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Naloxone Induced Micturition in Unanesthetized Paraplegic Cats
0022-5347 /83/1291-0202$02.00/0 Vol. 129, January
THE JOURNAL OF UROLOGY
Copyright© 1983 by The Williams & Wilkins Co.
Printed in U.S.A.
NALOXONE INDUCED MICTURITION IN UNANESTHETIZED PARAPLEGIC CATS KARL B. THOR,* JAMES R. ROPPOLO AND WILLIAM C. DEGROAT From the Department of Pharmacology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania
ABSTRACT
In chronic spinal cats 2 to 10 weeks after transection of the spinal cord at the lower thoracic level (T12-T13), the administration of naloxone, an opiate antagonist, (32-500 µg./kg. i.p.), stimulated micturition. The total quantity of urine released after administration of naloxone ranged from 10 to 70 per cent, (mean 39 per cent) of the initial bladder volume. The response to the drug occurred 5 to 10 minutes after injection and was characterized by repeated periodic expulsion of small quantities of urine (5 to 10 ml.) which coincided with a pattern of hind-limb movement which resembled walking behavior. The effects of naloxone persisted for about 1 hour. The motor activity following administration of naloxone was dependent upon activation of bladder afferents since it did not occur when the bladder was empty. Naloxone also facilitated the release of urine induced by stimulation of somatic afferents. With repeated administration of naloxone, tolerance developed which was evident for several days. These observations suggest that an endogenous opiate may have a tonic inhibitory role in regulation of micturition. Pharmacologic manipulation of this putative inhibitory mechanism may facilitate management of neurogenic bladder dysfunction. Recent immunohistochemical1 and pharmacologic 2 studies have suggested that enkephalin may be a transmitter in sacral parasympathetic reflex pathways to the urinary bladder of the cat. It was shown that leucine enkephalin containing nerve terminals surround neurons in both the sacral parasympathetic nucleus (SPN) 1· 3 and in ganglia in the bladder wall. 4 Leucine enkephalin was also identified in the soma of sacral preganglionic neurons innervating the bladder. 1· 4 In anesthetized cats exogenous enkephalins inhibit transmission in bladder ganglia4 and naloxone, an opiate antagonist, stimulates contractions of the urinary bladder. Stimulation of the bladder by naloxone was accompanied by a sustained increase in the parasympathetic efferent discharge to the bladder, indicative of a stimulatory effect in the central nervous system. 4 These results suggested that naloxone might be effective in facilitating micturition in neurogenic bladder disorders. Therefore, the present investigation was undertaken to examine the effect of naloxone on micturition in chronic spinal cats which exhibited urinary retention. MATERIALS AND METHODS
Nine female cats weighing 2.3 to 3.6 kg. were used in this study. Three of the cats studied were normal animals with an intact neuraxis. In 6 of the animals a laminectomy was performed at the T 12 segment under halothane anesthesia and the spinal cord was transected. The animals were treated with antibiotics and the bladder was expressed manually twice daily until automatic micturition developed. After a recovery period of 1 to 8 weeks, the pharmacologic investigation was started. One of the animals died prior to completion of the study and only partial results are presented. During each testing session, the animals' forepaws were restrained and the animals lay quietly on their side on a padded grid over a pan which collected the urine. After the initial 15minute observation period, the animals were given either an i.p. injection of sterile saline or naloxone hydrochloride. In the initial experiment, cumulative dose-response curves were ob-
tained in each animal by administering multiple injections of naloxone. Starting at a dose of 4 µg./kg., the dosage was doubled every 15 minutes until micturition occurred. Upon establishment of a dose that consistently produced micturition in each animal, single injections of that dose were tested in subsequent trials. The total volume of urine released during the period of observation was measured as well as the remaining urine in the bladder which was expelled by suprapubic pressure. In 6 experiments, after the naloxone-induced release of urine had subsided, the bladder was refilled with sterile saline via a hypodermic needle inserted into the bladder through the abdominal wall. The above tests were then repeated. The effects of naloxone were also studied by measuring intravesical pressure and by recording the electromyogram (EMG) of the external urethral sphincter. Cats with an intact neuraxis were not included in these studies. Bladder pressure was measured by passing a polyethylene cannula into the bladder through the urethra and attaching it to a Statham pressure transducer. The EMG was recorded simultaneously with bladder pressure through 2 needle electrodes inserted 5 mm. lateral to the urethral meatus. RESULTS
Chronic spinal cats exhibited many of the reflex changes observed in human paraplegics. After an approximately 4-day period of areflexia and bladder atony, the cats begin to show recovery of spinal cord function. Within 2 weeks hyperreflexia occurred in certain somatic reflexes (e.g., withdrawal reflex). At about this same time the animals developed some degree of spontaneous bladder activity and release of urine, however, it was not sufficient to empty the bladder. The 6 spinal cats studied in this series always had large volumes of urine in their bladders even after the appearance of spontaneous micturition. In addition to the development of automatic micturition, the animals also exhibited reflex excitation of the bladder by tactile stimulation of the perigenital region. 3 It was also noted that vigorous tactile stimulation of the skin overlying the LrS1 dorsal spinous processes provided sufficient excitation of the bladder to induce urine release. The amount of pressure necessary to evoke micturition by this method in spinal cats appeared to be aversive to cats with an intact neuraxis. Effects of naloxone on spontaneous bladder activity. Intra-
Accepted for publication October 11, 1982. Supported by grant 79-06093 from the National Science Foundation and grant NS18075 from the National Institutes of Health. Requests for reprints: Department of Pharmacology, 572 Scaife Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. 202
203of na!cxone :resulted in the
of
caL I--io-'0.1ever, the response vJas not
weeks after the transection. The re· sponses during the 3rd 4th week and remained stable thereafter. In the initial experiments the cats were cradled in the supine f'"'""'mu in the investigator's arm with his hand supporting animal's back. In this position, doses of naloxone of 32 to 64 /..tg./kg. were sufficient to induce micturition. When the cats were not held, the dose necessary to elicit micturition increased to 125 to 500 µg./kg. This discrep· ancy led us to discover that naloxone enhanced somatovesical reflexes. This will be discussed later. As our goal was to facilitate spontaneous bladder emptying the cats were maintained in a position which did not appear to evoke somatovesical reflexes (see Materials and Methods). In 5 cats that were subjected to multiple tests separated periods of at least one week naloxone consistently produced partial emptying of the bladder. The table gives the volume of urine excreted by the cats in the 35 rninutes following the injection of naloxone expressed as a percentage of the initial bladder volume (range 10 to 70 per cent, mean 39 per cent). Saline injection did not cause urine release. The manner in which micturition occurred deserves detailed description. Within 10 minutes of the injection of naloxone the first urination occurred. Usually the urine was released in 3 to 5 forceful streams over a period of 15 to 20 seconds with each ejection period lasting less than a second. The amount of urine released by the 3 to 5 streams totaled 5 to 8 mt After the 1st release, a period of 20 to 90 seconds elapsed before the next urination occurred. This and subsequent releases of urine again were achieved by short, forceful streams. Usually 5 to 7 of these urination episodes occurred following the naloxone injection. The frequency of the episodes gradually decreased as the bladder emptied. In the most sensitive animal the time between urinations was initially about 25 seconds and progressed to 3 rriinutes. In the least sensitive aninial the between rnicturition episodes v,ras i,,itially 1.5 minutes and progressed to 3 minutes. The episodes occurred over a period of approximately 35 minutes. In 6 experiments, the bladder was :refilled with sterile saline after spontaneous urination ceased. This resulted in a return of periodic urination until a volume of fluid slightly grnater than the amount injected was expelled. This indicated that the cessation of the urination vvas a function of the bladder volume and was not due to of the effect. o~w.~v."c on periodic urinations described were a series of ~~·""'"~" hindlimb movements. The 1st component of somatic activation occurred 7 to 9 seconds before urine release. ilmount ol urine relet?sed by razloxone expressed as ape/· cent of th,e urine in the bladder* Urine Released (%/ml.) Cat l
0.50
2
0.25
3
0.50 0.50 0.25
4 5 Group average
Test 1
Test 2
Test 3
37/85 60/60 11/51 38/78 40/48
37/52 56/55 24/75 40/42 32/49
38/61 69/72 10/51 61/40 35/37
Individual Averages (%) 37 62 15 46 36 39
* Data from 5 cats showing 3 different experiments separated by a period of at least 1 week during which the urine released following naloxone injection was measured. This volume was compared to the total volume in the bladder at the beginning of the experiment, which we determined by emptying the bladder manually at the end of spontaneous micturition. Following each percentage is the volume of urine initially contained in the bladder.
volume spontaneously released X lOO% = Per cent released volume spontaneously released
+ volume manually released
f
naloxone
f.,,IIN
SEC
Effects of naloxone on micturition in chronic spinal cats. A, recording of bladder pressure from an unanesthetized chronic spinal cat. B, recording of bladder pressure in same cat as in A, but after injection of 0.5 mg./kg. naloxone i.p. Arrow below record indicates the injection. Stars above contractions in A and B indicate contraction induced by tactile stimulation of skin overlying the L7 and S 1 vertebrae. The UR above the contractions in B indicates urine release. C, photograph of an oscilloscope record showing on the upper trace sphincter EMG and lower trace bladder pressure. Note the 4 periods of low sphincter activity at times when bladder pressure remains elevated. These periods coincided with the expulsion of urine. Note that the duration of the bladder contraction (20 seconds) corresponds to the time during which hindlimb extension, walking behavior and hindlimb adduction occurred. Vertical calibration equals 10 cm. H20 pressure in A and B and 40 cm. H 20 pressure in lower trace of C.
Usually this 1st motion was a strong bilateral leg extension which lasted approximately 5 seconds. Following leg extension, opposite legs alternately flexed and extended as if walking or running. This behavior lasted 7 to 9 seconds. Towards the end of walking behavior urine was released. As the final streams of urine occurred the legs bilaterally adducted and urination stopped. The somatic response occurred over a period of about 25 seconds. Each periodic urination was associated with the above behavioral episode. In the 20 second to 3 minute period between urinations, the hindlimbs relaxed. When the hind.limbs again bilaterally extended, one could predict walking behavior would follow with subsequent urination and bilateral adduction. When the urination stopped completely the somatic activity also ceased. In the 6 experiments previously described in which after urination had ceased the bladder was refilled, the initiation of urination was again accompanied the above pattern of skeletal muscle As the "···-··~··- motions were absent at low bladder volumes and could be generated simply by bladder, it is reasonable to assume that the "ff"''"'""'."' were responsible for generating the somatic response. To determine the state of the bladder and sphincter during the above ep1s()d,es, a cannula was inserted into the bladder the urethra and electrodes were inserted into the surrounding the urethral meatus. Fifteen minutes after insertion of the cannula and EMG electrodes, control records of bladder and sphincter activity were recorded. The figure, A shows a lack of strong bladder contractions during this control period. FollowLng intraperitoneal injections of naloxone (5 to 250 µg./kg.) (fig., B), strong bladder contractions occurred, each of which lasted approximately 25 seconds. Accompanying the bladder contractions were large increases in the EMG activity, indicative of dyssynergia (see fig., C). Shortly after the bladder pressure began to increase, we observed extension of the animals' hind limbs. At the peak of the contraction, walking behavior ensued followed by adduction of the legs at the end of the contraction. An examination of the EMG during the contractions (fig., C) revealed 3 to 5 brief periods of sphincter relaxation lasting about 1 second, during which urine was released around the cannula. These responses were identical to
204
THOR, ROPPOLO AND DEGROAT
the reflex patterns in the same animals before cannulation with the exception that the doses of naloxone to induce urination were considerably lower (i.e., 5 to 10 µg./kg. i.p. versus 125 to 250 µg./kg. i.p.) when cannula and EMG electrodes were in place. This discrepancy in doses may be attributed to stimulation of sensory afferents in the urethra and sphincter by the cannula and electrodes. Effects of naloxone on somatovesical reflexes. Naloxone facilitated somatovesical reflexes. As described earlier, urination in chronic spinal cats could be induced by tactile stimulation of the skin overlying the lumbosacral vertebrae. Prior to naloxone injection, vigorous stimulation over the dorsal processes of the vertebrae had to be maintained for 7 to 10 seconds to elicit micturition in the chronic spinal cats. The urination ended within 2 seconds after termination of the stimulation. Following the naloxone injections, urination could be induced by gentle stroking of the hairs in this region for 1 to 2 seconds. Often a puff of air on this region evoked urination. Urination continued for 5 to 6 seconds after the stimulation was terminated. Enhancement of the somatovesical reflex was noted with doses of naloxone as low as 5 µg./kg. The figure, A, shows the intravesical pressure recording during tactile stimulation of the skin over the L1, S1 region. The figure, B, shows the contraction produced by the same stimulation following naloxone injection. Reduction of the threshold doses of naloxone to induce urination when the animals were hand held and when recording apparatus were inserted provide additional evidence of increased somatic excitation of the bladder. It was also noticed that noxious forms of stimulation (e.g., pinch of hindpaws bilaterally) were effective in causing urination after naloxone whereas they were ineffective prior to naloxone. The above results were consistent when naloxone injections were separated by a 4-day period. However, if the injections were repeated on successive days, the effect was greatly attenuated or absent, indicative of tolerance. Tachyphylaxis to naloxone effects was also evident at a short dosing interval. For example, a dose of naloxone was much more effective when given as a single injection as opposed to initially injecting small multiple doses in an attempt to construct a cumulative dose response curve. Tolerance and tachyphylaxis to naloxone were evident in the effects on spontaneous bladder activity, vesicosomatic reflexes and somatovesical reflexes. None of the behavioral effects (i.e., urine release and/or activation of motor reflexes) seen in chronic spinal cats following naloxone injection were seen in three unanaesthetized cats with an intact neuraxis. DISCUSSION
Various evidence suggests a role of enkephalins as transmitters in reflex pathways to the urinary bladder of the cat. In the periphery, those neurons which directly innervate the smooth muscle of the bladder are surrounded by leucine enkephalin terminals which arise from preganglionic neurons in the sacral parasympathetic nucleus (SPN). 4 In vivo and in vitro studies have determined that enkephalins are inhibitory to synaptic transmission in the bladder ganglia. This inhibition is mediated by a decrease in the presynaptic release of excitatory neurotransmitters. 4' 5 In the sacral spinal cord, enkephalin is contained in interneurons which project to Lamina I and II of the dorsal horn. Lamina I also receives the strongest projection of processes from primary afferent neurons which innervate the bladder. 6 Additionally, leucine enkephalin terminals of unknown origin are heavily concentrated around preganglionic neurons of the SPN. 1• 4 As these preganglionic neurons also contain leucine enkephalin, it is possible these terminals are recurrent collaterals functioning in an inhibitory feedback mechanism. 7 .This point is unresolved at the present time. In addition to the morphologic association of enkephalins with sacral parasympathetic pathways to the bladder, recent demonstrations in anesthetized cats with an intact neuraxis of enhanced bladder activity upon injection of naloxone, and conversely inhibition of bladder activity following intrathecal
administration of Leu-enkephalin to the sacral spinal cord, indicate enkephalins play a physiological role in normal bladder function. 2 ' 4 The present paper demonstrates that at least part of the effect of naloxone can be attributed to an action on lumbosacral spinal cord and is not dependent upon centers in the brain. At the present time the most likely sites of naloxone's actions on spontaneous bladder activity are a blockade of the inhibitory action of endogenous leucine enkephalin, which is in the vicinity of the preganglionic neurons, or which is localized in the region of bladder afferent terminals in the dorsal horn. A 3rd possible site of action might be in the ganglia of the bladder; however, the effects of naloxone on ganglionic transmission are much less conspicuous than its central effects. In addition to the effects of enkephalins on bladder pathways, the present finding suggests that opiate peptides play a role in regulating somatic reflexes in the isolated spinal cord. This is evident by the ability of naloxone to enhance somatovesical and vesicosomatic reflexes. The ability of naloxone to enhance somatic activation of bladder pathways could arise in part from the heightened excitability of the preganglionic neurons due to antagonism of a tonic enkephalinergic inhibition. In addition the drug may be affecting the sensory input in the dorsal horn since the type of somatic stimulation required to evoke urination changed from noxious to nonnoxious following naloxone injection. It is interesting to note that the regions of tactile stimulation most capable of eliciting a bladder contraction (i.e., the perigenital region and the region overlying the dorsal processes of the lumbosacral vertebrae) are both areas of considerable overlap of tactile dermatomes (see fig. 6, p. 176, ref. 8). This suggests that convergence of somatic inputs from several segments might be necessary to cause excitation of bladder preganglionic neurons. The vesicosomatic mechanisms responsible for activation of complex patterns of motor movement during bladder distension is almost totally unexplored. Many theories exist for explaining patterned leg movement by the isolated spinal cord,9 but none of these theories include an activation of the reflexes by vesical afferents. The present data suggests that enkephalinergic neurons are responsible for inhibiting vesical afferent excitation of motor systems. Although a sparse labelling of enkephalin terminals is detected in the lumbar ventral horn, the much greater labelling of enkephalin terminals in the dorsal horn suggests that naloxone may enhance vesicosomatic reflexes by permitting weak vesical sensory inputs to be unmasked. Enkephalinergic inputs to sphincter motoneurons may also be affected by naloxone. Sphincter motor neurons are localized in Onuf's nucleus, which shows a very conspicuous labelling of enkephalin terminals in contrast to other lumbosacral motor nuclei. 1 It is possible that the twitching of the perinea! musculature and the pronounced EMG discharge of the sphincters during bladder contractions following naloxone are a result of blockade of enkephalinergic inhibition to this motor nucleus in addition to naloxone's effects in the dorsal horn. During the preparation of this manuscript a report was published 10 which described an enhancement by naloxone of bladder contractions in humans with neurogenic bladder. This study reported that in 5 patients with incomplete spinal lesions who exhibited urinary retention, the amount of bladder distension necessary to evoke detrusor reflex activity was reduced by an average of 150 ml. following naloxone (400 µg. i.v.). However, in 2 patients diagnosed as having complete spinal transections and areflexic bladders when distended to volumes as great as 600 ml., naloxone had no effect on bladder activity. These clinical findings would suggest that naloxone is only effective when descending inputs from the brain to the spinal cord remain partially intact. However, our observations in cats suggest otherwise. In this regard it should be noted that micturition in humans11-14 and cats 3 is mediated by a supraspinal pathway. However, during recovery from spinal injury, reflex pathways intrinsic to the sacral spinal cord develop which can initiate
205
NALOXONE INDUCED MICTURITION
automatic micturition. 3 In cats this recovery process requires 1 to 2 weeks, whereas in paraplegic patients recovery takes many weeks to months. As stated in the present report, the effects of naloxone in the cat were absent in the 1st 2 weeks following cord transection, and were not maximal until the 4th week. Possibly naloxone would induce micturition in human patients with complete cord transections provided sufficient time were allowed for recovery of spinal micturition reflexes. In addition, since dosages of 125 to 250 µg./kg. were required to induce urination in the spinal cats, it may be necessary to use larger doses in paraplegic patients than those employed in the aforementioned clinical trial. This should be possible since doses as large as 2.8 gm. have been given to human volunteers with no adverse affects, 15 although 2 deaths have been reported following administration of naloxone during surgery. 16 One of the characteristics of the action of naloxone that will affect possible clinical applications in urology is the development of tolerance. Administration of the drug on consecutive days resulted in a markedly attenuated response on the 2nd day. In the most sensitive cat (cat 2 in table) a doubling of the previous day's dose occasionally produced a small response, but in other animals even a small response was not observed. In the clinical study< 10) discussed earlier, tolerance was not mentioned, however, the authors do state that the naloxone effects were evanescent. Tachyphylaxis to naloxone has also been noted with the drug's effects on bladder activity in intact anesthetized cats (unpublished observations) and with its effects on monosynaptic and polysynaptic reflexes. 17 However, long term tolerance lasting days as seen following morphine administration has not been reported with naloxone until the present study. In conclusion, it appears that the effects of naloxone on bladder function warrant further investigation in humans. Experiments should include paraplegic patients with complete spinal transection allowing sufficient time for spinal shock to subside and for the development of some degree of bladder automaticity. In combination with pharmacologic agents that inhibit sphincter dyssynergia, sufficiently low doses ofnaloxone might be used to promote bladder emptying without the development of tolerance. Acknowledgments. The authors would like to thank Joseph Come and Susan Schellenberg for conscientiously caring for the spinal cats used in this study. Generous donations of naloxone hydrochloride were supplied by Endo Laboratories. Aircap SD240, cushion material to prevent ulceration of the skin of the cats at pressure points, was supplied by Sealed Air Corp. (Danbury, Connecticut).
REFERENCES
1. Glazer, E. J. and Basbaum, A. I.: Immunohistochemical localization of leucine-enkephalin in the spinal cord of the cat: enkephalincontaining marginal neurons and pain modulation. J. Comp. Neurol., 196: 377, 1981. 2. de Groat, W. C., Booth, A. M., Milne, R. J. and Roppolo, J. R.: Parasympathetic preganglionic neurons in the sacral spinal cord. J. Auton. Nerv. Syst., 5: 23, 1982. 3. de Groat, W. C., Nadelhaft, I., Milne, R. J., Booth, A. M., Morgan, C. and Thor, K. B.: Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine. J. Auton. Nerv. Syst., 3: 135, 1981. 4. de Groat, W. C., Kawatani, M., Hisamitsu, T., Lowe, I., Morgan, C., Roppolo, J., Booth, A., Nadelhaft, I., Kuo, D. and Thor, K. B.: The role of neuropeptides in the sacral autonomic reflex pathways of the cat. J. Autonomic Nerv. Syst., (In Press) 1982. 5. Konishi, S., Tsuno, A. and Otsuka, M.: Enkephalin as a transmitter for presynaptic inhibition in sympathetic ganglia. Nature, 294: 80, 1981. 6. Morgan, C., Nadelhaft, I. and de Groat, W. C.: The distribution of visceral primary afferents from the pelvic nerve to Lissauer's tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J. Comp. Neurol., 201: 415, 1981. 7. de Groat, W. C. and Ryall, R. W.: Recurrent inhibition in sacral parasympathetic pathways to the bladder. J. Physiol., 196: 579, 1968. 8. Kuhn, R. A.: Organization of tactile dermatomes in cat and monkey. J. Comp. Anat., 16: 169, 1953. 9. Grillner, S.: Locomotion in vertebrates: central mechanisms and reflex interactions. Physiol. Rev., 55: 247, 1975. 10. Vaidyanathan, S., Rao, M. S., Chary, K. S. N., Sharma, P. L. and Das, N.: Enhancement of detrusor reflex activity by naloxone in patients with chronic neurogenic bladder dysfunction. Preliminary report. J. Urol., 126: 500, 1981. 11. de Groat, W. C. and Booth, A. M.: Physiology of the urinary bladder and urethra. Ann. Intern. Med., 92: 312, 1980. 12. Kuru, M.: Nervous control of micturition. Physiol. Rev., 45: 425, 1965. 13. Wein, A. J. and Raezer, D. M.: Physiology of micturition. In: Clinical Neurology. Edited by R. J. Krane and M. B. Siroky. Boston: Little, Brown and Co., chapt. 1, p. 1, 1979. 14. Bradley, W. E.: Innervation of the male urinary bladder. Urol. Clin. North. Am., 5: 279, 1978. 15. Zaks, A., Jones, T., Fink, M. and Freedman, A. M.: Naloxone treatment of opiate dependence. J. A. M.A., 215: 2108, 1971. 16. Andree, R. A.: Sudden death following naloxone administration. Anesth. Analg., 59: 782, 1980. 17. Goldfarb, J. and Hu, J. W.: Enhancement of reflexes by naloxone in spinal cats. Neuropharmacology, 15: 785, 1976.
THE JOURNAL OF UROLOGY
Copyright© 1983 by The Williams & Wilkins Co.
Printed in U.S.A.
NALOXONE INDUCED MICTURITION IN UNANESTHETIZED PARAPLEGIC CATS KARL B. THOR,* JAMES R. ROPPOLO AND WILLIAM C. DEGROAT From the Department of Pharmacology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania
ABSTRACT
In chronic spinal cats 2 to 10 weeks after transection of the spinal cord at the lower thoracic level (T12-T13), the administration of naloxone, an opiate antagonist, (32-500 µg./kg. i.p.), stimulated micturition. The total quantity of urine released after administration of naloxone ranged from 10 to 70 per cent, (mean 39 per cent) of the initial bladder volume. The response to the drug occurred 5 to 10 minutes after injection and was characterized by repeated periodic expulsion of small quantities of urine (5 to 10 ml.) which coincided with a pattern of hind-limb movement which resembled walking behavior. The effects of naloxone persisted for about 1 hour. The motor activity following administration of naloxone was dependent upon activation of bladder afferents since it did not occur when the bladder was empty. Naloxone also facilitated the release of urine induced by stimulation of somatic afferents. With repeated administration of naloxone, tolerance developed which was evident for several days. These observations suggest that an endogenous opiate may have a tonic inhibitory role in regulation of micturition. Pharmacologic manipulation of this putative inhibitory mechanism may facilitate management of neurogenic bladder dysfunction. Recent immunohistochemical1 and pharmacologic 2 studies have suggested that enkephalin may be a transmitter in sacral parasympathetic reflex pathways to the urinary bladder of the cat. It was shown that leucine enkephalin containing nerve terminals surround neurons in both the sacral parasympathetic nucleus (SPN) 1· 3 and in ganglia in the bladder wall. 4 Leucine enkephalin was also identified in the soma of sacral preganglionic neurons innervating the bladder. 1· 4 In anesthetized cats exogenous enkephalins inhibit transmission in bladder ganglia4 and naloxone, an opiate antagonist, stimulates contractions of the urinary bladder. Stimulation of the bladder by naloxone was accompanied by a sustained increase in the parasympathetic efferent discharge to the bladder, indicative of a stimulatory effect in the central nervous system. 4 These results suggested that naloxone might be effective in facilitating micturition in neurogenic bladder disorders. Therefore, the present investigation was undertaken to examine the effect of naloxone on micturition in chronic spinal cats which exhibited urinary retention. MATERIALS AND METHODS
Nine female cats weighing 2.3 to 3.6 kg. were used in this study. Three of the cats studied were normal animals with an intact neuraxis. In 6 of the animals a laminectomy was performed at the T 12 segment under halothane anesthesia and the spinal cord was transected. The animals were treated with antibiotics and the bladder was expressed manually twice daily until automatic micturition developed. After a recovery period of 1 to 8 weeks, the pharmacologic investigation was started. One of the animals died prior to completion of the study and only partial results are presented. During each testing session, the animals' forepaws were restrained and the animals lay quietly on their side on a padded grid over a pan which collected the urine. After the initial 15minute observation period, the animals were given either an i.p. injection of sterile saline or naloxone hydrochloride. In the initial experiment, cumulative dose-response curves were ob-
tained in each animal by administering multiple injections of naloxone. Starting at a dose of 4 µg./kg., the dosage was doubled every 15 minutes until micturition occurred. Upon establishment of a dose that consistently produced micturition in each animal, single injections of that dose were tested in subsequent trials. The total volume of urine released during the period of observation was measured as well as the remaining urine in the bladder which was expelled by suprapubic pressure. In 6 experiments, after the naloxone-induced release of urine had subsided, the bladder was refilled with sterile saline via a hypodermic needle inserted into the bladder through the abdominal wall. The above tests were then repeated. The effects of naloxone were also studied by measuring intravesical pressure and by recording the electromyogram (EMG) of the external urethral sphincter. Cats with an intact neuraxis were not included in these studies. Bladder pressure was measured by passing a polyethylene cannula into the bladder through the urethra and attaching it to a Statham pressure transducer. The EMG was recorded simultaneously with bladder pressure through 2 needle electrodes inserted 5 mm. lateral to the urethral meatus. RESULTS
Chronic spinal cats exhibited many of the reflex changes observed in human paraplegics. After an approximately 4-day period of areflexia and bladder atony, the cats begin to show recovery of spinal cord function. Within 2 weeks hyperreflexia occurred in certain somatic reflexes (e.g., withdrawal reflex). At about this same time the animals developed some degree of spontaneous bladder activity and release of urine, however, it was not sufficient to empty the bladder. The 6 spinal cats studied in this series always had large volumes of urine in their bladders even after the appearance of spontaneous micturition. In addition to the development of automatic micturition, the animals also exhibited reflex excitation of the bladder by tactile stimulation of the perigenital region. 3 It was also noted that vigorous tactile stimulation of the skin overlying the LrS1 dorsal spinous processes provided sufficient excitation of the bladder to induce urine release. The amount of pressure necessary to evoke micturition by this method in spinal cats appeared to be aversive to cats with an intact neuraxis. Effects of naloxone on spontaneous bladder activity. Intra-
Accepted for publication October 11, 1982. Supported by grant 79-06093 from the National Science Foundation and grant NS18075 from the National Institutes of Health. Requests for reprints: Department of Pharmacology, 572 Scaife Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. 202
203of na!cxone :resulted in the
of
caL I--io-'0.1ever, the response vJas not
weeks after the transection. The re· sponses during the 3rd 4th week and remained stable thereafter. In the initial experiments the cats were cradled in the supine f'"'""'mu in the investigator's arm with his hand supporting animal's back. In this position, doses of naloxone of 32 to 64 /..tg./kg. were sufficient to induce micturition. When the cats were not held, the dose necessary to elicit micturition increased to 125 to 500 µg./kg. This discrep· ancy led us to discover that naloxone enhanced somatovesical reflexes. This will be discussed later. As our goal was to facilitate spontaneous bladder emptying the cats were maintained in a position which did not appear to evoke somatovesical reflexes (see Materials and Methods). In 5 cats that were subjected to multiple tests separated periods of at least one week naloxone consistently produced partial emptying of the bladder. The table gives the volume of urine excreted by the cats in the 35 rninutes following the injection of naloxone expressed as a percentage of the initial bladder volume (range 10 to 70 per cent, mean 39 per cent). Saline injection did not cause urine release. The manner in which micturition occurred deserves detailed description. Within 10 minutes of the injection of naloxone the first urination occurred. Usually the urine was released in 3 to 5 forceful streams over a period of 15 to 20 seconds with each ejection period lasting less than a second. The amount of urine released by the 3 to 5 streams totaled 5 to 8 mt After the 1st release, a period of 20 to 90 seconds elapsed before the next urination occurred. This and subsequent releases of urine again were achieved by short, forceful streams. Usually 5 to 7 of these urination episodes occurred following the naloxone injection. The frequency of the episodes gradually decreased as the bladder emptied. In the most sensitive animal the time between urinations was initially about 25 seconds and progressed to 3 rriinutes. In the least sensitive aninial the between rnicturition episodes v,ras i,,itially 1.5 minutes and progressed to 3 minutes. The episodes occurred over a period of approximately 35 minutes. In 6 experiments, the bladder was :refilled with sterile saline after spontaneous urination ceased. This resulted in a return of periodic urination until a volume of fluid slightly grnater than the amount injected was expelled. This indicated that the cessation of the urination vvas a function of the bladder volume and was not due to of the effect. o~w.~v."c on periodic urinations described were a series of ~~·""'"~" hindlimb movements. The 1st component of somatic activation occurred 7 to 9 seconds before urine release. ilmount ol urine relet?sed by razloxone expressed as ape/· cent of th,e urine in the bladder* Urine Released (%/ml.) Cat l
0.50
2
0.25
3
0.50 0.50 0.25
4 5 Group average
Test 1
Test 2
Test 3
37/85 60/60 11/51 38/78 40/48
37/52 56/55 24/75 40/42 32/49
38/61 69/72 10/51 61/40 35/37
Individual Averages (%) 37 62 15 46 36 39
* Data from 5 cats showing 3 different experiments separated by a period of at least 1 week during which the urine released following naloxone injection was measured. This volume was compared to the total volume in the bladder at the beginning of the experiment, which we determined by emptying the bladder manually at the end of spontaneous micturition. Following each percentage is the volume of urine initially contained in the bladder.
volume spontaneously released X lOO% = Per cent released volume spontaneously released
+ volume manually released
f
naloxone
f.,,IIN
SEC
Effects of naloxone on micturition in chronic spinal cats. A, recording of bladder pressure from an unanesthetized chronic spinal cat. B, recording of bladder pressure in same cat as in A, but after injection of 0.5 mg./kg. naloxone i.p. Arrow below record indicates the injection. Stars above contractions in A and B indicate contraction induced by tactile stimulation of skin overlying the L7 and S 1 vertebrae. The UR above the contractions in B indicates urine release. C, photograph of an oscilloscope record showing on the upper trace sphincter EMG and lower trace bladder pressure. Note the 4 periods of low sphincter activity at times when bladder pressure remains elevated. These periods coincided with the expulsion of urine. Note that the duration of the bladder contraction (20 seconds) corresponds to the time during which hindlimb extension, walking behavior and hindlimb adduction occurred. Vertical calibration equals 10 cm. H20 pressure in A and B and 40 cm. H 20 pressure in lower trace of C.
Usually this 1st motion was a strong bilateral leg extension which lasted approximately 5 seconds. Following leg extension, opposite legs alternately flexed and extended as if walking or running. This behavior lasted 7 to 9 seconds. Towards the end of walking behavior urine was released. As the final streams of urine occurred the legs bilaterally adducted and urination stopped. The somatic response occurred over a period of about 25 seconds. Each periodic urination was associated with the above behavioral episode. In the 20 second to 3 minute period between urinations, the hindlimbs relaxed. When the hind.limbs again bilaterally extended, one could predict walking behavior would follow with subsequent urination and bilateral adduction. When the urination stopped completely the somatic activity also ceased. In the 6 experiments previously described in which after urination had ceased the bladder was refilled, the initiation of urination was again accompanied the above pattern of skeletal muscle As the "···-··~··- motions were absent at low bladder volumes and could be generated simply by bladder, it is reasonable to assume that the "ff"''"'""'."' were responsible for generating the somatic response. To determine the state of the bladder and sphincter during the above ep1s()d,es, a cannula was inserted into the bladder the urethra and electrodes were inserted into the surrounding the urethral meatus. Fifteen minutes after insertion of the cannula and EMG electrodes, control records of bladder and sphincter activity were recorded. The figure, A shows a lack of strong bladder contractions during this control period. FollowLng intraperitoneal injections of naloxone (5 to 250 µg./kg.) (fig., B), strong bladder contractions occurred, each of which lasted approximately 25 seconds. Accompanying the bladder contractions were large increases in the EMG activity, indicative of dyssynergia (see fig., C). Shortly after the bladder pressure began to increase, we observed extension of the animals' hind limbs. At the peak of the contraction, walking behavior ensued followed by adduction of the legs at the end of the contraction. An examination of the EMG during the contractions (fig., C) revealed 3 to 5 brief periods of sphincter relaxation lasting about 1 second, during which urine was released around the cannula. These responses were identical to
204
THOR, ROPPOLO AND DEGROAT
the reflex patterns in the same animals before cannulation with the exception that the doses of naloxone to induce urination were considerably lower (i.e., 5 to 10 µg./kg. i.p. versus 125 to 250 µg./kg. i.p.) when cannula and EMG electrodes were in place. This discrepancy in doses may be attributed to stimulation of sensory afferents in the urethra and sphincter by the cannula and electrodes. Effects of naloxone on somatovesical reflexes. Naloxone facilitated somatovesical reflexes. As described earlier, urination in chronic spinal cats could be induced by tactile stimulation of the skin overlying the lumbosacral vertebrae. Prior to naloxone injection, vigorous stimulation over the dorsal processes of the vertebrae had to be maintained for 7 to 10 seconds to elicit micturition in the chronic spinal cats. The urination ended within 2 seconds after termination of the stimulation. Following the naloxone injections, urination could be induced by gentle stroking of the hairs in this region for 1 to 2 seconds. Often a puff of air on this region evoked urination. Urination continued for 5 to 6 seconds after the stimulation was terminated. Enhancement of the somatovesical reflex was noted with doses of naloxone as low as 5 µg./kg. The figure, A, shows the intravesical pressure recording during tactile stimulation of the skin over the L1, S1 region. The figure, B, shows the contraction produced by the same stimulation following naloxone injection. Reduction of the threshold doses of naloxone to induce urination when the animals were hand held and when recording apparatus were inserted provide additional evidence of increased somatic excitation of the bladder. It was also noticed that noxious forms of stimulation (e.g., pinch of hindpaws bilaterally) were effective in causing urination after naloxone whereas they were ineffective prior to naloxone. The above results were consistent when naloxone injections were separated by a 4-day period. However, if the injections were repeated on successive days, the effect was greatly attenuated or absent, indicative of tolerance. Tachyphylaxis to naloxone effects was also evident at a short dosing interval. For example, a dose of naloxone was much more effective when given as a single injection as opposed to initially injecting small multiple doses in an attempt to construct a cumulative dose response curve. Tolerance and tachyphylaxis to naloxone were evident in the effects on spontaneous bladder activity, vesicosomatic reflexes and somatovesical reflexes. None of the behavioral effects (i.e., urine release and/or activation of motor reflexes) seen in chronic spinal cats following naloxone injection were seen in three unanaesthetized cats with an intact neuraxis. DISCUSSION
Various evidence suggests a role of enkephalins as transmitters in reflex pathways to the urinary bladder of the cat. In the periphery, those neurons which directly innervate the smooth muscle of the bladder are surrounded by leucine enkephalin terminals which arise from preganglionic neurons in the sacral parasympathetic nucleus (SPN). 4 In vivo and in vitro studies have determined that enkephalins are inhibitory to synaptic transmission in the bladder ganglia. This inhibition is mediated by a decrease in the presynaptic release of excitatory neurotransmitters. 4' 5 In the sacral spinal cord, enkephalin is contained in interneurons which project to Lamina I and II of the dorsal horn. Lamina I also receives the strongest projection of processes from primary afferent neurons which innervate the bladder. 6 Additionally, leucine enkephalin terminals of unknown origin are heavily concentrated around preganglionic neurons of the SPN. 1• 4 As these preganglionic neurons also contain leucine enkephalin, it is possible these terminals are recurrent collaterals functioning in an inhibitory feedback mechanism. 7 .This point is unresolved at the present time. In addition to the morphologic association of enkephalins with sacral parasympathetic pathways to the bladder, recent demonstrations in anesthetized cats with an intact neuraxis of enhanced bladder activity upon injection of naloxone, and conversely inhibition of bladder activity following intrathecal
administration of Leu-enkephalin to the sacral spinal cord, indicate enkephalins play a physiological role in normal bladder function. 2 ' 4 The present paper demonstrates that at least part of the effect of naloxone can be attributed to an action on lumbosacral spinal cord and is not dependent upon centers in the brain. At the present time the most likely sites of naloxone's actions on spontaneous bladder activity are a blockade of the inhibitory action of endogenous leucine enkephalin, which is in the vicinity of the preganglionic neurons, or which is localized in the region of bladder afferent terminals in the dorsal horn. A 3rd possible site of action might be in the ganglia of the bladder; however, the effects of naloxone on ganglionic transmission are much less conspicuous than its central effects. In addition to the effects of enkephalins on bladder pathways, the present finding suggests that opiate peptides play a role in regulating somatic reflexes in the isolated spinal cord. This is evident by the ability of naloxone to enhance somatovesical and vesicosomatic reflexes. The ability of naloxone to enhance somatic activation of bladder pathways could arise in part from the heightened excitability of the preganglionic neurons due to antagonism of a tonic enkephalinergic inhibition. In addition the drug may be affecting the sensory input in the dorsal horn since the type of somatic stimulation required to evoke urination changed from noxious to nonnoxious following naloxone injection. It is interesting to note that the regions of tactile stimulation most capable of eliciting a bladder contraction (i.e., the perigenital region and the region overlying the dorsal processes of the lumbosacral vertebrae) are both areas of considerable overlap of tactile dermatomes (see fig. 6, p. 176, ref. 8). This suggests that convergence of somatic inputs from several segments might be necessary to cause excitation of bladder preganglionic neurons. The vesicosomatic mechanisms responsible for activation of complex patterns of motor movement during bladder distension is almost totally unexplored. Many theories exist for explaining patterned leg movement by the isolated spinal cord,9 but none of these theories include an activation of the reflexes by vesical afferents. The present data suggests that enkephalinergic neurons are responsible for inhibiting vesical afferent excitation of motor systems. Although a sparse labelling of enkephalin terminals is detected in the lumbar ventral horn, the much greater labelling of enkephalin terminals in the dorsal horn suggests that naloxone may enhance vesicosomatic reflexes by permitting weak vesical sensory inputs to be unmasked. Enkephalinergic inputs to sphincter motoneurons may also be affected by naloxone. Sphincter motor neurons are localized in Onuf's nucleus, which shows a very conspicuous labelling of enkephalin terminals in contrast to other lumbosacral motor nuclei. 1 It is possible that the twitching of the perinea! musculature and the pronounced EMG discharge of the sphincters during bladder contractions following naloxone are a result of blockade of enkephalinergic inhibition to this motor nucleus in addition to naloxone's effects in the dorsal horn. During the preparation of this manuscript a report was published 10 which described an enhancement by naloxone of bladder contractions in humans with neurogenic bladder. This study reported that in 5 patients with incomplete spinal lesions who exhibited urinary retention, the amount of bladder distension necessary to evoke detrusor reflex activity was reduced by an average of 150 ml. following naloxone (400 µg. i.v.). However, in 2 patients diagnosed as having complete spinal transections and areflexic bladders when distended to volumes as great as 600 ml., naloxone had no effect on bladder activity. These clinical findings would suggest that naloxone is only effective when descending inputs from the brain to the spinal cord remain partially intact. However, our observations in cats suggest otherwise. In this regard it should be noted that micturition in humans11-14 and cats 3 is mediated by a supraspinal pathway. However, during recovery from spinal injury, reflex pathways intrinsic to the sacral spinal cord develop which can initiate
205
NALOXONE INDUCED MICTURITION
automatic micturition. 3 In cats this recovery process requires 1 to 2 weeks, whereas in paraplegic patients recovery takes many weeks to months. As stated in the present report, the effects of naloxone in the cat were absent in the 1st 2 weeks following cord transection, and were not maximal until the 4th week. Possibly naloxone would induce micturition in human patients with complete cord transections provided sufficient time were allowed for recovery of spinal micturition reflexes. In addition, since dosages of 125 to 250 µg./kg. were required to induce urination in the spinal cats, it may be necessary to use larger doses in paraplegic patients than those employed in the aforementioned clinical trial. This should be possible since doses as large as 2.8 gm. have been given to human volunteers with no adverse affects, 15 although 2 deaths have been reported following administration of naloxone during surgery. 16 One of the characteristics of the action of naloxone that will affect possible clinical applications in urology is the development of tolerance. Administration of the drug on consecutive days resulted in a markedly attenuated response on the 2nd day. In the most sensitive cat (cat 2 in table) a doubling of the previous day's dose occasionally produced a small response, but in other animals even a small response was not observed. In the clinical study< 10) discussed earlier, tolerance was not mentioned, however, the authors do state that the naloxone effects were evanescent. Tachyphylaxis to naloxone has also been noted with the drug's effects on bladder activity in intact anesthetized cats (unpublished observations) and with its effects on monosynaptic and polysynaptic reflexes. 17 However, long term tolerance lasting days as seen following morphine administration has not been reported with naloxone until the present study. In conclusion, it appears that the effects of naloxone on bladder function warrant further investigation in humans. Experiments should include paraplegic patients with complete spinal transection allowing sufficient time for spinal shock to subside and for the development of some degree of bladder automaticity. In combination with pharmacologic agents that inhibit sphincter dyssynergia, sufficiently low doses ofnaloxone might be used to promote bladder emptying without the development of tolerance. Acknowledgments. The authors would like to thank Joseph Come and Susan Schellenberg for conscientiously caring for the spinal cats used in this study. Generous donations of naloxone hydrochloride were supplied by Endo Laboratories. Aircap SD240, cushion material to prevent ulceration of the skin of the cats at pressure points, was supplied by Sealed Air Corp. (Danbury, Connecticut).
REFERENCES
1. Glazer, E. J. and Basbaum, A. I.: Immunohistochemical localization of leucine-enkephalin in the spinal cord of the cat: enkephalincontaining marginal neurons and pain modulation. J. Comp. Neurol., 196: 377, 1981. 2. de Groat, W. C., Booth, A. M., Milne, R. J. and Roppolo, J. R.: Parasympathetic preganglionic neurons in the sacral spinal cord. J. Auton. Nerv. Syst., 5: 23, 1982. 3. de Groat, W. C., Nadelhaft, I., Milne, R. J., Booth, A. M., Morgan, C. and Thor, K. B.: Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine. J. Auton. Nerv. Syst., 3: 135, 1981. 4. de Groat, W. C., Kawatani, M., Hisamitsu, T., Lowe, I., Morgan, C., Roppolo, J., Booth, A., Nadelhaft, I., Kuo, D. and Thor, K. B.: The role of neuropeptides in the sacral autonomic reflex pathways of the cat. J. Autonomic Nerv. Syst., (In Press) 1982. 5. Konishi, S., Tsuno, A. and Otsuka, M.: Enkephalin as a transmitter for presynaptic inhibition in sympathetic ganglia. Nature, 294: 80, 1981. 6. Morgan, C., Nadelhaft, I. and de Groat, W. C.: The distribution of visceral primary afferents from the pelvic nerve to Lissauer's tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J. Comp. Neurol., 201: 415, 1981. 7. de Groat, W. C. and Ryall, R. W.: Recurrent inhibition in sacral parasympathetic pathways to the bladder. J. Physiol., 196: 579, 1968. 8. Kuhn, R. A.: Organization of tactile dermatomes in cat and monkey. J. Comp. Anat., 16: 169, 1953. 9. Grillner, S.: Locomotion in vertebrates: central mechanisms and reflex interactions. Physiol. Rev., 55: 247, 1975. 10. Vaidyanathan, S., Rao, M. S., Chary, K. S. N., Sharma, P. L. and Das, N.: Enhancement of detrusor reflex activity by naloxone in patients with chronic neurogenic bladder dysfunction. Preliminary report. J. Urol., 126: 500, 1981. 11. de Groat, W. C. and Booth, A. M.: Physiology of the urinary bladder and urethra. Ann. Intern. Med., 92: 312, 1980. 12. Kuru, M.: Nervous control of micturition. Physiol. Rev., 45: 425, 1965. 13. Wein, A. J. and Raezer, D. M.: Physiology of micturition. In: Clinical Neurology. Edited by R. J. Krane and M. B. Siroky. Boston: Little, Brown and Co., chapt. 1, p. 1, 1979. 14. Bradley, W. E.: Innervation of the male urinary bladder. Urol. Clin. North. Am., 5: 279, 1978. 15. Zaks, A., Jones, T., Fink, M. and Freedman, A. M.: Naloxone treatment of opiate dependence. J. A. M.A., 215: 2108, 1971. 16. Andree, R. A.: Sudden death following naloxone administration. Anesth. Analg., 59: 782, 1980. 17. Goldfarb, J. and Hu, J. W.: Enhancement of reflexes by naloxone in spinal cats. Neuropharmacology, 15: 785, 1976.