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Strain-Gauge Force Transducer Method for Evaluating Urethral Motility in Conscious Dogs
0022-5347/97/1573-1076$03.00/0 THE JOLIRNAL OF UROLOGY
Vol. 157. 1078-1082. March 1997 Printed
Copyright 0 1997 by A M E R ~ ~UROUKICAL AN ASXICIATION, lw.
In
U.S.A
STRAIN-GAUGE FORCE TRANSDUCER METHOD FOR EVALUATING URETHRAL MOTILITY IN CONSCIOUS DOGS NORIKATSU KATO," W J I IWANAGA, KOUJI MORIKAWA, HIDE0 KATO, YASUO I T 0 ZEN ITOH
AND
From the Research and Deiielopment Dwision, Hokuriku Seiyaku Co , Ltd , Katsuyama, Japan and the I n w u t r ot Molrc ulnr und Cellular Regulation. Gunma Uncoerstty, Maebashl, Japan
ABSTRACT
A new method has been developed that is suitable for physiological studies of urethral motility in the conscious state with a strain-gauge force transducer (force transducer). In anesthetized dogs, the contractile or relaxing responses of the urethral smooth muscle in the direction of the circular layers measured with the force transducer, elicited by either phenylephrine or isoproterenol, were significantly correlated to the increase or decrease in the intra-urethral pressure (r = 0.998 and 0.780, respectively). In conscious dogs, the fluctuation in the urethral tone was observed to be less than in the intra-urethral pressure, and a dose-dependent response to phenylephrine (1-30 pg./kg. i.v.) and isoproterenol (0.1-3 pg./kg. i.v.1 was clearly recognized. Moreover, this conscious model provided a fairly reproducible pattern with the urethral contraction or relaxation related to the filling or voiding phase of the cystometrogram, which was abolished by anesthetization. These results indicate that the present force transducer method makes i t possible to evaluate the further physiological characteristics of urethral motility in conscious dogs. KEY WOROS:urethral contraction, intra-urethral pressure, conscious dogs, strain-gauge transducer A strain-gauge force transducer (force transducer) was constructed by Walton et al. in 1950 to measure the contractile force of the heart,' and improved force transducers are now extensively used in conscious animals to evaluate gastroint e ~ t i n a l ,gallbladder' ~,~ and uterine5 motor activity. I n regard to the urethra, there are a few reports of studies in which Tsuchida et a1.6 and Nishizawa et al.7 investigated urethral response evoked by autonomic agents and electrical stimulation of autonomic nerves in anesthetized dogs fitted with the force transducer. Notoe also evaluated urethral motility during the micturition reflex by using the force transducer in decerebrated dogs, but the anesthetization caused a drop in intra-urethral pressure9 and abolished the reflex voiding;lO and the surgical decerebration induced facilitation of reflex voiding.11 The micturition reflex was readily subjected to the modulating influence of neural pathways, including the peripheral and the central nervous levels, so that suitable models allowing the study in conscious animals are needed for a complete evaluation of urethral motility, especially in the voiding cycles. The present experiments were designed to develop a new method for evaluating urethral motility in conscious dogs by means of a new experimental h I . the force transducer. MATERIALS AND METHODS
The following pharmacologic manipulation was composed of three different analytic sections: the first two were performed to compare the force transducer recording of urethral motility, urethral tone, versus (v/s) intra-urethral pressure; the last one, essential to our study, was the real recording of urethral tone in the conscious state. Analysis of the correlation of urethral tone u l s intraurethral pressure in anesthetized dogs. Nine female beagle dogs (7.4-12.0 kg.) were anesthetized with sodium pentobarbital, and ventilated artificially. The intra-urethral pressure
was measured by the method of Brown and Wickham'z as the perfusion pressure into a urodynamic urethral catheter (041312; 12 French, Create Medic Co., Ltd., Yokohama, Japan, the perfusion rate being 100 m1.h.); its side holes were placed at the point where the intra-urethral pressure was maximal (UPmax). The vesico-urethra was then exposed by midline suprapubic laparotomy. A force transducer (F-081S, Star Medical Corp., Tokyo, Japan, or constructed by us? 8 or 9 x 5 mm. (wh); these calibrations were trustworthy up to 100 gm. weight) was sutured onto the serosal surface of the urethra and oriented in the direction of the circular muscle layers at the point of UPmax (Figure I),while five of the dogs were implanted with another force transducer in the direction of the longitudinal muscle layers. The right femoral vein was cannulated for intravenous administration of drugs, and the force transducer was connected to an amplifier (FA-01, Star Medical Corp.). Comparison of fluctuation which occurred in urethral tone vls intra-urethral pressure in conscious dogs. Six female beagle dogs (7.4-10.9 kg.) were anesthetized with sodium pentobarbital, and a midline laparotomy was made. A force transducer was sutured onto the urethral serosa in the direction of the circular muscle layers a t the point of UPmax. A catheter (602-175, Dow Corning Corp., Midland, Michigan) for the measurement of intra-urethral pressure was inserted through a small incision made in the proximal urethra near the bladder neck and its tip was fixed at the point of UPmax. Two catheters (602-205, Dow Corning Corp.) for drainage of urine and intravenous administration of drugs were introduced into the bladder and the right jugular vein, respectively, and fixed. The lead wires for the force transducer and catheters were drawn out of the abdominal cavity through a stab wound made in the right abdominal wall, passed through a subcutaneous tunnel made on the right costal flank, and brought out through a skin incision between the scapulae. The incisions were closed in layers, and the lead wires and catheters were protected by a fitted jacket (FPJ-12, Star Medical Corp.). After a one week recovery period follow-
Accepted for publication July 8, 1996. * Re uests for reprints: Research and Development Division, Hokunxu Seiyaku Co., Katsuyama, Fukui 911, Japan. 1078
STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
1079
statistical analysis was performed by Student's t test; p <0.05 was considered significantly different for unpaired data. RESULTS
FIG. 1. Schematic diagram of implantation of strain-gauge force transducer on extraluminal canine urethra. The straingauge force transducer is placed at locus of maximum intra-urethral pressure (UPmax):length from vesico-urethral junction as well as functional urethral length are indicated in urethral pressure profile as mean ? S.E.M. (n = 25).Morphological details of urethral wall of UPmax are shown in light inset In = 5; excised urethral preparations from five dogs were fixed in neutral formalin after removal of force transducer, and tissues were then embedded in paraffin wax followed by cutting along circular axis, and dimensions of these were measured, while conditions of attachment of force transducer on urethra, e.g. sewed securely or not, adhered t o other parts of the gut, were checked on all twenty-five dogs after each examination).
ing the surgery, experiments were performed during the conscious state, while a pressure transducer (TP-ZOOT, Nihon Kohden Corp., Tokyo, Japan) for intra-urethral pressure was directly attached to the dog's jacket. Recording of response in urethral tone to adrenergic agonisfs and during micturition reflex in the conscious (and anesthetized) state of dogs. Ten female beagle dogs (8.3-11.2 kg.) which were chronically implanted with a n extraluminal force transducer and catheters for urine drainage and drug administration were prepared by the same procedures as described in the last paragraph. The micturition reflex was evoked, while dogs were conscious, with the constant infusion of saline into the bladder via the drainage catheter in the opposite way (at the rate of 6-10 ml./min.). Intra-vesical pressure was monitored as the infusion pressure while the bladder was filled (cystometrogram ). The dogs were then anesthetized with sodium pentobarbital, and the drug administration as well as the infusion for cystometrogram were performed. Preparation and administration of drugs. The following drugs were used: 1-phenylephrine hydrochloride (phenylephrine, Sigma, St. Louis, Missouri), 2-isoproterenol hydrochloride (isoproterenol, Sigma), sodium pentobarbital (pentobarbital, Abbott Lab., North Chicago, Illinois; 30 mg./kg.). Phenylephrine and isoproterenol were freshly dissolved in saline, and all drugs were administered intravenously. Analysis of data. In evaluating the relationship of urethral tone to intra-urethral pressure in the anesthetized dogs, a linear regression analysis was performed, and statistical significance of correlation was accepted at p <0.05. The data showing spontaneous changes in urethral tone as well as intra-urethral pressure were input into a computer (PC9801, NEC, Tokyo, Japan) for the measurement of fluctuation indices denoted by the area under the urethral response waves during one minute. And to evaluate the moveassociated fluctuations (the dogs were forcibly moved by us), fluctuation indices taken 1 min. after the change in the dog's posture were calculated. The urethral responses obtained in the comparative studies were calculated as percentages of the response evoked by 30 MJkg. i.v. of phenylephrine. The data were expressed as the mean 2 S.E.M., and the
Analysis of the correlation of urethral tone v l s intraurethral pressure in anesthetized dogs. In the anesthetized state, the injection of phenylephrine, a selective alpha,adrenergic agonist (30 pg./kg. i.v.), produced urethral muscle contraction, and isoproterenol, a beta-adrenergic agonist (3 pg./kg. i.v.i, caused relaxation (Figure 2). These responses which were coincident with the changes in intra-urethral pressure were clearly observed in the direction of the circular layers, whereas those in the longitudinal layers were slight (Figure 2). The same responses were obtained in four other dogs. By using dogs with one force transducer to measure circular urethral tone, phenylephrine (1-30 pg./kg. i.v.i produced urethral contraction as well as a n increase in intraurethral pressure, while isoproterenol (0.3-3 pg.kg. i.v.) caused urethral relaxation and a decrease in intra-urethral pressure, and these responses were dose-dependent. The quantitative comparison of the changes in these two methods revealed significant correlations (Figures 3A and 3B). Comparison of fluctuation which occurred in urethral tone u / s intra-urethral pressure in conscious dogs. By using the conscious dog, urethral tone and intra-urethral pressure were investigated simultaneously for eight successive hours. No matter what the position of each dog was, whether lying, sitting or standing, phasic fluctuation in the basic intraurethral pressure was observed irregularly and frequently (Figure 4Ai. The magnitude of a spontaneous fluctuation which occurred in urethral tone was obviously less than that in intra-urethral pressure (Table 1A).Furthermore, the basic intra-urethral pressure also fluctuated tonically accompanying changes in the body position of the dogs (Figure 4B). Especially when the dog moved from the sitting to the lying position, the fluctuation was significantly greater than that in urethral tone (Table 1B). Recording of response in urethral tone to adrenergic agonists and during micturition repex in the conscious (and anesthetized) state of dogs. While the dogs were conscious, the injection of phenylephrine (1-30 pg./kg. i.v.) produced dosedependent urethral contraction (Figures 5A and 5C), but no further increase in response was observed up to 50 pg./kg. i.v. The relaxing response to isoproterenol(O.1-3 pg./kg. i.v.) also was dose-dependent (Figures 5A and 5C). The administration of pentobarbital (30 mg./kg. i.v.) significantly induced tonic relaxation of the urethral muscle (0.9 -C 0.2 gm. of relaxation; n = 5). The responses elicited by phenylephrine and isoproterenol under anesthesia were quantitatively sim-
FIG 2. ResPnses in circular and longitudinal muscle layem of urethra and intra-urethral pressure to phenylephrine (let%)and kproterenol (right) in anesthetized dog. I.U.P.,intra-urethral pressure; U.T., urethral tone. For U.T., contractile response is indicated by upward change.
STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
1080
-E v
( A ) Phenylephrine
E 0
P.-8! c
-
= 3.929 +
( B ) lsoproterenol
;he micturition reflex was not achieved by filling the bladder with saline and there was consequently leakage from the 2xternal meatus (Figure 6B), these being observed in all five logs.
1.046 X
E
DISCUSSION
The present study is the first report demonstrating urethral motility in conscious dogs by means of a force trans5 40 ! ducer. The force transducer has recently been utilized to a 20 evaluate normal physiological motor activity of organs in c 0 conscious animals.2" Itoh et a1.2 developed a n excellent im8E - 0 o 2 0 4 0 6 0 a o i o o - 0 0 - 1 0 - 2 0 - 3 0 - 4 0 - 5 0 proved force transducer which is waterproof, insulated and flexible, and can be used to assess the gastrointestinal motor a activity in conscious dogs for a long period without disturbing Response of intra-urethral pressure (%) the flow of the intra-luminal contents. The investigation of FIG. 3. Relationship between changes in urethral tone of circular urethral motility employing the force transducer offers same muscle layer and intra-urethral pressure in anesthetized dogs. (A) advantages: it can avoid urethral intubation which may creshows phenylephrine-induced responses (n = 18 from 5 dogs), and ate artificial resistance and disturb urine flow; and it could (B) shows isoproterenol-induced responses (n = 11 from 4 dogs) at be utilized to measure motility in selected segments in sedose of 0.3 (reverse triangles), 1 (circles),3 (triangles),10 (squares) and 30 (lozenges) pg./kg. i.v. Each value is expressed as percentage lected directions,2 whereas the pressure recordings could not of response induced by 30 crg./kg. i.v. of phenylephrine, while 100% be evaluated without the unnecessary appendages which response in (A) is 26.2 f 2.6 cm. H,O increase in intra-urethral resulted from the pressure changes in its surroundings. pressure (1.U.P.)and 7.7 f 1.6 gm.for urethral contraction, and that Intra-urethral pressure has generally been evaluated as an in (B) is a 49.4 z 1.8 cm. H,O increase in I.U.P. and 9.5 z 0.8 gm.for index of urethral motility both in in vivo studiesI3 and clinurethral contraction, respectively. Linear regression analysis was utilized to assess correlation between changes in urethral tone and ical urodynamic examinations.9-l4 This customary recording I.U.P., and r is Pearson's statistical constant. of intra-urethral pressure was therefore first quantitatively compared to the new one of urethral tone. The contribution of the sympathetic nervous system to a substantial part of urethral activity is significant, and can be attributed to adrenoceptors in the urethral smooth muscles.13 We therefore utilized adrenergic agonists to induce the urethral response. The experiment was camed out on female dogs, and the force transducer was placed at the UPmax point on the urethra where there was the greatest contribution to urethral resistan~e.1~ In anesthetized dogs, phenylephrine and isoproterenol produced urethral contraction and relaxation, respectively, but only in the circumferential direction, and this finding was also reported by Nishizawa et al.7 In the in vitro comparison of the isometric tension generated in urethral transverse and longitudinal muscular strips in response to either adrenergic agonistsI5 or electrical field stimulations,lfi the transverse strips clearly responded to a greater degree than the longitudinal ones. And histologically, circularly oriented muscular fibers formed the muscularis layer of the proximal urethra in the vast majority as compared with longitudinal fibers which l6It were scattered in the submucous and subserous FIG.4. Occurrence of fluctuations in circular urethral tone and therefore seems that the contractile activity of the circular intra-urethral pressure in conscious dog. (A) shows that fluctuations muscle is closely involved in generating urethral resistance. in base-line occurred in keeping with posture, and (B) shows those in In addition, the circular muscular response to adrenergic movement of dog, while in (B), stippled columns indicate forcibly changed posture of dog. I.U.P., intra-urethral pressure; U.T.,ure- agonists was found to be a significant parallel response to thral tone, that for U.T.contractile response is indicated by upward intra-urethral pressure, which provides proof of that the change. Each response induced by 30 pg./kg. i.v. of phenylephrine change in circular urethral tone quantitatively reflects urewas comparatively uniform. thral motility as well as a change in intra-urethral pressure. With regard to the measurement in conscious dogs, the fluctuation occurring in urethral tone, both when moving ilar to those observed initially in the conscious state (Figure (sittingup from the lying position) and not moving conditions 5 ). (lying and sitting), was noticeably less than that observed in In order to further investigate the functional regulation of the intra-urethral pressure. The difference between these the urethral motility in the conscious dogs, the volume- fluctuations in frequency and quantity was attributed evoked micturition reflex was performed by constant and certain factors: in the measurement of the intra-urethral continuous infusion of saline into the bladder. When the pressure, the obstruction of the perfusion of saline by the bladder was filled, intra-vesical pressure slowly and steadily bending and straining of the urethral catheter owing to the increased, and voiding was accomplished by intra-vesical dog's movement, and the synergic addition of fluctuations in pressure augmentation, followed by a drop immediately after intra-abdominal pressure. These results suggest that the the expulsion of urine. With regard to the urethral tone, the force transducer method permits more quantitative analysis filling phase was characterized by progressive contraction of of urethral motility in conscious dogs than the customary the urethral muscle. And the voiding phase started with the intra-urethralpressure recording. transient relaxation of the urethral muscle followed by a conscious dogs, urethral contraction or relaxation By rapid return to the base-line (Figure 6A). Under anesthesia, in a dose-dependent manner in response to either phenyleph-
x
d
1081
STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
TABLE1. Comparison of fluctuations in the urethral tone and the intra-urethml pressure in COMCWUSdogs ( A ) In maintained posture
Lying Urethral tone (5) Intra-urethral pressure ($6) _- (B) In changing posture
17.3 0.2 -c -t 2.1 1'3
Sitting
1-
23.3 2 1.5 12.1 1.8
1-
27.9 17'2 Z 3'9 4.2
1
N.s
Standing fmm lying
Sitting fmm lying Urethral tone (%I
Standing
12.7 ? 3.2
1-
1
N,S,
15.3 7.7 2 2'1 1.6 Intra-urethral pressure (96) 10.2 2 1.6 The absolute fluctuation values were changed to percentages of the response induced by 30 pgsJlrs. i.v. of phenylephrine. end are indicated 811 the mean 2 S.E.M. (A) shows the fluctuation which occurred in the motionless conditions (n = 5 from 5 dogs), and (B) shows those in the movement of the dog (n = 15 from 5 dogs), whereas in (B), the 100%responses are 13.9 2 2.2 grn. for urethral contraction and a 54.2 2 6.8 em. H,O increase in intra-urethralpressure. reopeetively. The significance of difference between the urethral tone and the intra-urethral pressure is shorn as **; p <0.01, and N.S.means not sigtufcant.
I.V.P. .___-A
L
3
Dose ( pglkg i.v. )
FIG. 5. Contractile and relaxing response in circular urethral muscle to adrenergic agonists in conscious and anesthetized dogs. Typical responses to phenylephrine (left) and isoproterenol (right)in urethral tone (U.T.)in (A) conscious state and (B) anesthetized with sodium pentobarbital (30 mg./kg. i.v.) are shown. For U.T., contractile response is indicated by u ward change. (C) shows, dosedependent urethral response to pgenylephrine (circles) and isoproterenol (triangles), and each value is mean i_ S.E.M. for five dogs, whereas closed and open symbols indicate values recorded in conscious and anesthetized state, respectively.
rine or isoproterenol was recognized. And the response was unaltered after anesthesia with pentobarbital, which suggests that these exogenously applied agents exerted their actions through peripheral sites, not through the central nervous system. In addition, during the storage of saline (substituted for urine), progressive contraction of the urethral muscle was observed, and then voiding was accomplished by relaxation of the urethra, whereas the injection of pentobarbital resulted in complete blockade of the micturition reflex. Yaksh et a1.l" reported that the injection of anesthetics resulted in a rise in the threshold intra-vesical pressure to induce micturition, and subsequently overflow incontinence, which were attributed to their inhibitory effects on the synaptic transmission either at the peripheral or the spinal and supraspinal levels in the micturition reflex pathway. The volume-evoked micturition reflex is observed in the animal decerebrated a t the level of the intercolliculus, while transection of the neuraxis at any point below the colliculi abolishes that reflex." But in an experiment using decerebrated dogs, NotoRdid not notice a definite tendency to urethral response, occasionally showing an absence of or a contractile response in the circular urethral muscle accompanied by the expulsion of urine. It thus appears that decerebration, removing almost all of the brain matter, causes some systematic confusion in the regular innervation of the urethral tone controlled in a coordinated manner during normal micturition, possibly by a dog's
-------
FIG.6. Changes in circular urethral tone during micturition reflex in conscious dog and anesthetic-induced urinary incontinence. Cystometrogram of do was obtained during continuous infusion of saline into bladder (incficated by broken arrows) a t rate of 6 ml./min. in (A) the conscious state or (B)anesthetized with sodium pentobarbital (30 mg./kg. i.v.). I.V.P., intra-vesical pressure; U.T., urethral tone. That for U.T. contractile response is indicated by upward change. Arrows pointin upward and downward indicate standing up and micturition of jog, respectively. Broken underline means leakage of infused saline.
critical condition characterized by, e.g., a considerable decrease in blood pressure. In conclusion, it was found that our force transducer method-the conscious and continuous recording obtained with a force transducer chronically implanted on canine urethrawill provide an improved approach to the evaluation of the physiological characteristics of urethral motility. REFERENCES
1. Walton, R. P., Leary, J . S . and Jones, H. P.: Comparative increase in ventricular contractile force produced by several cardiac glycosides. J . Pharmacol. Exp. Ther., 98:346. 1950. 2. Itoh, Z.: Motility of the gut. In: Endocrinology of the Gut. Tokyo: Nanzando Company, chapt. 9, pp. 173-204, 1976. 3. Itoh, Z., Mizumoto, A., Iwanaga, Y., Yoshida. N., Toni, K and Wakabayashi, K: Involvement of 5-hydrotryptamine 3 receptors in regulation of interdigestive gastric contractions by motilin in the dog. Gastroenterology, 100: 901, 1991. 4. Fujimura, M., Sakamoto, T., Lluis, F., Beauchamp, R. D., Townsend, C. M., Gleeley, J r . and Thompson, J. C.: Effect of a cholecystokinin antagonist, proglumide, on cholecystohnin-8induced gallbladder contraction in conscious dogs. Biol. Signals, 1: 94, 1992. 5. Hawk, H. W. and Conley, H. H.: Effects of prostaglandin F, phenylephrine and ergonovine on uterine contractions in the ewe. J. Animal. Sci., 60:537, 1985. 6. Tsuchida. S., Koinuma, N., Nishizawa, 0.. Moriya, I., Satoh, s., Noto, H., Harada, T. and Satoh, K: Urethral response to nerve
1082
STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
stimulation measured by strain gauge force transducer. Urol. Int., 38:300, 1983. 7. Nishizawa, O.,Monya, I., Satoh, S., Fukuda, T. and Tsuchida, S.:Effects of autonomic agonists on in viva female canine urethral motility. Urol. Int., 40: 320, 1985. 8. Noto, H.: Urethral behaviors during mictural reflex on the decerebrated female dog. Nippon Hinyokika Gakkai Zasshl, 7 5 104, 1984. 9. Doyle, P. T. and Bnscoe, C. E.: The effects of drugs and anaesthetic agents on the urinary bladder and sphincters. Br. J. Urol., 48:329, 1976. 10. Yaksh, T. L.,Durant, P. A. C. and Brent, C. R.: Micturition in rats: a chronic model for study of bladder function and effect of anesthetics. Am. J. Physiol., 251: R1177, 1986. 11. Tang, P.C.: Levels of brain stem and diencephalon controlling
micturition reflex, J. Neurophysiol., 18: 583, 1955. 12. Brown, M. and Wickham, J. E. A,: The urethral pressure profile. Br. J. Urol., 41: 211, 1969. 13. Awad, S. A. and Downie, J. W.: Relative contributions of smooth and striated muscles to the canine urethral pressure profile. Br. J. Urol., 48:347, 1976. 14. MacGuire, E. J., Fitzpatrick, C. C., Wan, J., Bloom, D., Sanvordenker, J., Ritchey, M. and Gormley, E. A.: Clinical assessment ofurethral sphincter function. J. U d , 150: 1452,1993. 15. Itoh, K.: Pharmacological and anatomical studies of urethral function in the female dog proximal urethra. Nippon Hinyokika Gakkai Zasshi, 78: 917, 1987. 16. Anderson, P. O.,Malmgren, A. and Uvelius, B.: Functional responses of different muscle types of the female rat urethra in vitro. Acta Physiol. Scand., 140 365, 1990.
Vol. 157. 1078-1082. March 1997 Printed
Copyright 0 1997 by A M E R ~ ~UROUKICAL AN ASXICIATION, lw.
In
U.S.A
STRAIN-GAUGE FORCE TRANSDUCER METHOD FOR EVALUATING URETHRAL MOTILITY IN CONSCIOUS DOGS NORIKATSU KATO," W J I IWANAGA, KOUJI MORIKAWA, HIDE0 KATO, YASUO I T 0 ZEN ITOH
AND
From the Research and Deiielopment Dwision, Hokuriku Seiyaku Co , Ltd , Katsuyama, Japan and the I n w u t r ot Molrc ulnr und Cellular Regulation. Gunma Uncoerstty, Maebashl, Japan
ABSTRACT
A new method has been developed that is suitable for physiological studies of urethral motility in the conscious state with a strain-gauge force transducer (force transducer). In anesthetized dogs, the contractile or relaxing responses of the urethral smooth muscle in the direction of the circular layers measured with the force transducer, elicited by either phenylephrine or isoproterenol, were significantly correlated to the increase or decrease in the intra-urethral pressure (r = 0.998 and 0.780, respectively). In conscious dogs, the fluctuation in the urethral tone was observed to be less than in the intra-urethral pressure, and a dose-dependent response to phenylephrine (1-30 pg./kg. i.v.) and isoproterenol (0.1-3 pg./kg. i.v.1 was clearly recognized. Moreover, this conscious model provided a fairly reproducible pattern with the urethral contraction or relaxation related to the filling or voiding phase of the cystometrogram, which was abolished by anesthetization. These results indicate that the present force transducer method makes i t possible to evaluate the further physiological characteristics of urethral motility in conscious dogs. KEY WOROS:urethral contraction, intra-urethral pressure, conscious dogs, strain-gauge transducer A strain-gauge force transducer (force transducer) was constructed by Walton et al. in 1950 to measure the contractile force of the heart,' and improved force transducers are now extensively used in conscious animals to evaluate gastroint e ~ t i n a l ,gallbladder' ~,~ and uterine5 motor activity. I n regard to the urethra, there are a few reports of studies in which Tsuchida et a1.6 and Nishizawa et al.7 investigated urethral response evoked by autonomic agents and electrical stimulation of autonomic nerves in anesthetized dogs fitted with the force transducer. Notoe also evaluated urethral motility during the micturition reflex by using the force transducer in decerebrated dogs, but the anesthetization caused a drop in intra-urethral pressure9 and abolished the reflex voiding;lO and the surgical decerebration induced facilitation of reflex voiding.11 The micturition reflex was readily subjected to the modulating influence of neural pathways, including the peripheral and the central nervous levels, so that suitable models allowing the study in conscious animals are needed for a complete evaluation of urethral motility, especially in the voiding cycles. The present experiments were designed to develop a new method for evaluating urethral motility in conscious dogs by means of a new experimental h I . the force transducer. MATERIALS AND METHODS
The following pharmacologic manipulation was composed of three different analytic sections: the first two were performed to compare the force transducer recording of urethral motility, urethral tone, versus (v/s) intra-urethral pressure; the last one, essential to our study, was the real recording of urethral tone in the conscious state. Analysis of the correlation of urethral tone u l s intraurethral pressure in anesthetized dogs. Nine female beagle dogs (7.4-12.0 kg.) were anesthetized with sodium pentobarbital, and ventilated artificially. The intra-urethral pressure
was measured by the method of Brown and Wickham'z as the perfusion pressure into a urodynamic urethral catheter (041312; 12 French, Create Medic Co., Ltd., Yokohama, Japan, the perfusion rate being 100 m1.h.); its side holes were placed at the point where the intra-urethral pressure was maximal (UPmax). The vesico-urethra was then exposed by midline suprapubic laparotomy. A force transducer (F-081S, Star Medical Corp., Tokyo, Japan, or constructed by us? 8 or 9 x 5 mm. (wh); these calibrations were trustworthy up to 100 gm. weight) was sutured onto the serosal surface of the urethra and oriented in the direction of the circular muscle layers at the point of UPmax (Figure I),while five of the dogs were implanted with another force transducer in the direction of the longitudinal muscle layers. The right femoral vein was cannulated for intravenous administration of drugs, and the force transducer was connected to an amplifier (FA-01, Star Medical Corp.). Comparison of fluctuation which occurred in urethral tone vls intra-urethral pressure in conscious dogs. Six female beagle dogs (7.4-10.9 kg.) were anesthetized with sodium pentobarbital, and a midline laparotomy was made. A force transducer was sutured onto the urethral serosa in the direction of the circular muscle layers a t the point of UPmax. A catheter (602-175, Dow Corning Corp., Midland, Michigan) for the measurement of intra-urethral pressure was inserted through a small incision made in the proximal urethra near the bladder neck and its tip was fixed at the point of UPmax. Two catheters (602-205, Dow Corning Corp.) for drainage of urine and intravenous administration of drugs were introduced into the bladder and the right jugular vein, respectively, and fixed. The lead wires for the force transducer and catheters were drawn out of the abdominal cavity through a stab wound made in the right abdominal wall, passed through a subcutaneous tunnel made on the right costal flank, and brought out through a skin incision between the scapulae. The incisions were closed in layers, and the lead wires and catheters were protected by a fitted jacket (FPJ-12, Star Medical Corp.). After a one week recovery period follow-
Accepted for publication July 8, 1996. * Re uests for reprints: Research and Development Division, Hokunxu Seiyaku Co., Katsuyama, Fukui 911, Japan. 1078
STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
1079
statistical analysis was performed by Student's t test; p <0.05 was considered significantly different for unpaired data. RESULTS
FIG. 1. Schematic diagram of implantation of strain-gauge force transducer on extraluminal canine urethra. The straingauge force transducer is placed at locus of maximum intra-urethral pressure (UPmax):length from vesico-urethral junction as well as functional urethral length are indicated in urethral pressure profile as mean ? S.E.M. (n = 25).Morphological details of urethral wall of UPmax are shown in light inset In = 5; excised urethral preparations from five dogs were fixed in neutral formalin after removal of force transducer, and tissues were then embedded in paraffin wax followed by cutting along circular axis, and dimensions of these were measured, while conditions of attachment of force transducer on urethra, e.g. sewed securely or not, adhered t o other parts of the gut, were checked on all twenty-five dogs after each examination).
ing the surgery, experiments were performed during the conscious state, while a pressure transducer (TP-ZOOT, Nihon Kohden Corp., Tokyo, Japan) for intra-urethral pressure was directly attached to the dog's jacket. Recording of response in urethral tone to adrenergic agonisfs and during micturition reflex in the conscious (and anesthetized) state of dogs. Ten female beagle dogs (8.3-11.2 kg.) which were chronically implanted with a n extraluminal force transducer and catheters for urine drainage and drug administration were prepared by the same procedures as described in the last paragraph. The micturition reflex was evoked, while dogs were conscious, with the constant infusion of saline into the bladder via the drainage catheter in the opposite way (at the rate of 6-10 ml./min.). Intra-vesical pressure was monitored as the infusion pressure while the bladder was filled (cystometrogram ). The dogs were then anesthetized with sodium pentobarbital, and the drug administration as well as the infusion for cystometrogram were performed. Preparation and administration of drugs. The following drugs were used: 1-phenylephrine hydrochloride (phenylephrine, Sigma, St. Louis, Missouri), 2-isoproterenol hydrochloride (isoproterenol, Sigma), sodium pentobarbital (pentobarbital, Abbott Lab., North Chicago, Illinois; 30 mg./kg.). Phenylephrine and isoproterenol were freshly dissolved in saline, and all drugs were administered intravenously. Analysis of data. In evaluating the relationship of urethral tone to intra-urethral pressure in the anesthetized dogs, a linear regression analysis was performed, and statistical significance of correlation was accepted at p <0.05. The data showing spontaneous changes in urethral tone as well as intra-urethral pressure were input into a computer (PC9801, NEC, Tokyo, Japan) for the measurement of fluctuation indices denoted by the area under the urethral response waves during one minute. And to evaluate the moveassociated fluctuations (the dogs were forcibly moved by us), fluctuation indices taken 1 min. after the change in the dog's posture were calculated. The urethral responses obtained in the comparative studies were calculated as percentages of the response evoked by 30 MJkg. i.v. of phenylephrine. The data were expressed as the mean 2 S.E.M., and the
Analysis of the correlation of urethral tone v l s intraurethral pressure in anesthetized dogs. In the anesthetized state, the injection of phenylephrine, a selective alpha,adrenergic agonist (30 pg./kg. i.v.), produced urethral muscle contraction, and isoproterenol, a beta-adrenergic agonist (3 pg./kg. i.v.i, caused relaxation (Figure 2). These responses which were coincident with the changes in intra-urethral pressure were clearly observed in the direction of the circular layers, whereas those in the longitudinal layers were slight (Figure 2). The same responses were obtained in four other dogs. By using dogs with one force transducer to measure circular urethral tone, phenylephrine (1-30 pg./kg. i.v.i produced urethral contraction as well as a n increase in intraurethral pressure, while isoproterenol (0.3-3 pg.kg. i.v.) caused urethral relaxation and a decrease in intra-urethral pressure, and these responses were dose-dependent. The quantitative comparison of the changes in these two methods revealed significant correlations (Figures 3A and 3B). Comparison of fluctuation which occurred in urethral tone u / s intra-urethral pressure in conscious dogs. By using the conscious dog, urethral tone and intra-urethral pressure were investigated simultaneously for eight successive hours. No matter what the position of each dog was, whether lying, sitting or standing, phasic fluctuation in the basic intraurethral pressure was observed irregularly and frequently (Figure 4Ai. The magnitude of a spontaneous fluctuation which occurred in urethral tone was obviously less than that in intra-urethral pressure (Table 1A).Furthermore, the basic intra-urethral pressure also fluctuated tonically accompanying changes in the body position of the dogs (Figure 4B). Especially when the dog moved from the sitting to the lying position, the fluctuation was significantly greater than that in urethral tone (Table 1B). Recording of response in urethral tone to adrenergic agonists and during micturition repex in the conscious (and anesthetized) state of dogs. While the dogs were conscious, the injection of phenylephrine (1-30 pg./kg. i.v.) produced dosedependent urethral contraction (Figures 5A and 5C), but no further increase in response was observed up to 50 pg./kg. i.v. The relaxing response to isoproterenol(O.1-3 pg./kg. i.v.) also was dose-dependent (Figures 5A and 5C). The administration of pentobarbital (30 mg./kg. i.v.) significantly induced tonic relaxation of the urethral muscle (0.9 -C 0.2 gm. of relaxation; n = 5). The responses elicited by phenylephrine and isoproterenol under anesthesia were quantitatively sim-
FIG 2. ResPnses in circular and longitudinal muscle layem of urethra and intra-urethral pressure to phenylephrine (let%)and kproterenol (right) in anesthetized dog. I.U.P.,intra-urethral pressure; U.T., urethral tone. For U.T., contractile response is indicated by upward change.
STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
1080
-E v
( A ) Phenylephrine
E 0
P.-8! c
-
= 3.929 +
( B ) lsoproterenol
;he micturition reflex was not achieved by filling the bladder with saline and there was consequently leakage from the 2xternal meatus (Figure 6B), these being observed in all five logs.
1.046 X
E
DISCUSSION
The present study is the first report demonstrating urethral motility in conscious dogs by means of a force trans5 40 ! ducer. The force transducer has recently been utilized to a 20 evaluate normal physiological motor activity of organs in c 0 conscious animals.2" Itoh et a1.2 developed a n excellent im8E - 0 o 2 0 4 0 6 0 a o i o o - 0 0 - 1 0 - 2 0 - 3 0 - 4 0 - 5 0 proved force transducer which is waterproof, insulated and flexible, and can be used to assess the gastrointestinal motor a activity in conscious dogs for a long period without disturbing Response of intra-urethral pressure (%) the flow of the intra-luminal contents. The investigation of FIG. 3. Relationship between changes in urethral tone of circular urethral motility employing the force transducer offers same muscle layer and intra-urethral pressure in anesthetized dogs. (A) advantages: it can avoid urethral intubation which may creshows phenylephrine-induced responses (n = 18 from 5 dogs), and ate artificial resistance and disturb urine flow; and it could (B) shows isoproterenol-induced responses (n = 11 from 4 dogs) at be utilized to measure motility in selected segments in sedose of 0.3 (reverse triangles), 1 (circles),3 (triangles),10 (squares) and 30 (lozenges) pg./kg. i.v. Each value is expressed as percentage lected directions,2 whereas the pressure recordings could not of response induced by 30 crg./kg. i.v. of phenylephrine, while 100% be evaluated without the unnecessary appendages which response in (A) is 26.2 f 2.6 cm. H,O increase in intra-urethral resulted from the pressure changes in its surroundings. pressure (1.U.P.)and 7.7 f 1.6 gm.for urethral contraction, and that Intra-urethral pressure has generally been evaluated as an in (B) is a 49.4 z 1.8 cm. H,O increase in I.U.P. and 9.5 z 0.8 gm.for index of urethral motility both in in vivo studiesI3 and clinurethral contraction, respectively. Linear regression analysis was utilized to assess correlation between changes in urethral tone and ical urodynamic examinations.9-l4 This customary recording I.U.P., and r is Pearson's statistical constant. of intra-urethral pressure was therefore first quantitatively compared to the new one of urethral tone. The contribution of the sympathetic nervous system to a substantial part of urethral activity is significant, and can be attributed to adrenoceptors in the urethral smooth muscles.13 We therefore utilized adrenergic agonists to induce the urethral response. The experiment was camed out on female dogs, and the force transducer was placed at the UPmax point on the urethra where there was the greatest contribution to urethral resistan~e.1~ In anesthetized dogs, phenylephrine and isoproterenol produced urethral contraction and relaxation, respectively, but only in the circumferential direction, and this finding was also reported by Nishizawa et al.7 In the in vitro comparison of the isometric tension generated in urethral transverse and longitudinal muscular strips in response to either adrenergic agonistsI5 or electrical field stimulations,lfi the transverse strips clearly responded to a greater degree than the longitudinal ones. And histologically, circularly oriented muscular fibers formed the muscularis layer of the proximal urethra in the vast majority as compared with longitudinal fibers which l6It were scattered in the submucous and subserous FIG.4. Occurrence of fluctuations in circular urethral tone and therefore seems that the contractile activity of the circular intra-urethral pressure in conscious dog. (A) shows that fluctuations muscle is closely involved in generating urethral resistance. in base-line occurred in keeping with posture, and (B) shows those in In addition, the circular muscular response to adrenergic movement of dog, while in (B), stippled columns indicate forcibly changed posture of dog. I.U.P., intra-urethral pressure; U.T.,ure- agonists was found to be a significant parallel response to thral tone, that for U.T.contractile response is indicated by upward intra-urethral pressure, which provides proof of that the change. Each response induced by 30 pg./kg. i.v. of phenylephrine change in circular urethral tone quantitatively reflects urewas comparatively uniform. thral motility as well as a change in intra-urethral pressure. With regard to the measurement in conscious dogs, the fluctuation occurring in urethral tone, both when moving ilar to those observed initially in the conscious state (Figure (sittingup from the lying position) and not moving conditions 5 ). (lying and sitting), was noticeably less than that observed in In order to further investigate the functional regulation of the intra-urethral pressure. The difference between these the urethral motility in the conscious dogs, the volume- fluctuations in frequency and quantity was attributed evoked micturition reflex was performed by constant and certain factors: in the measurement of the intra-urethral continuous infusion of saline into the bladder. When the pressure, the obstruction of the perfusion of saline by the bladder was filled, intra-vesical pressure slowly and steadily bending and straining of the urethral catheter owing to the increased, and voiding was accomplished by intra-vesical dog's movement, and the synergic addition of fluctuations in pressure augmentation, followed by a drop immediately after intra-abdominal pressure. These results suggest that the the expulsion of urine. With regard to the urethral tone, the force transducer method permits more quantitative analysis filling phase was characterized by progressive contraction of of urethral motility in conscious dogs than the customary the urethral muscle. And the voiding phase started with the intra-urethralpressure recording. transient relaxation of the urethral muscle followed by a conscious dogs, urethral contraction or relaxation By rapid return to the base-line (Figure 6A). Under anesthesia, in a dose-dependent manner in response to either phenyleph-
x
d
1081
STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
TABLE1. Comparison of fluctuations in the urethral tone and the intra-urethml pressure in COMCWUSdogs ( A ) In maintained posture
Lying Urethral tone (5) Intra-urethral pressure ($6) _- (B) In changing posture
17.3 0.2 -c -t 2.1 1'3
Sitting
1-
23.3 2 1.5 12.1 1.8
1-
27.9 17'2 Z 3'9 4.2
1
N.s
Standing fmm lying
Sitting fmm lying Urethral tone (%I
Standing
12.7 ? 3.2
1-
1
N,S,
15.3 7.7 2 2'1 1.6 Intra-urethral pressure (96) 10.2 2 1.6 The absolute fluctuation values were changed to percentages of the response induced by 30 pgsJlrs. i.v. of phenylephrine. end are indicated 811 the mean 2 S.E.M. (A) shows the fluctuation which occurred in the motionless conditions (n = 5 from 5 dogs), and (B) shows those in the movement of the dog (n = 15 from 5 dogs), whereas in (B), the 100%responses are 13.9 2 2.2 grn. for urethral contraction and a 54.2 2 6.8 em. H,O increase in intra-urethralpressure. reopeetively. The significance of difference between the urethral tone and the intra-urethral pressure is shorn as **; p <0.01, and N.S.means not sigtufcant.
I.V.P. .___-A
L
3
Dose ( pglkg i.v. )
FIG. 5. Contractile and relaxing response in circular urethral muscle to adrenergic agonists in conscious and anesthetized dogs. Typical responses to phenylephrine (left) and isoproterenol (right)in urethral tone (U.T.)in (A) conscious state and (B) anesthetized with sodium pentobarbital (30 mg./kg. i.v.) are shown. For U.T., contractile response is indicated by u ward change. (C) shows, dosedependent urethral response to pgenylephrine (circles) and isoproterenol (triangles), and each value is mean i_ S.E.M. for five dogs, whereas closed and open symbols indicate values recorded in conscious and anesthetized state, respectively.
rine or isoproterenol was recognized. And the response was unaltered after anesthesia with pentobarbital, which suggests that these exogenously applied agents exerted their actions through peripheral sites, not through the central nervous system. In addition, during the storage of saline (substituted for urine), progressive contraction of the urethral muscle was observed, and then voiding was accomplished by relaxation of the urethra, whereas the injection of pentobarbital resulted in complete blockade of the micturition reflex. Yaksh et a1.l" reported that the injection of anesthetics resulted in a rise in the threshold intra-vesical pressure to induce micturition, and subsequently overflow incontinence, which were attributed to their inhibitory effects on the synaptic transmission either at the peripheral or the spinal and supraspinal levels in the micturition reflex pathway. The volume-evoked micturition reflex is observed in the animal decerebrated a t the level of the intercolliculus, while transection of the neuraxis at any point below the colliculi abolishes that reflex." But in an experiment using decerebrated dogs, NotoRdid not notice a definite tendency to urethral response, occasionally showing an absence of or a contractile response in the circular urethral muscle accompanied by the expulsion of urine. It thus appears that decerebration, removing almost all of the brain matter, causes some systematic confusion in the regular innervation of the urethral tone controlled in a coordinated manner during normal micturition, possibly by a dog's
-------
FIG.6. Changes in circular urethral tone during micturition reflex in conscious dog and anesthetic-induced urinary incontinence. Cystometrogram of do was obtained during continuous infusion of saline into bladder (incficated by broken arrows) a t rate of 6 ml./min. in (A) the conscious state or (B)anesthetized with sodium pentobarbital (30 mg./kg. i.v.). I.V.P., intra-vesical pressure; U.T., urethral tone. That for U.T. contractile response is indicated by upward change. Arrows pointin upward and downward indicate standing up and micturition of jog, respectively. Broken underline means leakage of infused saline.
critical condition characterized by, e.g., a considerable decrease in blood pressure. In conclusion, it was found that our force transducer method-the conscious and continuous recording obtained with a force transducer chronically implanted on canine urethrawill provide an improved approach to the evaluation of the physiological characteristics of urethral motility. REFERENCES
1. Walton, R. P., Leary, J . S . and Jones, H. P.: Comparative increase in ventricular contractile force produced by several cardiac glycosides. J . Pharmacol. Exp. Ther., 98:346. 1950. 2. Itoh, Z.: Motility of the gut. In: Endocrinology of the Gut. Tokyo: Nanzando Company, chapt. 9, pp. 173-204, 1976. 3. Itoh, Z., Mizumoto, A., Iwanaga, Y., Yoshida. N., Toni, K and Wakabayashi, K: Involvement of 5-hydrotryptamine 3 receptors in regulation of interdigestive gastric contractions by motilin in the dog. Gastroenterology, 100: 901, 1991. 4. Fujimura, M., Sakamoto, T., Lluis, F., Beauchamp, R. D., Townsend, C. M., Gleeley, J r . and Thompson, J. C.: Effect of a cholecystokinin antagonist, proglumide, on cholecystohnin-8induced gallbladder contraction in conscious dogs. Biol. Signals, 1: 94, 1992. 5. Hawk, H. W. and Conley, H. H.: Effects of prostaglandin F, phenylephrine and ergonovine on uterine contractions in the ewe. J. Animal. Sci., 60:537, 1985. 6. Tsuchida. S., Koinuma, N., Nishizawa, 0.. Moriya, I., Satoh, s., Noto, H., Harada, T. and Satoh, K: Urethral response to nerve
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STRAIN GAUGE FORCE TRANSDUCER FOR URETHRAL MOTILITY
stimulation measured by strain gauge force transducer. Urol. Int., 38:300, 1983. 7. Nishizawa, O.,Monya, I., Satoh, S., Fukuda, T. and Tsuchida, S.:Effects of autonomic agonists on in viva female canine urethral motility. Urol. Int., 40: 320, 1985. 8. Noto, H.: Urethral behaviors during mictural reflex on the decerebrated female dog. Nippon Hinyokika Gakkai Zasshl, 7 5 104, 1984. 9. Doyle, P. T. and Bnscoe, C. E.: The effects of drugs and anaesthetic agents on the urinary bladder and sphincters. Br. J. Urol., 48:329, 1976. 10. Yaksh, T. L.,Durant, P. A. C. and Brent, C. R.: Micturition in rats: a chronic model for study of bladder function and effect of anesthetics. Am. J. Physiol., 251: R1177, 1986. 11. Tang, P.C.: Levels of brain stem and diencephalon controlling
micturition reflex, J. Neurophysiol., 18: 583, 1955. 12. Brown, M. and Wickham, J. E. A,: The urethral pressure profile. Br. J. Urol., 41: 211, 1969. 13. Awad, S. A. and Downie, J. W.: Relative contributions of smooth and striated muscles to the canine urethral pressure profile. Br. J. Urol., 48:347, 1976. 14. MacGuire, E. J., Fitzpatrick, C. C., Wan, J., Bloom, D., Sanvordenker, J., Ritchey, M. and Gormley, E. A.: Clinical assessment ofurethral sphincter function. J. U d , 150: 1452,1993. 15. Itoh, K.: Pharmacological and anatomical studies of urethral function in the female dog proximal urethra. Nippon Hinyokika Gakkai Zasshi, 78: 917, 1987. 16. Anderson, P. O.,Malmgren, A. and Uvelius, B.: Functional responses of different muscle types of the female rat urethra in vitro. Acta Physiol. Scand., 140 365, 1990.