 30.3 ⫾ 7.2 17.5 ⫾ 1.9 20.0 ⫾ 12.9 98.0 ⫾ 28.6
48.9 ⫾ 12.3 26.6 ⫾ 3.1 16.3 ⫾ 2.4 34.4 ⫾ 12.3 50.0 ⫾ 13.3
51.7 ⫾ 18.3 37.0 ⫾ 3.0 16.0 ⫾ 1.4 39.5 ⫾ 12.8 46.5 ⫾ 17.0
24.9 ⫾ 7.6 38.0 ⫾ 2.4* 17.6 ⫾ 2.2 50.4 ⫾ 5.6 12.6 ⫾ 4.7*
15.5 ⫾ 8.2* 40.7 ⫾ 2.0 17.3 ⫾ 1.1 34.2 ⫾ 12.6 13.5 ⫾ 7.4*
12.6 ⫾ 3.3* 40.0 ⫾ 3.7† 19.0 ⫾ 2.0 62.4 ⫾ 4.9‡ 10.1 ⫾ 4.3†
MICTURITION IN SPINAL CORD INJURED CATS WITH AUTOGRAFT
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FIG. 1. Continuous cystometrography (CMG) and electromyography (EMG) of external urethral sphincter. In control cat detrusorsphincter dyssynergia persisted from weeks 2 to 6. In adrenal grafted cat detrusor-sphincter coordination was present from weeks 2 to 6. FIG. 3. In adrenal grafted cat 6 and 3 tyrosine hydroxylase immunoreactive cells (filled circles) were evident in S1 and S2, respectively. Many tyrosine hydroxylase immunoreactive cells were located in intermediate gray matter. PSN, parasympathetic nucleus. N, nucleus.
FIG. 2. Photomicrographs of pons (A) and sacral spinal cord (B) of adrenal grafted cat. In dorsolateral pontine tegmentum (A) almost all cells in nucleus locus coeruleus (LC) and many in nucleus locus coeruleus ␣ (LCa) were tyrosine hydroxylase immunoreactive cells (dark brown areas). BC, brachium conjunctivum. MTV, trigeminal nerve mesencephalic tract. 4V, fourth ventricle. Bar represents 500 m. In intermediate gray matter of S1 (B) there are 2 spindle-shaped tyrosine hydroxylase immunoreactive cells are visible (arrows). Bar represents 100 m.
immunoreactivity of a few dendrite or axon-like fibers, indicating that they were neurons (figs. 2, B and 3). Tyrosine hydroxylase immunoreactive cells in the small nodules on the surface of the sacral spinal cord were cuboidal. There were no tyrosine hydroxylase immunoreactive cells in the sacral spinal cord in control cats. DISCUSSION
Detrusor-external sphincter dyssynergia was identified during micturition in our thoracic spinal cord injured cats. Our study also demonstrated that transplantation of the adrenal medulla to the sacral cord increased detrusorexternal sphincter coordination during bladder contraction. Therefore, noradrenergic projections from the pontine micturition center to the sacral spinal cord may inhibit external urethral sphincter activity during bladder contraction. The pontine micturition center in cats and dogs is the nucleus locus coeruleus ␣1, 2 but the pontine micturition center in rats is Barrington’s nucleus, which is located just inside the locus coeruleus.13 In rats the neurons of Barrington’s nucleus do not use catecholamines as a neurotrans-
mitter.3, 14 Therefore, there may be species difference in the location of the pontine micturition center and the neurotransmitter of this center. Regarding the role of noradrenergic projections from the upper central nervous system to the sacral cord there have been various conflicting reports.5–9 It has been reported that systemic or intrathecal injection of glutamate receptor antagonists can strongly inhibit the bladder contraction elicited by filling the bladder or by electrical stimulation of the pontine micturition center and can markedly reduce external urethral sphincter activity in rats with and without spinal cord injury.15–17 Therefore, glutaminergic transmission to the sacral spinal cord may mainly induce bladder and urethral contraction, while noradrenergic transmission to the sacral spinal cord may be involved in bladder and urethral contraction or relaxation. After spinal cord injury the spinal micturition reflex appears and gradually develops as a result of neuronal plasticity.18 Neuronal plasticity may have gradually induced a decrease in threshold bladder volume and post-void residual urine as well as an increase in maximum bladder contraction pressure in our spinal cord injured cats. Detrusor-external sphincter dyssynergia also improved gradually but it was persistent, especially in control spinal cats. Detrusorsphincter dyssynergia is a characteristic of patients with suprasacral spinal cord injury.19 Galeano et al reported that the main reasons for such detrusor-sphincter dyssynergia were an increase in the urethro-urethral contraction reflex and weakness of the vesicourethral relaxation reflex.20 In addition, we have shown that blocking the urethro-urethral contraction reflex by urethral anesthesia can improve detrusor-external sphincter dyssynergia and decrease postvoid residual urine in spinal cord injured cats as well as in patients with spinal cord disorders.21 Therefore, it seems possible that projections from the pontine micturition center to the sacral cord may inhibit the urethro-urethral contraction reflex pathway. In our autografted spinal cord injured cats it seems possible that catecholamines from the adrenal medulla grafts acted on the inhibitory ␣2 receptors of motoneurons in the pudendal nerve or interneurons activating these motoneurons and, thus, inhibited this pathway.7 However, it is also possible that motoneuron inhibition developed as a result of neuronal plasticity18 caused by an influence of the implant.
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This study showed a large differences in mean values of threshold bladder volume and post-void residual urine in the 2 groups during weeks 2 and 4 but the differences were not significant because of the large SE. Despite this finding it seems possible that catecholamines from the autografted adrenal medulla acted to decrease threshold bladder volume or post-void residual urine volume. However, these parameters became almost equal in the 2 groups during week 6, and so the effect of neuronal plasticity after spinal cord injury may be greater than that of autografting the adrenal medulla. Also, maximum bladder contraction pressure did not differ in the 2 groups and a reason may have been the existence of detrusor-internal sphincter dyssynergia.22, 23 Electrical stimulation of the pontine micturition center has been reported to evoke relaxation of the internal urethral sphincter in decerebrate dogs with the urethra separated from the bladder.24 The internal urethral sphincter is innervated by the hypogastric nerves, which are sympathetic nerves originating from the upper and middle lumbar cord segments in cats.25 Therefore, autografting adrenal medulla pieces to the lumbar and sacral spinal cord may improve urodynamic parameters by abolishing detrusor-internal sphincter and detrusorexternal sphincter dyssynergia. We found tyrosine hydroxylase immunoreactive cells in the intermediate gray matter of the sacral spinal cord. Although cell bodies for motoneurons of the pudendal nerves innervating the external urethral sphincter are located in the ventrolateral part (Onuf’s nucleus) of the ventral horn of the sacral cord, the neurons in this nucleus project their dendrites into the intermediate gray matter.11 Therefore, it may be possible that the tyrosine hydroxylase immunoreactive cells detected in our cats were connected to the dendrites of neurons in Onuf’s nucleus. However, small nodules of tyrosine hydroxylase immunoreactive cells were also located on the surface of the sacral cord in each autografted cat. These nodules were thought to arise from the 3 pieces of adrenal medulla that were placed between the dura and sacral spinal cord. Since the number of tyrosine hydroxylase immunoreactive cells in the sacral spinal cord was small, most catecholamines acting on the sacral cord may have been produced by these tyrosine hydroxylase immunoreactive cells on the surface of the spinal cord. However, the concentration of catecholamines produced by these cells on the sacral spinal cord may have been low in the upper and mid segments of the lumbar spinal cord, where the sympathetic nucleus innevating the internal urethral sphincter is located, because catecholamine metabolism is rapid in the central nervous system.26 Transplanting sympathetic ganglionic neurons, cells of the adrenal medulla or fetal brain tissue into the brain has been performed in animals27 and in patients with Parkinson’s disease.28 The grafted cells have been reported to change shape,27 as did the adrenal cells grafted to the sacral spinal cord in our subjects. Our study suggests that autografting sympathetic ganglionic neurons or adrenal medulla cells to the spinal cord may eventually be done to treat micturition disorders in patients with spinal cord injury and severe detrusor-sphincter dyssynergia.
CONCLUSIONS
Autografting adrenal medulla pieces to the sacral spinal cord can increase the duration of detrusor-external sphincter coordination during bladder contraction in thoracic spinal cord cats. Noradrenergic projections to the sacral spinal cord may relax the external urethral sphincter during bladder contraction. Although more detailed experiments are necessary, autografting catecholamine secreting cells to the lumbosacral spinal cord may represent possible treatment for severe detrusor-sphincter dyssynergia in patients with spinal cord injury.
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