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Somatovesical and vesicovesical excitatory reflexes in urethane-anaesthetized rats
Brain Research. 380 (1986) 83-93 Elsevier
83
BRE 11913
Somatovesical and Vesicovesical Excitatory Reflexes in Urethane-Anaesthetized Rats CARLO ALBERTO MAGGI, PAOLO SANTICIOLI and ALBERTO MELI Department of Pharmacology, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Florence (Italy)
(Accepted December 24th, 1985) Key words: rat - - micturition reflex - - somatovesical reflex - - vesicovesical reflex - - urethane - - bladder voiding
The effect of spinal cord transection on excitatory somato- and vesicovesical micturition reflexes have been investigated in urethane-anaesthetized rats. In adult rats, 3 distinct types of excitatory reflexes to the bladder may be observed: a somatovesical reflex organized at spinal level and two vesicovesical reflexes organized at spinal and supraspinal level, respectively. In agreement with results of lesion experiments (Neurosci. Lett., 8 (1978) 27-33), bladder voiding is abolished following spinal cord transection although both somato- and vesicovesical reflexes may be demonstrated in acute spinal rats. Occurrence of the spinal vesicovesical reflex during the collecting phase of the cystometrogram appears to he inhibited by a supraspinal inhibitory pathway.
INTROD UCTION It is widely accepted that both in cats 2-4,7-9,22 and humans 5,35 micturition is produced through a reflex organized at supraspinal level. These studies led to the identification, at pontine level, of a distinct micturition center which subserves bladder voiding2-4,8,9~22. On the other hand, in newborn cats, bladder voiding is mediated through a somatovesical excitatory reflex, activated when the mother licks the perineal area s,l° which is replaced, during postnatal development, by the supraspinal vesicovesical reflex s'l°. In cats, after a time lag from spinal cord transection, a spinal, 'short loop' vesicovesical reflex subserves partial bladder voiding s'11,t2,14. Electrophysiological experiments demonstrated that 'automatic micturition' in spinal animals depends upon the emergence of spinal autonomic reflex(es) 14. As pointed out by De Groat et al. 8'11 the question remains open as to whether or not appearance of this spinal reflex(es) depends upon formation of new pathways by axonal sprouting or unmasking of an existing pathway which, following spinal cord transection, would escape from the inhibitory control
of descending pathways. This question appears of particular interest since, if a spinal vesicovesical reflex exists in normal adult animals (and humans) then disinhibition of this 'short-loop' reflex may lead to occurrence of 'involuntary" (spinal) reflex contractions of the detrusor during the collecting phase of the cystometrogram, that is, a finding typical of detrusor instability in humans. Recent anatomical studies suggest the presence, in normal adult animals, of monosynaptic connections between primary afferents in the pelvic nerves and neurons of the sacral parasympathetic nucleus 32'34 which provides the preganglionic neural input to the bladder8,11,t2A 4. In rats, micturition reflexes (excitatory and inhibitory) are organized at both spinal and supraspinal level 31,37-40. However, in normal adult rats bladder voiding appears to be produced through a reflex of supraspinal origin 31,37.39. The aim of the present study was to assess the relative contribution of spinal and supraspinal micturition reflexes to bladder voiding in adult rats. Functional evidence will be presented suggesting the existence, in normal adult rats, of 3 distinct vesicoexcitatory reflexes, that is, a somatovesical reflex, orga-
Correspondence: C.A. Maggi, Department of Pharmacology, Smooth Muscle Division, Research Laboratories, Via Sette Santi 3, 50131, Florence, Italy.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
84 nized at spinal level and two vesicovesical reflexes which are organized at spinal and supraspinal levels, respectively. MATERIALS AND METHODS Male albino rats, Wistar Morini strain, weighing 340-360 g were anaesthetized with subcutaneous urethane (1.2 g/kg) and the left jugular vein was cannulated for drug injection. Reasons for choosing urethane as anaesthetic were detailed elsewhere 31. In some experiments tetrodotoxin (20/~g in saline) was applied topically on the bladder dome as described previously24,27. Through a midline incision of the abdomen the urinary bladder was exposed, emptied of urine by application of a slight manual pressure, cannulated with polythene tubing and prepared for recording intraluminal pressure variations to saline infusion (cystometrogram, CMG) as described previously 26'28. Warm saline-soaked cotton wool swabs were laid around the exteriorized organ to maintain its temperature and keep it moist. In some experiments the animals were ventilated by means of an Harvard apparatus for small rodents. In other experiments (transvesical CMG) intraluminal pressure was monitored through a needle inserted in the bladder dome to make the bladder outlet free to void. Through a midline incision of the abdomen the urinary bladder was exposed and emptied of urine by application of slight manual pressure. A 20-gauge needle was inserted through the apex of the bladder dome for 3 - 4 mm into its lumen. The needle was connected to an H.P. 1280 pressure transducer by means of a polythene tubing (1.5 mm o.d. and 1.0 mm i.d.) and the whole system filled with saline. The tubing was provided with an internal coaxial polyethylene tubing (0.6 mm o.d. and 0.3 mm i.d.) inserted through a side hole and sealed by a drop of epoxy resin. This second tubing served for intravesical infusion of fluid and was connected through a peristaltic pump to a saline reservoir.
Somatovesical excitatory reflex In a first series of experiments (intraluminal pressure recorded either by the transurethral or transvesical route) we assessed as to whether or not a typical excitatory cutaneovesical reflex organized at spinal
level could be observed in our experimental conditions. This was obtained, as described by Sato et al. 37 by a 5-s pinching of a localized area (about 5 x 5 mm) of the perineal skin by means of a forceps. In these experiments the bladder was filled with a small amount (0.2 ml) of saline which was insufficient to elicit the micturition reflex. Pinching at 10-15 rain intervals gave fairly reproducible bladder contractions for at least 2 h.
Transurethral cystometrogram After a 15-rain equilibration period at zero volume, variations in intraluminal pressure were recorded in response to continuous transurethral infusion of warm (37 °C) saline for 30 min by means of a De Saga 131900 6-channel peristaltic pump connected to a polyethylene tubing inserted into the bladder. Infusion rate (0.046 ml/min) was chosen to simulate a maximal hourly diuresis value in the physiological range 28. The rhythmic contractions of the detrusor muscle elicited by saline loading have been assumed to represent a micturition reflex 24'2s'3°. The repetitive nature of the reflex depends upon the occlusive ligature of the urethra which allows the stretching stimulus to be maintained 29. The volume of infused saline required to elicit rhythmic contractions of at least 4 mm Hg amplitude (this value was chosen because the tetrodotoxin-resistant phasic contractile activity does not exceed it) which were followed by rhythmic contractions of increasing amplitude was assumed to be the effective stimulus for triggering the micturition reflex in each animal 28.
Transvesical CMG After a 15-min equilibration period at zero volume, variations in intraluminal pressure were recorded in response to continuous infusion of saline (0.046-0.1 ml/min) at 37 °C for 30-40 min by means of a De Saga 131900 6-channel peristaltic pump connected to the polyethylene tubing inserted into the bladder. In each preparation the infusion of saline continued until micturition (end point) occurred. Micturition will be thereafter referred to as the emission of several drops of fluid during a sustained phasic contraction of the detrusor muscle which was followed by return of intraluminal pressure to a value near to
85 zero or, in any case, to a value lower than that recorded just before micturition. In each experiment 3 parameters were evaluated: (a) the intraluminal pressure value recorded just before micturition (this value will be thereafter referred to as pressure threshold); (b) the volume of infused saline required to obtain micturition (this value will be thereafter referred to as volume threshold) and (c) maximal amplitude of micturition contraction. To have an estimate of residual volume the intravesical fluid was collected, following micturition, by puncturing the bladder with a 1-ml syringe. Volume threshold and residual volume were calculated on the assumption that no significant leakage of fluid occurred at the point of insertion of the needle in the bladder dome. This was verified in preparations whose pelvic nerves were bilaterally divided before the CMG. In these preparations no micturition occurred during a 40 min infusion period. At the end of this period the bladder content amounted to 96 + 4% (n = 5) of infused volume.
Additional surgical procedures Some experiments were performed in acute spinal rats. Spinalization was performed, under urethane anaesthesia, by severing the cord at the level of the intervertebral space C2-C3. The skin was closed
with wound clips and the animals allowed to recover for 30-60 min before the CMG. The hypogastric nerves were divided bilaterally at the level of the iliac crest to prevent bladder depression due to hyperactivity of the sympathetic nervous system following intraspinal surgery 17'31. In some animals i.v. naloxone (0.2 mg/kg) was administered either before (10 min) or after (10-30 min) the spinalization and, in any case, at least 30 min before saline infusion to prevent the depressant effect of endogenous opioids on micturition reflex activation 36.
Statistical analysis All data in the text are mean _+ S.E.M. Statistical analysis of the data was performed by means of the Student's t-test for paired or unpaired data, when applicable. Statistical analysis of nonparametric data was made by means of the chi-square test (Yeates correction).
Drugs Drugs used were: tetrodotoxin (Sankyo), atropine-HCl (Serva), hexamethonium bromide (Serva), strychnine nitrate (Sandoz) naloxone-HC1 (Sigma), physostigmine sulphate (Sigma) and acetylcholineHCI (Fluka). RESULTS
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Fig. 1. Upper panel: typical tracings showing the effect of intravenous atropine (1 mg/kg, i.v., 5 min before) and topical tetrodotoxin (TTX, 20 ~g in 0.1 ml, 5 rain before) on amplitude of the pinching-induced phasic bladder contractions. Lower panel: typical tracings showing the effect of intravenous hexamethonium (20 mg/kg, 5 rain before) and topical tetrodotoxin (TTX, 20 #g in 0.1 ml, 5 min before) on amplitude of the pinching-induced phasic bladder contractions.
Somatovesical excitatory reflex These experiments were performed in bladders filled with an amount of saline (0.2 ml) insufficient to trigger rhythmic phasic contractions (micturition reflex). Pinching of the perineal skin elicited rapid phasic contractions in 28 out of 35 (80%) preparations (Figs. 1 and 2). Response to pinching ensued within 1-2 s from application of the stimulus and reached a maximum within 2-3 s. Thereafter contractions subsided even if the stimulus was not removed. It was noted that the amplitude of pinching-induced bladder contractions was influenced markedly by the depth of anaesthesia. To assess this point, in some experiments, after having recorded two or more reproducible responses to pinching, the dose of urethane was increased by i.v. injection of the anaesthetic in amounts of 0.15 g/kg. Urethane decreased in a dose-dependent manner amplitude of pinching-induced contractions; at a total dose of 1.5 g/kg (1.2
86 g/kg s.c. plus 0.3 g/kg i.v.) response to pinching was greatly reduced (over 80% inhibition) or even abolished (n = 4; Fig. 2). In accordance with previous observations in cats 7, i.v. strychnine (0.5 mg/kg) produced the appearance of a small somatovesical response in previously unresponsive preparations (n = 4). Moreover, in some preparations, i.v. strychnine reverted the urethaneinduced depression of the somatovesical reflex (Fig. 2). Pinching-induced bladder contractions were suppressed by topical tetrodotoxin (20 ~g in 0.1 ml, n = 6) or intravenous hexamethonium (20 mg/kg, n = 4), thus indicating their neurogenic reflex origin (Figs. 1 and 2). On the other hand, intravenous atropine (0.1-1 mg/kg, n = 6) had only a slight inhibitory effect ( 5 - 1 5 % ) on the amplitude of the pinching-induced phasic bladder contractions (Fig. 1, upper panel). Pinching-induced phasic bladder contractions were observed when pressure was recorded either from the transvesical (6.8 + 1.3 m m Hg, n = 10) or the transurethral (7.4 _+ 0.6 m m Hg, n = 14, n.s.) route demonstrating that both types of recording were equally suitable for measuring low-amplitude phasic contractions of reflex origin. The amplitude of pinching-induced phasic bladder contraction amounted to 1 5 - 2 0 % of maximal contractile response elicited by topical acetylcholine (0.1 ml of a 1 mM solution). Amplitude of the pinching-induced phasic bladder contraction was unaffected by i.v. naloxone (0.2 mg/kg, 20 min before) (from 8.5 + 1 to 8.4 + 1.9 m m Hg, n = 6).
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In some experiments we tried to assess to what extent the pinching-induced somatovesical reflex could release urine in adult animals. In these experiments the bladders were filled via the transvesical route (0.1 ml/min) in a non-stop manner, so that repetitive voiding cycles were obtained at regular intervals (440 + 51 s, n = 8). In some experiments, when the intravesical fluid amounted to 0.2 ml (which was insufficient to elicit micturition; Fig. 3, upper panel) pinching of perineal skin produced a response whose characteristics are similar of that described above; this contraction (2-12 mm Hg, n = 10) was insufficient to produce micturition. Likewise, when the pinching was done 2 0 - 3 0 s before the expected next voiding cycle (Fig. 3, lower panel) this maneuver failed to elicit micturition.
Spinal origin of the excitatory somatovesical reflex The pinching-induced phasic bladder contraction could be observed within 60 min from spinal cord transection ( C 2 - C 3 ) thus confirming the notion that it represents a somatovesical excitatory reflex organized at spinal level 37. This response was observed in 12 out of 18 preparations tested (67%, n.s. as compared to controls). Amplitude of the somatovesical excitatory reflex was slightly (6.1 + 1 mm Hg, n = 8) but not significantly reduced as compared to controls. Administration of naloxone (0.2 mg/kg, i.v.) either before (6.2 + I m m H g , n = 5) or after (6 + 0.5 mm Hg, n = 8) spinal cord transection did not modify amplitude of the pinching-induced phasic bladder contraction.
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Fig. 2. Effect of urethane and strychnine on the amplitude of the pinching-induced phasic bladder contraction in urethane-anaesthetized rats. After having recorded two or more control responses, urethane 0.1 g/kg was injected intravenously which decreased the amplitude of the response and finally suppressed it. At this time intravenous strychnine (0.5 mg/kg) induced a partial recovery in amplitude of the pinching-induced phasic response which was finally suppressed by topical tetrodotoxin (20 f~g in 0.1 ml).
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Fig. 3. Effect of pinching-induced phasic bladder contraction (somatovesical spinal reflex) on bladder voiding. Upper panel: a series of rhythmic voiding cycles (A, B) was obtained in response to the transvesical infusion of saline. In C, when the bladder was filled with an amount of fluid (0.2 ml) lower than volume threshold, pinching of the perineal skin induced a phasic contraction which was insufficient to release urine. In D the next voiding cycle was shown. Lower panel: a series of rhythmic voiding cycles (A-C) was obtained in response to transvesical infusion of saline. In B, about 20 s before the expected next voiding cycle, pinching of the perineal skin induced a phasic contraction but failed to elicit micturition which occurred a few s after this maneuver.
Bladder response to transurethral filling A representative e x a m p l e of transurethral C M G o b t a i n e d in normal rats is shown in Fig. 4. A t a filling rate of 0.046 ml/min the m e a n volume of saline required to elicit neurogenic rhythmic contractions was 0.541 + 0.06 ml (n = 20). T h e intraluminal pressure value for rhythmic contractions to occur was 5.0 + 0.5 m m Hg. In about 7 0 - 8 0 % of p r e p a r a t i o n s (type ' A ' transurethral C M G ) the a m p l i t u d e of the rhythmic contractions was 4 - 8 m m H g at their first a p p e a r a n c e and their frequency was 1.5-2 contractions/rain. Duration of each one of these 'early' contractions did not exceed 15 s. The a m p l i t u d e of rhythmic contractions increased t h e r e a f t e r for 3 - 1 0 min to reach a steady value comprised between 18 and 50 m m Hg (Fig. 4). A t steady state the maximal a m p l i t u d e of rhythmic contractions elicited in response to the transurethral c y s t o m e t r o g r a m resulted 25.8 + 2 m m Hg (n = 20). W h e n the amplitude of these 'late' rhythmic contractions had reached a steady value their frequency was in the range of 0 . 8 - 1 . 5 contractions/min and the duration of each phasic contraction was in the range of 60-120 s. In about 2 0 - 3 0 % of p r e p a r a t i o n s (type 'B' transurethral C M G ) saline filling-induced rhythmic contractions were of high a m p l i t u d e (above 16 m m Hg) from their first a p p e a r a n c e ; in these p r e p a r a t i o n s the
rhythmic contractions occurred at a volume slightly higher ( 0 . 6 - 0 . 7 ml) than that r e q u i r e d in the remaining preparations.
Bladder response to transvesical filling R e p r e s e n t a t i v e examples of cystometric recordings o b t a i n e d in response to the transvesical infusion of saline are shown in Fig. 4. Micturition was characterized by the emission of several drops of fluid at the top of a phasic contraction. Following micturition intravesical pressure r e t u r n e d below pressure threshold and in most cases, n e a r to zero (Fig. 4). A t a physiological-like filling rate (0.046 ml/min) the mean values of pressure and v o l u m e threshold were 3.8 + 0.4 m m H g and 0.826 + 0.08 ml, respectively (n = 41). M e a n a m p l i t u d e of micturition contraction in control rats was 25 + 2 m m Hg. Only in 3 out of 41 p r e p a r a t i o n s was micturition p r o d u c e d by an increase of intraluminal pressure lower than 20 mm Hg. The d u r a t i o n of micturition contractions was 29 + 2 s. The m e a n residual v o l u m e after micturition a m o u n t e d to 33 + 6% (n = 10) of the infused volume. In 73% (30 out of 41) of p r e p a r a t i o n s a phasic contractile activity higher than 4 - 6 m m H g could be recorded only for the last 2 - 3 rain before micturition. In only 27% of p r e p a r a t i o n s (11 out of 41) a series of rhythmic contractions was o b s e r v e d for 3 - 1 0 min before micturition.
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Fig. 4. Typical tracings showing the bladder response to transurethral (upper panel) and transvesical (lower panel) CMG. Note that the 'early' response to transurethral CMG consisted of high-frequency low-amplitude contractions which were thereafter replaced by a series of low-frequency high-amplitude rhythmic contractions ('late' response). On the other hand the response to transvesical CMG was characterized by a flat volume-response curve without any significant (>4-6 mm Hg) contractile activity before the time at which micturition occurred. Micturition contraction was almost invariably higher than 20 mm Hg. The dots indicate the start of saline infusion (0.046 ml/min). The arrows indicate start of neurogenic reflex contractions (tetrodotoxin- and hexamethonium-sensitive, upper panel) or micturition (lower panel).
Neurogenic nature of phasic bladder contractions in response to distension The reflex neurogenic nature of the contractile response to the transurethral C M G was proven by suppression of rhythmic bladder contractions following topical tetrodotoxin (20/~g in 0.1 ml) or intravenous hexamethonium (40 mg/kg, i.v.). In 6 preparations receiving topical tetrodotoxin 10 min before the start of saline infusion no rhythmic contractile activity higher than 4 m m Hg could be elicited during a 30-rain infusion period. Topical tetrodotoxin prevented bladder voiding in response to transvesical infusion of saline (n = 8). The neurogenic nature of phasic contractile activity recorded in response to either transurethral (n = 5) or transvesical (n = 6) C M G was further demonstrated in preparations of which the pelvic nerves were bilaterally divided 10 min before start of saline infusion. In these preparations no phasic contraction higher than 4 - 6 mm H g could be observed during a 30 rain-infusion period.
Transurethral infusion of saline in rats spinalized at upper cervical ( C 2 - C 3 ) level ( 0 . 5 - 2 h before) induced a series of rhythmic phasic contractions whose amplitude ranged between 6 and 18 m m Hg and frequency between 1.7 and 2.5 contractions/rain. Maximal amplitude of rhythmic contractions produced by transurethral saline filling in acute spinal rats was reduced by about 5 0 - 6 0 % as compared to controls (see below). The duration of each contractile wave did not exceed 15 s. These rhythmic bladder contractions were observed in 12 out of 19 (63%) preparations receiving i.v. naloxone (0.2 mg/kg) prior to spinalization and in 5 out of 22 (23%, P < 0.05) naloxone-untreated preparations. Characteristics of frequency amplitude and duration of rhythmic contractions were similar in both groups. Maximal amplitude, of rhythmic contractions was 11.9 _+ 0,7 and 12.2 _+ 1.5 mm Hg in naloxone-pretreated and control animals, respectively (n = 12 and 5, n.s.). Therefore, while naloxone administration prior to spinalization increased the excitability of the spinal reflex, its administration is no 'conditio sine qua non" to observe the presence of a spinal vesicovesical reflex in rats (see also ref. 31). In 6 animals which did not develop rhythmic bladder contractions higher than 4 - 6 m m Hg during the first C M G following spinal cord transection the bladder was emptied, naloxone was administered (0.2 mg/kg, i.v.) and a new C M G performed 10 min later, The second C M G occurred with characteristics similar to those observed before administration of naloxone. It is apparent that the characteristics of the rhyth-
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Fig. 5. Typical tracing showing the response to the transurethral CMG (0.046 ml/min) in acute (60 min before) spinal (C2-C3) rats. Note that the transurethral infusion of saline elicited a series of low-amplitude, high-frequency neurogenic (tetrodotoxin (TTX)-sensitive, 20 ktg in 0.1 ml) contractions which closely resemble the 'early' response to the transurethral CMG in normal rats. The dot indicates start of saline infusion.
89 mic bladder contractions observed in response to transurethral filling in acute spinal rats are similar to those of the 'low-amplitude, high-frequency' 'early' rhythmic contractions observed in type ' A ' transurethral C M G (Fig. 5). In acute spinal rats the amplitude and duration of the rhythmic contraction did not increase over 18 m m Hg even when the amount of fluid into the bladder was greater than 1.0 ml. Therefore only the 'late' component of the type ' A ' response to the transurethral C M G was lacking in acute spinal rats suggesting that it may represent an activation of a reflex of supraspinal origin. Rhythmic bladder contractions activated by transurethral infusion of saline in acute spinal rats were promptly abolished by topical tetrodotoxin (20 ~g in 0.1 ml) as well as by intravenous hexamethonium (20 mg/kg), thereby indicating their neurogenic reflex origin. Bladder response to transvesical filling in acute spinal rats These experiments were performed in naloxonepretreated (0.2 mg/kg prior to spinal cord transection) animals. Transvesical infusion of saline in rats spinalized at the upper cervical level ( C 2 - C 3 ) elicited in 9 out of 16 (56%) preparations a series of rhythmic phasic contractions of the urinary bladder. Amplitude of these contractions ranged between 6 and 16 m m Hg and frequency between 2 and 3 contractions/min. Duration of each contractile wave did not exceed 15 s. None of these rhythmic contractions was sufficient to produce bladder voiding and this may account for their rhythmic behaviour 29. The rhythmic bladder contractions elicited in response to transvesical C M G in acute spinal rats were promptly suppressed following intravenous hexamethonium (20-40 mg/kg, n = 6), thus indicating their reflex neurogenic origin. Effect o f physostigmine pretreatment on bladder response to transvesical C M G in acute spinal rats Experiments described in the preceding section indicate that, in a certain percentage of preparations, shortly after spinal cord transection, a spinal vesicovesical reflex could be observed in response to transvesical CMG. The question was raised as to whether or not failure to observe a rhythmic phasic contractile activity in about 40% of preparations could be as-
cribed to the fact that urethane exerts a depressant effect on spinal reflexes as'38 and, therefore, reflex contractions were too weak to be recorded. To assess this point after having recorded a first C M G , the bladder was emptied and the preparations received intravenous physostigmine (0.1 mg/kg). After 20 min a new C M G was made; this time lag allowed us to obtain reproducible cystometric recordings in controls (saline-treated). These experiments were performed in naloxone-pretreated (0.2 mg/kg, i.v.) animals. In 6 out of 6 preparations pretreatment with physostigmine induced appearance of rhythmic bladder contractions (Fig. 6, upper and middle panel) having an amplitude comprised between 9 and 29 m m Hg at a frequency of 2 - 3 contractions/rain. In none of these preparations the first C M G had elicited rhythmic bladder contractions higher than 4 m m Hg (Fig. 6 up-
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Fig. 6. Upper and middle panel: the bladder responses to transvesical CMG in one acute (1 h before the first CMG) spinal (C2-C3) rat before and after the administration of intravenous physostigmine (0.1 mg/kg 10 min before the second CMG). Note that following physostigmine, saline filling induced the appearance of rhythmic bladder contraction having sufficient amplitude and duration to produce, at the top of each phasic contraction, the emission of one or two drops of fluid (dripping). These rhythmic contractions were promptly abolished by intravenous atropine (0.1 mg/kg). Lower panel: potentiating effect of intravenous physostigmine (0.1 mg/kg) on the rhythmic bladder contraction produced by transvesical infusion of saline in one acute (1 h before) spinal (C2-C3) rat. The reflex nature of the phasic bladder contractions was proven by their suppression following intravenous hexamethonium (20 mg/kg).
90 per panel). Duration of the physostigmine-activated rhythmic bladder contractions did not exceed 15 s in 3 out of 6 preparations while in the remainder it was greater than 20 s. In these latter preparations one or two drops of fluid were expelled at the top of each phasic contraction (Fig. 6, middle panel). The rhythmic behaviour of the phasic, contractile activity elicited by transvesical C M G in physostigmine-pretreated spinal rats indicated that bladder voiding was largely incomplete (Fig. 6, upper panel). Moreover, after each phasic contraction intraluminal pressure did not return below pressure threshold as was observed following micturition in normal rats (Fig. 6, middle panel). In a further series of experiments we studied the effect of physostigmine (0.1 mg/kg, i.v.) in spinal preparations which developed, in response to transvesical CMG, a series of rhythmic bladder contractions. In these experiments amplitude of rhythmic bladder contractions was enhanced by physostigmine (fromll +2to18+2mmHg, n = 6 , P < 0 . 0 1 ) as was the duration of each contractile wave (from 10 + 1 to 18 + 2 s, P < 0.01) while the frequency of rhythmic contractions was not significantly affected (from 2.3 + 0.2 to 2.6 + 0.3 contractions/min; Fig. 6, lower panel). Rhythmic bladder contractions elicited in physostigmine-pretreated spinal rats were promptly suppressed by intravenous hexamethonium (20 mg/kg) or atropine (1 mg/kg; n = 6, Fig. 6). DISCUSSION Supraspinal origin of micturition contraction in normal adult rats There is evidence suggesting that, in rats, both anatomical and neurophysiological organization of the micturition reflex is similar 33'37'39 to that described in catsS.9,22,23. Cystometric experiments revealed that high-amplitude (over 20 mm Hg) vesicovesical reflex contractions could be observed in response to bladder distension in adult anaesthetized r a t s 24'26'3°'37'40 but not in acute spinal preparations 31"37'4°. Transection experiments 4° revealed that, in rats, the supraspinal center activating the distension-induced bladder contractions is most likely located at pontine level suggesting its identity with the pontine micturition center de-
scribed by Satoh et al. 39. It appears conceivable that the high amplitude (>20 mm Hg) vesicovesical reflex which produces bladder voiding in response to transvesical saline filling in urethane-anaesthetized rats which was demonstrated in the present study is of supraspinal origin, since it is abolished by spinal cord transection. However, a simple reduction in amplitude of the reflex contractions in acute spinal rats does not in itself establish a supraspinal origin of the reflex since a depression due to spinal shock may have been implicated. Electrophysiological experiments are needed to establish firmly whether the vesicovesical reflex which produces bladder voiding in anaesthetized rats is of supraspinal origin; however, 3 lines of evidence support this hypothesis i.e., (a) destruction of the pontine micturition center produces urine retention in rats 39 indicating that, in this species, spinal reflex(es) are insufficient to produce bladder voiding and (b) intracisternal administration of chemicals (acetylcholine, glycine, substance P) activates a series of rhythmic bladder contractions whose characteristics of amplitude, frequency and duration are the same of the high-amplitude bladder contractions elicited by saline filling (ref. 25 and M. Furio, unpublished data) and (c) reduction in amplitude (15-20%) of the spinal 37 somatovesical reflex following spinal cord transection is much lower than that (50-60%) of the rhythmic contractions observed in response to transurethral saline filling. This latter point indicates that the 'spinal shock' alone cannot explain the absence, in response to transurethral CMG in spinal rats, of the high-amplitude contractions which may represent activation of the supraspinal vesicovesical reflex. Our findings in urethane-anaesthetized rats establish that attainment of an intraluminal pressure of at least 20 mm Hg is required for producing bladder voiding. Therefore it is not surprising that all types of reflex contractile activity (notably both somatovesical and vesicovesical spinal reflexes) which do not attain such critical pressure values are 'per se' insufficient to induce bladder voiding in normal adult animals. Duration of micturition contraction appears also relevant in determining an efficient bladder voiding; in fact, duration of micturition contraction in response to transvesical filling (usually over 20 s) out-
91 lasts that of both somato- and vesicovesical reflexes which can be observed in spinal rats.
Spinal vesicovesical excitatory reflex and its potential relationship with transurethral CMG De Groat and Ryal114 described that, in cats, a short latency C-fiber reflex could be detected as early as 3 days after spinal cord transection. This reflex increased thereafter parallel to development of automatic micturition 14. Appearance of this vesicovesical reflex following spinal cord transection could be explained either by axonal sprouting (and consequent formationof new pathways) or removal of a descending bulbospinal inhibitory pathway 7's'13. Subsequent experiments indicated that the C-fiber reflex may be present also in normal cats which supports the second hypothesis (inhibition of the spinal reflex by descending inhibitory pathways)11. Our present findings provide evidence indicating that in adult rats a vesicovesical excitatory reflex, organized at a spinal level, can be observed within a short time (0.5-2 h) from spinal cord transection. It is unlikely that formation of new pathways by axonal sprouting occurred in such a short lag of time. Therefore we must conclude that anatomical connections responsible for a vesicovesical spinal reflex are already present in normal adult rats. This is supported by anatomical observations indicating that primary afferents from the detrusor may come into close contact with sacral parasympathetic neurons at spinal level either directly (monosynaptic interaction) or through interneurons 33. In rats a certain recovery of bladder function was observed within 3 weeks from destruction of the ponfine micturition center 39. It is conceivable that the vesicovesical spinal reflex demonstrated in our experiments subserves automatic micturition in chronic spinal rats. Failure to observe this spinal vesicovesical reflex during the collecting phase of the transvesical CMG suggests that this 'short-loop' reflex may be suppressed by some inhibitory mechanism(s) of supraspinal origin. Emergency of the vesicovesical spinal reflex shortly after spinal cord transection in response to transvesical filling cannot be simply ascribed to administration of naloxone (see below) and suggests a supraspinal origin of these inhibitory mechanism(s) as also indicated by electrophysiologi-
cal findings in cats 1134. It appears conceivable that, if a similar organization of micturition reflexes exists in humans (i.e. supraspinal inhibition of the spinal vesicovesical reflex), then neurological or even functional disturbances of these inhibitory mechanism(s) might lead to emergency of involuntary reflex detrusot contractions. In most of the present experiments the spinal vesicovesical reflex was demonstrated in naloxone-pretreated animals. Recent findings from various laboratories indicate that endogenous opioids may be involved in regulating micturition in c a t s 6'21 and r a t s 15A6'36. However, administration of naloxone does not seem necessary to observe the spinal vesicovesical reflex although it may have increased its excitability. In fact a vesicovesical reflex was demonstrated in 5 out of 22 (23%) and in 6 out of 18 preparations (33 %) whose spinal cord was transected at C2C3 (present study) or T12-L131 level, respectively. It is worth mentioning that the excitatory effect of naloxone pretreatment may be observed when this drug was administered prior to but not following spinal cord transection. Failure to observe the reflex in a certain percentage of preparations is presumably ascribable to some depressant effect of anaesthesia or spinal shock since: (a) urethane depresses amplitude of the spinal reflex and (b) the spinal reflex could be demonstrated in all preparations in appropriate conditions (naloxone and physostigmine pretreatment). This suggests that failure of naloxone to increase excitability of the spinal reflex when it is administered to spinal rats does not depend upon the existence of a population of 'non-responders'. Further studies are required to clarify the mechanisms underlying the mechanisms through which naloxone increases excitability of the spinal vesicovesical reflex when it is administered prior to spinalization. It is noteworthy that the characteristics (amplitude, frequency, duration of each contractile wave) of the vesicovesical reflex observed in acute spinal rats closely mimicked those of the 'early' phase of the transurethral CMG of normal animals. On the other hand, in the majority of transvesical CMGs no significant contractile activity is recorded until micturition. In view of the above it might be speculated that, in response to transurethral CMG the spinal vesicovesical reflex is elicited at low filling volume and is there-
92 after replaced by a supraspinal vesicovesical reflex. If this were true we should assume that factors, such as presence of the urethral ligature, increase the excitability of the vesicovesical spinal reflex leading to its emergency in the 'early' phase of the transurethral CMG. It is well known that urethral afferents modulate reflex activation of the detrusor muscle. For instance the seventh Barrington's reflex (having both afferent and efferent limb in pelvic nerves) is a spinal reflex producing bladder contraction in response to stimuli arising in the urethra; such a reflex is thought to play a role in reinforcing bladder contraction during micturifion 19.
urethane on excitability of the somatovesical reflex. Such a finding recalls some unpublished data from De Groat (quoted in ref. 8) who observed that excitability of perineal and bladder reflexes was increased following i.v. strychnine in cats. A strychnine-sensitive recurrent inhibition in sacral parasympathetic pathways to the bladder was demonstrated previously by De Groat and Ryal113. Therefore it is conceivable that such an inhibitory mechanism, presumably potentiated by deepening of anaesthesia, plays a role in reducing excitability of the excitatory somatovesical reflex in adult rats.
CONCLUSIONS Spinal somatovesical excitatory reflex in normal adult rats Some characteristics of the somatovesical excitatory reflex deserve consideration. This type of response is of primary relevance in producing bladder voiding in newborn kittens s and rats 1'2°'41. In normal
adult cats stimulation of the perineum or sex organs either inhibits micturition or produces small bladder contractions which are too weak to release urine 8'9'n. In adult rats somatovesical reflex was found to have opposite effects on bladder contractility depending upon the degree of bladder filling 37,38. However, as already observed in cats, bladder contractions produced in this way are too weak to release urine. We also observed that the somatovesical reflex was influenced markedly by depth of anaesthesia. This is consistent with previous observations on the depressant effect of urethane on spinal reflexes in the isolated frog spinal cord 18. Interestingly, intravenous strychnine antagonized the depressant effect of
REFERENCES 1 Adolph, E.F., Ontogeny of physiological regulations in the rat, Q. Rev. Biol., 32 (1957) 89-137. 2 Barrington, F.J.F., The nervous mechanisms of micturition, Q. J. Exp. Physiol., 9 (1914) 261-264. 3 Barrington, F.J.F., The relation of the hindbrain to micturition, Brain, 44 (1921) 23-53. 4 Barrington, FJ.F., The effect of lesions in the hind brain on micturition in the cat, Q. J. Exp. Physiol., 15 (1925) 81-102. 5 Blaivas, J., The neurophysiology of micturition: a clinical study of 550 patients, J. Urol., 127 (1982) 958-963. 6 Booth, A.M., Hisamitsu, T., Kawatani, M. and De Groat,
In conclusion, our findings suggest that at least 3 distinct vesicoexcitatory reflexes are present in urethane-anaesthetized adult rats. Of these only the vesicovesical reflex observed in animals with an intact spinal cord appears to produce an increase of intraluminal pressure having sufficient intensity and duration to induce efficacious bladder voiding. This reflex could be similar or even identical to the supraspinal reflex which subserves bladder voiding in normal animals 39. Available evidence suggests that a spinal vesicovesical reflex could be selectively elicited when CMGs are performed through the transurethral route. Supraspinal inhibition of such a vesicovesical 'short-loop' reflex could contribute to urine continence before micturition, The set of experimental models described in this paper may be suitable for studying the physiology and pharmacology of the mechanisms regulating micturition at spinal and supraspinal level.
W.C., Regulation of bladder capacity by endogenous opioid peptides, J. Urol., 133 (1985) 339-342. 7 Bradley, W.E. and Conway, C.J., The bladder representation in the pontine-mesencephalic reticular formation, Exp. Neurol., 16 (1966) 237-249. 8 De Groat, W.C., Nervous control of the urinary bladder in the cat, Brain Research, 87 (1975) 201-211. 9 De Groat, W.C., Booth, A.M., Krier, J., Milne, J., Morgan, C. and Nadelhaft, I., Neural control of the urinary bladder and large intestine. In C.M. Brooks, K. Koizumi and A. Sato (Eds.), Integrative Functions of the Autonomic Nervous System, Tokyo University Press, Elsevier, 1979, pp. 50-67. 10 De Groat, W.C., Douglas, J.W., Glass, J., Simonds, W.,
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Weimer, B. and Wermer, P., Changes in somato-vesical reflexes during postnatal development in the kitten, Brain Research, 94 (1975) 150-154. De Groat, W.C., Nadelhaft, I., Milne, R.J., Booth, A.M., Morgan, C. and Thor, K., Organization of the sacral parasympathetic reflex pathway to the urinary bladder and large intestine, J. Auton. Nerv. Syst., 4 (1981) 135-160. De Groat, W.C. and Ryall, R.W., The identification and characteristics of sacral parasympathetic preganglionic neurons, J. Physiol. (London), 196 (1968) 563-577. De Groat, W.C. and Ryall, R.W., Recurrent inhibition in sacral parasympathetic pathways to the bladder, J. Physiol. (London), 196 (1968) 579-591. De Groat, W.C. and Ryall, R.W., Reflexes to sacral parasympathetic neurons concerned with micturition in the cat, J. Physiol. (London), 200 (1969) 87-108. Dray, A. and Metsch, R., Morphine and the centrally mediated inhibition of urinary bladder motility in the rat, Brain Research, 297 (1984) 191-195. Dray, A. and Nunan, Opioid inhibition of reflex urinary bladder contractions: dissociation of supraspinal and spinal mechanisms, Brain Research, 337 (1985) 142-145. Edvardsen, P., Nervous control of the urinary bladder in cats. I. The collecting phase, Acta Physiol. Scand., 72 (1968) 157-171. Evans, R.H. and Smith, D.A.S., Effect of urethane on synaptic and amino acid-induced excitation in isolated spinal cord preparation, Neuropharmacology, 21 (1982) 857-860. Garry, R.C., Roberts, T.D.M. and Todd, J.K., Reflexes involving the external urethral sphincter in the cat, J. Physiol. (London), 149 (1959) 653-665. Henning, S.J., Postnatal development: coordination of feeding, digestion and metabolism, Am. J. Physiol., 241 (1981) G199-214. Hisamitsu, T. and De Groat, W.C., The inhibitory effect of opioid peptides and morphine applied intrathecally and intracerebroventricularly on the micturition reflex, Brain Research, 298 (1984) 51-65. Kuru, M., Nervous control of micturition, Physiol. Rev., 45 (1965) 425-494. MacMahon, S.B. and Morrison, J.F.B., Factors that determine the excitability of parasympathetic reflexes to the cat bladder, J. Physiol. (London), 322 (1982) 35-43. Maggi, C.A., Evangelista, S., Grimaldi, G., Santicioli, P., Giolitti, A. and Meli, A., Evidence for the involvement of arachidonic acid metabolites in spontaneous and drug induced contractions of the rat urinary bladder, J. Pharrnacol. Exp. Ther., 230 (1984) 500-514. Maggi, C.A., Furio, M., Santicioli, P. and Meli, A., Intracisternal glycine activates the micturition reflex in urethane-anaesthetized rats, J. Pharm. Pharmacol., 37 (1985) 517-520. Maggi, C.A. and Meli, A., Modulation by beta adrenoreceptors of spontaneous contractions of rat urinary bladder, J. Auton. Pharmacol., 2 (1982) 255-260. Maggi, C.A. and Meli, A., An in vivo procedure for estimating spasmolytic activity in the rat by measuring smooth
28
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muscle contractions to topically applied acetylchohne, J. Pharmacol. Meth., 8 (1982) 39-46. Maggi, C.A. and Meli, A., Reserpine-induced detrusor hyperreflexia: an in vivo model for studying smooth muscle relaxants at urinary bladder level, J. Pharmacol. Meth., 10 (1983) 79-93. Maggi, C.A., Santicioli, P., Grimaldi, G. and Meli, A., The effect of peripherally administered G A B A on spontaneous motility of rat urinary bladder in vivo, Gen. Pharmacol., 14 (1983) 455-458. Maggi, C.A., Santicioli, P. and Meli, A., The effect of capsaicin on rat urinary bladder motility in vivo, Eur. J. Pharmacol., 103 (1984) 41-50. Maggi, C.A., Santicioli, P. and Meli, A., Sympathetic inhibition of reflex activation of bladder motility during filling at a physiological-like rate in urethane anaesthetized rats, Neurourol. Urodynamics, 4 (1985) 37-45. Morgan, C., Nadelhaft, I. and De Groat, W.C., The distribution of visceral primary afferents from the pelvic nerve within the Lissauer's tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus, J. Comp. Neurol., 201 (1981) 415-440. Nadelhaft, I. and Booth, A.M., The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study, J. Comp. Neurol., 226 (1984) 238-245. Nadelhaft, I., Roppolo, J., Morgan, C. and De Groat, W.C., Parasympathetic preganglionic neurons and visceral primary afferents in monkey sacral spinal cord revealed following application of horseradish peroxidase to pelvic nerve, J. Comp. Neurol., 216 (1983) 36-52. Nesbit, R.M. and Lapides, J., Tonus of the bladder during spinal shock, Arch. Surg., 56 (1948) 138-144. Roppolo, J.R., Haley, L., Smith, M. and De Groat, W.C., The effect of naloxone on reflexes to the urinary bladder in the intact and chronic spinal rats, Pharmacologist, 25 (1983) 152. Sato, A., Sato, Y., Shimada, F. and Torigata, Y., Changes in vesical function produced by cutaneous stimulation in rats, Brain Research, 94 (1975) 465-474. Sato, A., Sato, Y. and Schmidt, R.F., Somatic afferents and their effects on bladder function. In M. Brooks, K. Koizumi, A. Sato (Eds.), Integrative Function of the Autonomic Nervous System, Tokyo University Press, Elsevier, 1979, pp. 309-318. Satoh, K., Shimizu, N., Tohyama, M. and Maeda, T., Localization of the micturition reflex center at dorsolateral pontine tegmentum of the rat, Neurosci. Lett., 8 (1978) 27-33. Sillen, U., Central neurotransmitter mechanisms involved in the control of urinary bladder function, Scand. J. Urol. Nephrol., Suppl. 58 (1980) 1-45. Stelzner, D.J., The normal postnatal development of synaptic end-feet in the lumbosacral spinal cord and of responses in the hind limbs of the albino rat, Exp. Neurol., 31 (1971) 337-357.
83
BRE 11913
Somatovesical and Vesicovesical Excitatory Reflexes in Urethane-Anaesthetized Rats CARLO ALBERTO MAGGI, PAOLO SANTICIOLI and ALBERTO MELI Department of Pharmacology, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Florence (Italy)
(Accepted December 24th, 1985) Key words: rat - - micturition reflex - - somatovesical reflex - - vesicovesical reflex - - urethane - - bladder voiding
The effect of spinal cord transection on excitatory somato- and vesicovesical micturition reflexes have been investigated in urethane-anaesthetized rats. In adult rats, 3 distinct types of excitatory reflexes to the bladder may be observed: a somatovesical reflex organized at spinal level and two vesicovesical reflexes organized at spinal and supraspinal level, respectively. In agreement with results of lesion experiments (Neurosci. Lett., 8 (1978) 27-33), bladder voiding is abolished following spinal cord transection although both somato- and vesicovesical reflexes may be demonstrated in acute spinal rats. Occurrence of the spinal vesicovesical reflex during the collecting phase of the cystometrogram appears to he inhibited by a supraspinal inhibitory pathway.
INTROD UCTION It is widely accepted that both in cats 2-4,7-9,22 and humans 5,35 micturition is produced through a reflex organized at supraspinal level. These studies led to the identification, at pontine level, of a distinct micturition center which subserves bladder voiding2-4,8,9~22. On the other hand, in newborn cats, bladder voiding is mediated through a somatovesical excitatory reflex, activated when the mother licks the perineal area s,l° which is replaced, during postnatal development, by the supraspinal vesicovesical reflex s'l°. In cats, after a time lag from spinal cord transection, a spinal, 'short loop' vesicovesical reflex subserves partial bladder voiding s'11,t2,14. Electrophysiological experiments demonstrated that 'automatic micturition' in spinal animals depends upon the emergence of spinal autonomic reflex(es) 14. As pointed out by De Groat et al. 8'11 the question remains open as to whether or not appearance of this spinal reflex(es) depends upon formation of new pathways by axonal sprouting or unmasking of an existing pathway which, following spinal cord transection, would escape from the inhibitory control
of descending pathways. This question appears of particular interest since, if a spinal vesicovesical reflex exists in normal adult animals (and humans) then disinhibition of this 'short-loop' reflex may lead to occurrence of 'involuntary" (spinal) reflex contractions of the detrusor during the collecting phase of the cystometrogram, that is, a finding typical of detrusor instability in humans. Recent anatomical studies suggest the presence, in normal adult animals, of monosynaptic connections between primary afferents in the pelvic nerves and neurons of the sacral parasympathetic nucleus 32'34 which provides the preganglionic neural input to the bladder8,11,t2A 4. In rats, micturition reflexes (excitatory and inhibitory) are organized at both spinal and supraspinal level 31,37-40. However, in normal adult rats bladder voiding appears to be produced through a reflex of supraspinal origin 31,37.39. The aim of the present study was to assess the relative contribution of spinal and supraspinal micturition reflexes to bladder voiding in adult rats. Functional evidence will be presented suggesting the existence, in normal adult rats, of 3 distinct vesicoexcitatory reflexes, that is, a somatovesical reflex, orga-
Correspondence: C.A. Maggi, Department of Pharmacology, Smooth Muscle Division, Research Laboratories, Via Sette Santi 3, 50131, Florence, Italy.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
84 nized at spinal level and two vesicovesical reflexes which are organized at spinal and supraspinal levels, respectively. MATERIALS AND METHODS Male albino rats, Wistar Morini strain, weighing 340-360 g were anaesthetized with subcutaneous urethane (1.2 g/kg) and the left jugular vein was cannulated for drug injection. Reasons for choosing urethane as anaesthetic were detailed elsewhere 31. In some experiments tetrodotoxin (20/~g in saline) was applied topically on the bladder dome as described previously24,27. Through a midline incision of the abdomen the urinary bladder was exposed, emptied of urine by application of a slight manual pressure, cannulated with polythene tubing and prepared for recording intraluminal pressure variations to saline infusion (cystometrogram, CMG) as described previously 26'28. Warm saline-soaked cotton wool swabs were laid around the exteriorized organ to maintain its temperature and keep it moist. In some experiments the animals were ventilated by means of an Harvard apparatus for small rodents. In other experiments (transvesical CMG) intraluminal pressure was monitored through a needle inserted in the bladder dome to make the bladder outlet free to void. Through a midline incision of the abdomen the urinary bladder was exposed and emptied of urine by application of slight manual pressure. A 20-gauge needle was inserted through the apex of the bladder dome for 3 - 4 mm into its lumen. The needle was connected to an H.P. 1280 pressure transducer by means of a polythene tubing (1.5 mm o.d. and 1.0 mm i.d.) and the whole system filled with saline. The tubing was provided with an internal coaxial polyethylene tubing (0.6 mm o.d. and 0.3 mm i.d.) inserted through a side hole and sealed by a drop of epoxy resin. This second tubing served for intravesical infusion of fluid and was connected through a peristaltic pump to a saline reservoir.
Somatovesical excitatory reflex In a first series of experiments (intraluminal pressure recorded either by the transurethral or transvesical route) we assessed as to whether or not a typical excitatory cutaneovesical reflex organized at spinal
level could be observed in our experimental conditions. This was obtained, as described by Sato et al. 37 by a 5-s pinching of a localized area (about 5 x 5 mm) of the perineal skin by means of a forceps. In these experiments the bladder was filled with a small amount (0.2 ml) of saline which was insufficient to elicit the micturition reflex. Pinching at 10-15 rain intervals gave fairly reproducible bladder contractions for at least 2 h.
Transurethral cystometrogram After a 15-rain equilibration period at zero volume, variations in intraluminal pressure were recorded in response to continuous transurethral infusion of warm (37 °C) saline for 30 min by means of a De Saga 131900 6-channel peristaltic pump connected to a polyethylene tubing inserted into the bladder. Infusion rate (0.046 ml/min) was chosen to simulate a maximal hourly diuresis value in the physiological range 28. The rhythmic contractions of the detrusor muscle elicited by saline loading have been assumed to represent a micturition reflex 24'2s'3°. The repetitive nature of the reflex depends upon the occlusive ligature of the urethra which allows the stretching stimulus to be maintained 29. The volume of infused saline required to elicit rhythmic contractions of at least 4 mm Hg amplitude (this value was chosen because the tetrodotoxin-resistant phasic contractile activity does not exceed it) which were followed by rhythmic contractions of increasing amplitude was assumed to be the effective stimulus for triggering the micturition reflex in each animal 28.
Transvesical CMG After a 15-min equilibration period at zero volume, variations in intraluminal pressure were recorded in response to continuous infusion of saline (0.046-0.1 ml/min) at 37 °C for 30-40 min by means of a De Saga 131900 6-channel peristaltic pump connected to the polyethylene tubing inserted into the bladder. In each preparation the infusion of saline continued until micturition (end point) occurred. Micturition will be thereafter referred to as the emission of several drops of fluid during a sustained phasic contraction of the detrusor muscle which was followed by return of intraluminal pressure to a value near to
85 zero or, in any case, to a value lower than that recorded just before micturition. In each experiment 3 parameters were evaluated: (a) the intraluminal pressure value recorded just before micturition (this value will be thereafter referred to as pressure threshold); (b) the volume of infused saline required to obtain micturition (this value will be thereafter referred to as volume threshold) and (c) maximal amplitude of micturition contraction. To have an estimate of residual volume the intravesical fluid was collected, following micturition, by puncturing the bladder with a 1-ml syringe. Volume threshold and residual volume were calculated on the assumption that no significant leakage of fluid occurred at the point of insertion of the needle in the bladder dome. This was verified in preparations whose pelvic nerves were bilaterally divided before the CMG. In these preparations no micturition occurred during a 40 min infusion period. At the end of this period the bladder content amounted to 96 + 4% (n = 5) of infused volume.
Additional surgical procedures Some experiments were performed in acute spinal rats. Spinalization was performed, under urethane anaesthesia, by severing the cord at the level of the intervertebral space C2-C3. The skin was closed
with wound clips and the animals allowed to recover for 30-60 min before the CMG. The hypogastric nerves were divided bilaterally at the level of the iliac crest to prevent bladder depression due to hyperactivity of the sympathetic nervous system following intraspinal surgery 17'31. In some animals i.v. naloxone (0.2 mg/kg) was administered either before (10 min) or after (10-30 min) the spinalization and, in any case, at least 30 min before saline infusion to prevent the depressant effect of endogenous opioids on micturition reflex activation 36.
Statistical analysis All data in the text are mean _+ S.E.M. Statistical analysis of the data was performed by means of the Student's t-test for paired or unpaired data, when applicable. Statistical analysis of nonparametric data was made by means of the chi-square test (Yeates correction).
Drugs Drugs used were: tetrodotoxin (Sankyo), atropine-HCl (Serva), hexamethonium bromide (Serva), strychnine nitrate (Sandoz) naloxone-HC1 (Sigma), physostigmine sulphate (Sigma) and acetylcholineHCI (Fluka). RESULTS
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Fig. 1. Upper panel: typical tracings showing the effect of intravenous atropine (1 mg/kg, i.v., 5 min before) and topical tetrodotoxin (TTX, 20 ~g in 0.1 ml, 5 rain before) on amplitude of the pinching-induced phasic bladder contractions. Lower panel: typical tracings showing the effect of intravenous hexamethonium (20 mg/kg, 5 rain before) and topical tetrodotoxin (TTX, 20 #g in 0.1 ml, 5 min before) on amplitude of the pinching-induced phasic bladder contractions.
Somatovesical excitatory reflex These experiments were performed in bladders filled with an amount of saline (0.2 ml) insufficient to trigger rhythmic phasic contractions (micturition reflex). Pinching of the perineal skin elicited rapid phasic contractions in 28 out of 35 (80%) preparations (Figs. 1 and 2). Response to pinching ensued within 1-2 s from application of the stimulus and reached a maximum within 2-3 s. Thereafter contractions subsided even if the stimulus was not removed. It was noted that the amplitude of pinching-induced bladder contractions was influenced markedly by the depth of anaesthesia. To assess this point, in some experiments, after having recorded two or more reproducible responses to pinching, the dose of urethane was increased by i.v. injection of the anaesthetic in amounts of 0.15 g/kg. Urethane decreased in a dose-dependent manner amplitude of pinching-induced contractions; at a total dose of 1.5 g/kg (1.2
86 g/kg s.c. plus 0.3 g/kg i.v.) response to pinching was greatly reduced (over 80% inhibition) or even abolished (n = 4; Fig. 2). In accordance with previous observations in cats 7, i.v. strychnine (0.5 mg/kg) produced the appearance of a small somatovesical response in previously unresponsive preparations (n = 4). Moreover, in some preparations, i.v. strychnine reverted the urethaneinduced depression of the somatovesical reflex (Fig. 2). Pinching-induced bladder contractions were suppressed by topical tetrodotoxin (20 ~g in 0.1 ml, n = 6) or intravenous hexamethonium (20 mg/kg, n = 4), thus indicating their neurogenic reflex origin (Figs. 1 and 2). On the other hand, intravenous atropine (0.1-1 mg/kg, n = 6) had only a slight inhibitory effect ( 5 - 1 5 % ) on the amplitude of the pinching-induced phasic bladder contractions (Fig. 1, upper panel). Pinching-induced phasic bladder contractions were observed when pressure was recorded either from the transvesical (6.8 + 1.3 m m Hg, n = 10) or the transurethral (7.4 _+ 0.6 m m Hg, n = 14, n.s.) route demonstrating that both types of recording were equally suitable for measuring low-amplitude phasic contractions of reflex origin. The amplitude of pinching-induced phasic bladder contraction amounted to 1 5 - 2 0 % of maximal contractile response elicited by topical acetylcholine (0.1 ml of a 1 mM solution). Amplitude of the pinching-induced phasic bladder contraction was unaffected by i.v. naloxone (0.2 mg/kg, 20 min before) (from 8.5 + 1 to 8.4 + 1.9 m m Hg, n = 6).
20
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In some experiments we tried to assess to what extent the pinching-induced somatovesical reflex could release urine in adult animals. In these experiments the bladders were filled via the transvesical route (0.1 ml/min) in a non-stop manner, so that repetitive voiding cycles were obtained at regular intervals (440 + 51 s, n = 8). In some experiments, when the intravesical fluid amounted to 0.2 ml (which was insufficient to elicit micturition; Fig. 3, upper panel) pinching of perineal skin produced a response whose characteristics are similar of that described above; this contraction (2-12 mm Hg, n = 10) was insufficient to produce micturition. Likewise, when the pinching was done 2 0 - 3 0 s before the expected next voiding cycle (Fig. 3, lower panel) this maneuver failed to elicit micturition.
Spinal origin of the excitatory somatovesical reflex The pinching-induced phasic bladder contraction could be observed within 60 min from spinal cord transection ( C 2 - C 3 ) thus confirming the notion that it represents a somatovesical excitatory reflex organized at spinal level 37. This response was observed in 12 out of 18 preparations tested (67%, n.s. as compared to controls). Amplitude of the somatovesical excitatory reflex was slightly (6.1 + 1 mm Hg, n = 8) but not significantly reduced as compared to controls. Administration of naloxone (0.2 mg/kg, i.v.) either before (6.2 + I m m H g , n = 5) or after (6 + 0.5 mm Hg, n = 8) spinal cord transection did not modify amplitude of the pinching-induced phasic bladder contraction.
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Fig. 2. Effect of urethane and strychnine on the amplitude of the pinching-induced phasic bladder contraction in urethane-anaesthetized rats. After having recorded two or more control responses, urethane 0.1 g/kg was injected intravenously which decreased the amplitude of the response and finally suppressed it. At this time intravenous strychnine (0.5 mg/kg) induced a partial recovery in amplitude of the pinching-induced phasic response which was finally suppressed by topical tetrodotoxin (20 f~g in 0.1 ml).
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Fig. 3. Effect of pinching-induced phasic bladder contraction (somatovesical spinal reflex) on bladder voiding. Upper panel: a series of rhythmic voiding cycles (A, B) was obtained in response to the transvesical infusion of saline. In C, when the bladder was filled with an amount of fluid (0.2 ml) lower than volume threshold, pinching of the perineal skin induced a phasic contraction which was insufficient to release urine. In D the next voiding cycle was shown. Lower panel: a series of rhythmic voiding cycles (A-C) was obtained in response to transvesical infusion of saline. In B, about 20 s before the expected next voiding cycle, pinching of the perineal skin induced a phasic contraction but failed to elicit micturition which occurred a few s after this maneuver.
Bladder response to transurethral filling A representative e x a m p l e of transurethral C M G o b t a i n e d in normal rats is shown in Fig. 4. A t a filling rate of 0.046 ml/min the m e a n volume of saline required to elicit neurogenic rhythmic contractions was 0.541 + 0.06 ml (n = 20). T h e intraluminal pressure value for rhythmic contractions to occur was 5.0 + 0.5 m m Hg. In about 7 0 - 8 0 % of p r e p a r a t i o n s (type ' A ' transurethral C M G ) the a m p l i t u d e of the rhythmic contractions was 4 - 8 m m H g at their first a p p e a r a n c e and their frequency was 1.5-2 contractions/rain. Duration of each one of these 'early' contractions did not exceed 15 s. The a m p l i t u d e of rhythmic contractions increased t h e r e a f t e r for 3 - 1 0 min to reach a steady value comprised between 18 and 50 m m Hg (Fig. 4). A t steady state the maximal a m p l i t u d e of rhythmic contractions elicited in response to the transurethral c y s t o m e t r o g r a m resulted 25.8 + 2 m m Hg (n = 20). W h e n the amplitude of these 'late' rhythmic contractions had reached a steady value their frequency was in the range of 0 . 8 - 1 . 5 contractions/min and the duration of each phasic contraction was in the range of 60-120 s. In about 2 0 - 3 0 % of p r e p a r a t i o n s (type 'B' transurethral C M G ) saline filling-induced rhythmic contractions were of high a m p l i t u d e (above 16 m m Hg) from their first a p p e a r a n c e ; in these p r e p a r a t i o n s the
rhythmic contractions occurred at a volume slightly higher ( 0 . 6 - 0 . 7 ml) than that r e q u i r e d in the remaining preparations.
Bladder response to transvesical filling R e p r e s e n t a t i v e examples of cystometric recordings o b t a i n e d in response to the transvesical infusion of saline are shown in Fig. 4. Micturition was characterized by the emission of several drops of fluid at the top of a phasic contraction. Following micturition intravesical pressure r e t u r n e d below pressure threshold and in most cases, n e a r to zero (Fig. 4). A t a physiological-like filling rate (0.046 ml/min) the mean values of pressure and v o l u m e threshold were 3.8 + 0.4 m m H g and 0.826 + 0.08 ml, respectively (n = 41). M e a n a m p l i t u d e of micturition contraction in control rats was 25 + 2 m m Hg. Only in 3 out of 41 p r e p a r a t i o n s was micturition p r o d u c e d by an increase of intraluminal pressure lower than 20 mm Hg. The d u r a t i o n of micturition contractions was 29 + 2 s. The m e a n residual v o l u m e after micturition a m o u n t e d to 33 + 6% (n = 10) of the infused volume. In 73% (30 out of 41) of p r e p a r a t i o n s a phasic contractile activity higher than 4 - 6 m m H g could be recorded only for the last 2 - 3 rain before micturition. In only 27% of p r e p a r a t i o n s (11 out of 41) a series of rhythmic contractions was o b s e r v e d for 3 - 1 0 min before micturition.
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Fig. 4. Typical tracings showing the bladder response to transurethral (upper panel) and transvesical (lower panel) CMG. Note that the 'early' response to transurethral CMG consisted of high-frequency low-amplitude contractions which were thereafter replaced by a series of low-frequency high-amplitude rhythmic contractions ('late' response). On the other hand the response to transvesical CMG was characterized by a flat volume-response curve without any significant (>4-6 mm Hg) contractile activity before the time at which micturition occurred. Micturition contraction was almost invariably higher than 20 mm Hg. The dots indicate the start of saline infusion (0.046 ml/min). The arrows indicate start of neurogenic reflex contractions (tetrodotoxin- and hexamethonium-sensitive, upper panel) or micturition (lower panel).
Neurogenic nature of phasic bladder contractions in response to distension The reflex neurogenic nature of the contractile response to the transurethral C M G was proven by suppression of rhythmic bladder contractions following topical tetrodotoxin (20/~g in 0.1 ml) or intravenous hexamethonium (40 mg/kg, i.v.). In 6 preparations receiving topical tetrodotoxin 10 min before the start of saline infusion no rhythmic contractile activity higher than 4 m m Hg could be elicited during a 30-rain infusion period. Topical tetrodotoxin prevented bladder voiding in response to transvesical infusion of saline (n = 8). The neurogenic nature of phasic contractile activity recorded in response to either transurethral (n = 5) or transvesical (n = 6) C M G was further demonstrated in preparations of which the pelvic nerves were bilaterally divided 10 min before start of saline infusion. In these preparations no phasic contraction higher than 4 - 6 mm H g could be observed during a 30 rain-infusion period.
Transurethral infusion of saline in rats spinalized at upper cervical ( C 2 - C 3 ) level ( 0 . 5 - 2 h before) induced a series of rhythmic phasic contractions whose amplitude ranged between 6 and 18 m m Hg and frequency between 1.7 and 2.5 contractions/rain. Maximal amplitude of rhythmic contractions produced by transurethral saline filling in acute spinal rats was reduced by about 5 0 - 6 0 % as compared to controls (see below). The duration of each contractile wave did not exceed 15 s. These rhythmic bladder contractions were observed in 12 out of 19 (63%) preparations receiving i.v. naloxone (0.2 mg/kg) prior to spinalization and in 5 out of 22 (23%, P < 0.05) naloxone-untreated preparations. Characteristics of frequency amplitude and duration of rhythmic contractions were similar in both groups. Maximal amplitude, of rhythmic contractions was 11.9 _+ 0,7 and 12.2 _+ 1.5 mm Hg in naloxone-pretreated and control animals, respectively (n = 12 and 5, n.s.). Therefore, while naloxone administration prior to spinalization increased the excitability of the spinal reflex, its administration is no 'conditio sine qua non" to observe the presence of a spinal vesicovesical reflex in rats (see also ref. 31). In 6 animals which did not develop rhythmic bladder contractions higher than 4 - 6 m m Hg during the first C M G following spinal cord transection the bladder was emptied, naloxone was administered (0.2 mg/kg, i.v.) and a new C M G performed 10 min later, The second C M G occurred with characteristics similar to those observed before administration of naloxone. It is apparent that the characteristics of the rhyth-
20" , 5 rain
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Fig. 5. Typical tracing showing the response to the transurethral CMG (0.046 ml/min) in acute (60 min before) spinal (C2-C3) rats. Note that the transurethral infusion of saline elicited a series of low-amplitude, high-frequency neurogenic (tetrodotoxin (TTX)-sensitive, 20 ktg in 0.1 ml) contractions which closely resemble the 'early' response to the transurethral CMG in normal rats. The dot indicates start of saline infusion.
89 mic bladder contractions observed in response to transurethral filling in acute spinal rats are similar to those of the 'low-amplitude, high-frequency' 'early' rhythmic contractions observed in type ' A ' transurethral C M G (Fig. 5). In acute spinal rats the amplitude and duration of the rhythmic contraction did not increase over 18 m m Hg even when the amount of fluid into the bladder was greater than 1.0 ml. Therefore only the 'late' component of the type ' A ' response to the transurethral C M G was lacking in acute spinal rats suggesting that it may represent an activation of a reflex of supraspinal origin. Rhythmic bladder contractions activated by transurethral infusion of saline in acute spinal rats were promptly abolished by topical tetrodotoxin (20 ~g in 0.1 ml) as well as by intravenous hexamethonium (20 mg/kg), thereby indicating their neurogenic reflex origin. Bladder response to transvesical filling in acute spinal rats These experiments were performed in naloxonepretreated (0.2 mg/kg prior to spinal cord transection) animals. Transvesical infusion of saline in rats spinalized at the upper cervical level ( C 2 - C 3 ) elicited in 9 out of 16 (56%) preparations a series of rhythmic phasic contractions of the urinary bladder. Amplitude of these contractions ranged between 6 and 16 m m Hg and frequency between 2 and 3 contractions/min. Duration of each contractile wave did not exceed 15 s. None of these rhythmic contractions was sufficient to produce bladder voiding and this may account for their rhythmic behaviour 29. The rhythmic bladder contractions elicited in response to transvesical C M G in acute spinal rats were promptly suppressed following intravenous hexamethonium (20-40 mg/kg, n = 6), thus indicating their reflex neurogenic origin. Effect o f physostigmine pretreatment on bladder response to transvesical C M G in acute spinal rats Experiments described in the preceding section indicate that, in a certain percentage of preparations, shortly after spinal cord transection, a spinal vesicovesical reflex could be observed in response to transvesical CMG. The question was raised as to whether or not failure to observe a rhythmic phasic contractile activity in about 40% of preparations could be as-
cribed to the fact that urethane exerts a depressant effect on spinal reflexes as'38 and, therefore, reflex contractions were too weak to be recorded. To assess this point after having recorded a first C M G , the bladder was emptied and the preparations received intravenous physostigmine (0.1 mg/kg). After 20 min a new C M G was made; this time lag allowed us to obtain reproducible cystometric recordings in controls (saline-treated). These experiments were performed in naloxone-pretreated (0.2 mg/kg, i.v.) animals. In 6 out of 6 preparations pretreatment with physostigmine induced appearance of rhythmic bladder contractions (Fig. 6, upper and middle panel) having an amplitude comprised between 9 and 29 m m Hg at a frequency of 2 - 3 contractions/rain. In none of these preparations the first C M G had elicited rhythmic bladder contractions higher than 4 m m Hg (Fig. 6 up-
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Fig. 6. Upper and middle panel: the bladder responses to transvesical CMG in one acute (1 h before the first CMG) spinal (C2-C3) rat before and after the administration of intravenous physostigmine (0.1 mg/kg 10 min before the second CMG). Note that following physostigmine, saline filling induced the appearance of rhythmic bladder contraction having sufficient amplitude and duration to produce, at the top of each phasic contraction, the emission of one or two drops of fluid (dripping). These rhythmic contractions were promptly abolished by intravenous atropine (0.1 mg/kg). Lower panel: potentiating effect of intravenous physostigmine (0.1 mg/kg) on the rhythmic bladder contraction produced by transvesical infusion of saline in one acute (1 h before) spinal (C2-C3) rat. The reflex nature of the phasic bladder contractions was proven by their suppression following intravenous hexamethonium (20 mg/kg).
90 per panel). Duration of the physostigmine-activated rhythmic bladder contractions did not exceed 15 s in 3 out of 6 preparations while in the remainder it was greater than 20 s. In these latter preparations one or two drops of fluid were expelled at the top of each phasic contraction (Fig. 6, middle panel). The rhythmic behaviour of the phasic, contractile activity elicited by transvesical C M G in physostigmine-pretreated spinal rats indicated that bladder voiding was largely incomplete (Fig. 6, upper panel). Moreover, after each phasic contraction intraluminal pressure did not return below pressure threshold as was observed following micturition in normal rats (Fig. 6, middle panel). In a further series of experiments we studied the effect of physostigmine (0.1 mg/kg, i.v.) in spinal preparations which developed, in response to transvesical CMG, a series of rhythmic bladder contractions. In these experiments amplitude of rhythmic bladder contractions was enhanced by physostigmine (fromll +2to18+2mmHg, n = 6 , P < 0 . 0 1 ) as was the duration of each contractile wave (from 10 + 1 to 18 + 2 s, P < 0.01) while the frequency of rhythmic contractions was not significantly affected (from 2.3 + 0.2 to 2.6 + 0.3 contractions/min; Fig. 6, lower panel). Rhythmic bladder contractions elicited in physostigmine-pretreated spinal rats were promptly suppressed by intravenous hexamethonium (20 mg/kg) or atropine (1 mg/kg; n = 6, Fig. 6). DISCUSSION Supraspinal origin of micturition contraction in normal adult rats There is evidence suggesting that, in rats, both anatomical and neurophysiological organization of the micturition reflex is similar 33'37'39 to that described in catsS.9,22,23. Cystometric experiments revealed that high-amplitude (over 20 mm Hg) vesicovesical reflex contractions could be observed in response to bladder distension in adult anaesthetized r a t s 24'26'3°'37'40 but not in acute spinal preparations 31"37'4°. Transection experiments 4° revealed that, in rats, the supraspinal center activating the distension-induced bladder contractions is most likely located at pontine level suggesting its identity with the pontine micturition center de-
scribed by Satoh et al. 39. It appears conceivable that the high amplitude (>20 mm Hg) vesicovesical reflex which produces bladder voiding in response to transvesical saline filling in urethane-anaesthetized rats which was demonstrated in the present study is of supraspinal origin, since it is abolished by spinal cord transection. However, a simple reduction in amplitude of the reflex contractions in acute spinal rats does not in itself establish a supraspinal origin of the reflex since a depression due to spinal shock may have been implicated. Electrophysiological experiments are needed to establish firmly whether the vesicovesical reflex which produces bladder voiding in anaesthetized rats is of supraspinal origin; however, 3 lines of evidence support this hypothesis i.e., (a) destruction of the pontine micturition center produces urine retention in rats 39 indicating that, in this species, spinal reflex(es) are insufficient to produce bladder voiding and (b) intracisternal administration of chemicals (acetylcholine, glycine, substance P) activates a series of rhythmic bladder contractions whose characteristics of amplitude, frequency and duration are the same of the high-amplitude bladder contractions elicited by saline filling (ref. 25 and M. Furio, unpublished data) and (c) reduction in amplitude (15-20%) of the spinal 37 somatovesical reflex following spinal cord transection is much lower than that (50-60%) of the rhythmic contractions observed in response to transurethral saline filling. This latter point indicates that the 'spinal shock' alone cannot explain the absence, in response to transurethral CMG in spinal rats, of the high-amplitude contractions which may represent activation of the supraspinal vesicovesical reflex. Our findings in urethane-anaesthetized rats establish that attainment of an intraluminal pressure of at least 20 mm Hg is required for producing bladder voiding. Therefore it is not surprising that all types of reflex contractile activity (notably both somatovesical and vesicovesical spinal reflexes) which do not attain such critical pressure values are 'per se' insufficient to induce bladder voiding in normal adult animals. Duration of micturition contraction appears also relevant in determining an efficient bladder voiding; in fact, duration of micturition contraction in response to transvesical filling (usually over 20 s) out-
91 lasts that of both somato- and vesicovesical reflexes which can be observed in spinal rats.
Spinal vesicovesical excitatory reflex and its potential relationship with transurethral CMG De Groat and Ryal114 described that, in cats, a short latency C-fiber reflex could be detected as early as 3 days after spinal cord transection. This reflex increased thereafter parallel to development of automatic micturition 14. Appearance of this vesicovesical reflex following spinal cord transection could be explained either by axonal sprouting (and consequent formationof new pathways) or removal of a descending bulbospinal inhibitory pathway 7's'13. Subsequent experiments indicated that the C-fiber reflex may be present also in normal cats which supports the second hypothesis (inhibition of the spinal reflex by descending inhibitory pathways)11. Our present findings provide evidence indicating that in adult rats a vesicovesical excitatory reflex, organized at a spinal level, can be observed within a short time (0.5-2 h) from spinal cord transection. It is unlikely that formation of new pathways by axonal sprouting occurred in such a short lag of time. Therefore we must conclude that anatomical connections responsible for a vesicovesical spinal reflex are already present in normal adult rats. This is supported by anatomical observations indicating that primary afferents from the detrusor may come into close contact with sacral parasympathetic neurons at spinal level either directly (monosynaptic interaction) or through interneurons 33. In rats a certain recovery of bladder function was observed within 3 weeks from destruction of the ponfine micturition center 39. It is conceivable that the vesicovesical spinal reflex demonstrated in our experiments subserves automatic micturition in chronic spinal rats. Failure to observe this spinal vesicovesical reflex during the collecting phase of the transvesical CMG suggests that this 'short-loop' reflex may be suppressed by some inhibitory mechanism(s) of supraspinal origin. Emergency of the vesicovesical spinal reflex shortly after spinal cord transection in response to transvesical filling cannot be simply ascribed to administration of naloxone (see below) and suggests a supraspinal origin of these inhibitory mechanism(s) as also indicated by electrophysiologi-
cal findings in cats 1134. It appears conceivable that, if a similar organization of micturition reflexes exists in humans (i.e. supraspinal inhibition of the spinal vesicovesical reflex), then neurological or even functional disturbances of these inhibitory mechanism(s) might lead to emergency of involuntary reflex detrusot contractions. In most of the present experiments the spinal vesicovesical reflex was demonstrated in naloxone-pretreated animals. Recent findings from various laboratories indicate that endogenous opioids may be involved in regulating micturition in c a t s 6'21 and r a t s 15A6'36. However, administration of naloxone does not seem necessary to observe the spinal vesicovesical reflex although it may have increased its excitability. In fact a vesicovesical reflex was demonstrated in 5 out of 22 (23%) and in 6 out of 18 preparations (33 %) whose spinal cord was transected at C2C3 (present study) or T12-L131 level, respectively. It is worth mentioning that the excitatory effect of naloxone pretreatment may be observed when this drug was administered prior to but not following spinal cord transection. Failure to observe the reflex in a certain percentage of preparations is presumably ascribable to some depressant effect of anaesthesia or spinal shock since: (a) urethane depresses amplitude of the spinal reflex and (b) the spinal reflex could be demonstrated in all preparations in appropriate conditions (naloxone and physostigmine pretreatment). This suggests that failure of naloxone to increase excitability of the spinal reflex when it is administered to spinal rats does not depend upon the existence of a population of 'non-responders'. Further studies are required to clarify the mechanisms underlying the mechanisms through which naloxone increases excitability of the spinal vesicovesical reflex when it is administered prior to spinalization. It is noteworthy that the characteristics (amplitude, frequency, duration of each contractile wave) of the vesicovesical reflex observed in acute spinal rats closely mimicked those of the 'early' phase of the transurethral CMG of normal animals. On the other hand, in the majority of transvesical CMGs no significant contractile activity is recorded until micturition. In view of the above it might be speculated that, in response to transurethral CMG the spinal vesicovesical reflex is elicited at low filling volume and is there-
92 after replaced by a supraspinal vesicovesical reflex. If this were true we should assume that factors, such as presence of the urethral ligature, increase the excitability of the vesicovesical spinal reflex leading to its emergency in the 'early' phase of the transurethral CMG. It is well known that urethral afferents modulate reflex activation of the detrusor muscle. For instance the seventh Barrington's reflex (having both afferent and efferent limb in pelvic nerves) is a spinal reflex producing bladder contraction in response to stimuli arising in the urethra; such a reflex is thought to play a role in reinforcing bladder contraction during micturifion 19.
urethane on excitability of the somatovesical reflex. Such a finding recalls some unpublished data from De Groat (quoted in ref. 8) who observed that excitability of perineal and bladder reflexes was increased following i.v. strychnine in cats. A strychnine-sensitive recurrent inhibition in sacral parasympathetic pathways to the bladder was demonstrated previously by De Groat and Ryal113. Therefore it is conceivable that such an inhibitory mechanism, presumably potentiated by deepening of anaesthesia, plays a role in reducing excitability of the excitatory somatovesical reflex in adult rats.
CONCLUSIONS Spinal somatovesical excitatory reflex in normal adult rats Some characteristics of the somatovesical excitatory reflex deserve consideration. This type of response is of primary relevance in producing bladder voiding in newborn kittens s and rats 1'2°'41. In normal
adult cats stimulation of the perineum or sex organs either inhibits micturition or produces small bladder contractions which are too weak to release urine 8'9'n. In adult rats somatovesical reflex was found to have opposite effects on bladder contractility depending upon the degree of bladder filling 37,38. However, as already observed in cats, bladder contractions produced in this way are too weak to release urine. We also observed that the somatovesical reflex was influenced markedly by depth of anaesthesia. This is consistent with previous observations on the depressant effect of urethane on spinal reflexes in the isolated frog spinal cord 18. Interestingly, intravenous strychnine antagonized the depressant effect of
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In conclusion, our findings suggest that at least 3 distinct vesicoexcitatory reflexes are present in urethane-anaesthetized adult rats. Of these only the vesicovesical reflex observed in animals with an intact spinal cord appears to produce an increase of intraluminal pressure having sufficient intensity and duration to induce efficacious bladder voiding. This reflex could be similar or even identical to the supraspinal reflex which subserves bladder voiding in normal animals 39. Available evidence suggests that a spinal vesicovesical reflex could be selectively elicited when CMGs are performed through the transurethral route. Supraspinal inhibition of such a vesicovesical 'short-loop' reflex could contribute to urine continence before micturition, The set of experimental models described in this paper may be suitable for studying the physiology and pharmacology of the mechanisms regulating micturition at spinal and supraspinal level.
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