0022-5347/03/1692-0735/0 THE JOURNAL OF UROLOGY® Copyright © 2003 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 169, 735–739, February 2003 Printed in U.S.A.
DOI: 10.1097/01.ju.0000045680.05119.84
RESPONSE OF THE FETAL SHEEP BLADDER TO URINARY DIVERSION SEIJI MATSUMOTO, BARRY A. KOGAN, ROBERT M. LEVIN, PAMELA S. HOWARD AND EDWARD J. MACARAK From the Division of Urology, Albany Medical College, Division of Basic and Pharmaceutical Sciences, Albany College of Pharmacy and Stratton Veterans Affairs Medical Center, Albany, New York, Department of Anatomy and Histology, University of Pennsylvania Dental School, Philadelphia, Pennsylvania, and Department of Urology, Kinki University School of Medicine, Osaka, Japan
ABSTRACT
Purpose: In adults urinary diversion results in bladder atrophy and a rapid decrease in contractile function. Little is known about the effects of urinary diversion on bladder development. In this regard we characterized the responses of fetal sheep bladder strips obtained from animals that underwent urinary diversion. Materials and Methods: Urinary diversion was performed on fetal sheep after 90 days of gestation (term 147 days) and bladder tissue was obtained 2 weeks later. Contractile and relaxant responses of full-thickness bladder strips were tested. Results: Bladders from fetal sheep subjected to urinary diversion weighed significantly less than control fetal bladders. Histological studies demonstrated marked connective tissue infiltration and the reorganization of smooth muscle elements. Carbachol stimulated a tonic contraction, while field stimulation administered during the tonic contraction elicited a phasic relaxation or a biphasic response, consisting of an initial relaxation and then a phasic contraction in control and diverted bladders. Contractile responses of defunctionalized strips to carbachol were significantly less than those of control bladder strips. Contractile responses of defunctionalized bladder strips to field stimulation at 1 Hz. were significantly greater than those of control strips. Responses of the 2 sets of fetal bladder strips to higher frequencies were similar, as were the contractile responses to adenosine triphosphate and KCl. Field stimulated relaxations in the presence of carbachol stimulated contraction of defunctionalized bladder strips were significantly greater than those of control strips, while the relaxant responses of each set of fetal bladder strips to isoproterenol and nitroprusside were similar. Conclusions: Urinary diversion in normal fetal sheep resulted in marked structural changes, reduced carbachol stimulation and increased field stimulation relaxation. KEY WORDS: urinary diversion, sheep, fetus, bladder, growth
In adults bladder defunctionalization results in the rapid atrophy of the bladder smooth muscle and a reduction in contractile function.1, 2 It is not known if bladder defunctionalization has the same effect on fetal bladder development. This condition is exemplified by infants who have undergone urinary diversion for posterior urethral valves.1 However, some groups contend that temporary bladder defunctionalization has no adverse effect on later bladder function and any dysfunction is attributable not to bladder defunctionalization, but to bladder obstruction during embryogenesis.1, 2 To begin to investigate this matter we performed experiments in a fetal sheep model of urinary diversion. A well characterized sheep fetal model was used for this study. Although the structural and urodynamic aspects of fetal sheep bladder development have been studied,3–5 little is known about the pharmacology of bladder development. To our knowledge the pharmacological response of the fetal sheep bladder to urinary diversion has not been studied. Micturition is a complex neuromuscular process.6, 7 Bladder function depends on several interrelated factors, including the effective integration of autonomic innervation and receptor function of detrusor smooth muscle, and simultaneous, coordinated contraction of the bladder and relaxation of the urethra.6 – 8 Although control mechanisms have been identified in the central nervous system and spinal cord,
the final pathway in neural control of micturition involves the autonomic innervation of the bladder and related structures.6, 7 The hypothesis of the current investigation was that the fetal bladder rapidly responds structurally and functionally to urinary diversion. In this regard we studied the response after a relatively short period, that is 2 weeks. Future studies are planned to evaluate the long-term response to diversion. MATERIALS AND METHODS
Pregnant sheep (Thomas Morris, Reisterstown, Maryland) at 90 days of pregnancy underwent surgery using strict aseptic techniques under ketamine anesthesia. Hysterotomy was performed and the fetal flank was exposed via a posterior lumbar incision. The ureters were exposed just distal to the kidney. The proximal ureter was catheterized using a 3Fr feeding tube and the distal ureter was ligated. The procedure was performed bilaterally, resulting in complete diversion of urine away from the bladder. After urinary diversion was created the wound was closed in layers and the fetus was replaced in the uterus. Fetal urine production emptied directly into the amnion via the catheters. The animals were sacrificed 2 weeks later. At that time bladder tissue was obtained from the fetuses. At sacrifice the bladder was removed and muscle strips were dissected free of the bladder and placed in chilled culture medium. Bladder tissue from 10 control fetal sheep (4 males and 6 females) and 9 fetal sheep that underwent urinary diversion
Accepted for publication August 30, 2002. Supported in part by National Institutes of Health Grants RO1 DK 54987, DK 26508, DK53965 and DK 48215, and a Veterans Affairs Merit Grant. 735
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(4 males and 5 females) was used. Some individual fullthickness strips fixed in buffered formalin were used for routine histological evaluation. Other full-thickness strips were frozen in OTC compound for the preparation of frozen sections. The remaining isolated strips were mounted in separate 15 ml. baths containing Tyrode’s solution (124.9 mM. NaCl, 12.5 mM. KCl, 23.8 mM. NaHCO3, 0.5 mM. MgCl2 6H2O, 0.4 mM. NaH2PO4 H2O and 1.8 mM. CaCl with 1 mg./ml. glucose), maintained at 37C and equilibrated with 95% O2 ⫽ 5% CO2. After 1 hour of equilibration 2 gm. resting tension were placed on each strip. Contractile responses to field stimulation at 1, 2, 4, 8, 16 and 32 Hz. at 80 V. 1 millisecond in duration, carbachol (10 M.), adenosine triphosphate (ATP) (1 mM.) and KCl (120 mM.) were determined. The relaxant responses of carbachol (10 M.) precontracted strips to field stimulation were assessed. In addition, the relaxant effects of isoproterenol (20 M.) and nitroprusside (100 M.) on tension in the presence of carbachol pre-contraction were measured. Analog signals from Polygraph (Grass Instruments, Quincy, Massachusetts) were digitized and analyzed using a PolyView (Astro-Med, W. Warwick, Rhode Island) data acquisition and analysis system. Data are shown as the mean ⫾ SEM. Comparisons among groups were made using ANOVA, followed by the Newman-Keuls test for individual differences with p ⬍0.05 considered significant. RESULTS
Figure 1 shows bladder and isolated strip weight of the control fetal bladders and that of sheep that underwent urinary diversion. Bladder weight in diverted sheep was significantly less than in controls. Interestingly the mean weight of the isolated strips from diverted fetal sheep was significantly greater than that of strips from control fetal sheep. Care was taken to maintain the length and width of the strips the same. The thickness of the strips from diverted bladders was visibly greater than that from control bladders, which accounted for the increased mass. Figures 2 and 3 show representative photomicrographs of a control and diverted bladder, respectively. The muscle layer in the control fetal bladder appeared uniform and well-defined, whereas in the diverted bladder the whole muscle layer was thinner with additional muscle fibers proximal to the mucosa unevenly dispersed with considerable connective tissue infiltration. Figure 4 shows the contractile responses of control and defunctionalized fetal bladder strips to field stimulation. The maximal contractile responses of defunctionalized bladder strips were significantly greater than those of controls at 1
FIG. 1. Effect of urinary diversion on fetal bladder-to-fetal sheep weight (Bld Wt/Ft Wt) and isolated bladder strip weights. Bars represent mean ⫾ SEM of 5 or 6 bladders. Asterisk indicates significantly different from control fetal bladder response (p ⬍0.05).
FIG. 2. Representative photomicrograph of control fetal bladder cross-sectional area.
FIG. 3. Representative photomicrograph of diverted fetal bladder cross-sectional area.
RESPONSE OF FETAL SHEEP BLADDER TO URINARY DIVERSION
FIG. 4. Effect of urinary diversion on fetal bladder contractile responses to field stimulation. Bars represent mean ⫾ SEM of 5 or 6 bladders. Asterisk indicates significantly different from control fetal bladder response (p ⬍0.05).
Hz. stimulation, while at higher frequencies responses did not differ. Figure 5 shows the characteristic responses of a carbachol pre-contracted defunctionalized fetal bladder strip to intermittent field stimulation. All field stimulation frequencies elicited a biphasic response consisting of initial relaxation followed by a phasic contraction or a phasic relaxation (fig. 5). However, the relaxant responses of defunctionalized bladder strips were significantly greater than those of controls at all frequencies (fig. 6). Figure 7 shows the contractile responses to ATP, carbachol and KCl of control and defunctionalized fetal bladder strips. The responses of defunctionalized bladder strips to carbachol were significantly less than those of controls. There were no differences in responses to ATP and KCl (fig. 7). Figure 8 shows the relaxant responses to isoproterenol and nitroprusside of carbachol pre-contracted fetal bladders strips. There were no significant differences in the responses of control and defunctionalized bladder strips. For control and defunctionalized bladders we compared the responses from strips isolated from male and female fetal sheep. These comparisons showed no differences in the qualitative or quantitative contractile or relaxant responses in males and females. DISCUSSION
Neonates with posterior urethral valves are generally treated by valve ablation shortly after diagnosis. However,
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because of neonatal size or clinical picture, some of these patients are treated with some type of urinary diversion, that is vesicostomy or ureterostomy. There is considerable debate on the renal and growth benefits of higher, complete urinary diversion in severe cases. Furthermore, there is debate on whether there is a bladder benefit of the cycling that occurs after valve ablation or vesicostomy compared with high complete diversion.1, 2, 9 The function of the bladder is to store urine at low pressure and then to expel periodically the urine via coordinated and sustained contractions.10 However, if the ureters are diverted, the bladder fails to perform cyclic filling and emptying, that is it becomes defunctionalized. We used a fetal sheep preparation to study the result of short-term bladder defunctionalization during normal development. Our results show that the weight of defunctionalized fetal bladders was significantly less than that of controls, although there was an increase in the thickness of the bladder wall. Histological analysis revealed marked thinning of the muscle layer and increased mucosal thickness. There appeared to be connective tissue infiltration and dispersal of the smooth muscle elements, of which many were not arranged into tightly compacted bundles. This observation suggests that diversion results in rapid alterations, not only in the rate of bladder growth, but also in the structural architecture of the bladder. The detrusor layer was less substantial than in controls, suggesting atrophy of the muscle due to a lack of cycling. However, it is curious that the thickness of noncycling bladders was greater than in controls. This finding suggests that, while muscle mass appears to be directly related the lack of cycling, the mucosal response is not regulated in coordinated fashion with that of muscle. In contrast, the mucosa appeared to grow to a greater degree in the absence of cycling since it is thicker than the detrusor. Chang et al noted that the mucosa is the capacitance layer during bladder filling since it thinned during bladder filling to a greater extent than the detrusor.11 Because the mucosa in a defunctionalized bladder does not experience tension, these data suggest that excessive growth cannot be related to stretch or tension in the wall. Rather, tension or stretch under normal conditions appears to be necessary to generate normal architecture, not only of the detrusor layer, but also of the mucosa. These structural changes, muscle atrophy and increased mucosal thickening strongly suggest that these 2 layers are operating under different control mechanisms. It is not unlikely since the predominant cell type in the detrusor is the smooth muscle cell, while that in the mucosa is the fibroblast. It is likely that the structure of these bladders
FIG. 5. Representative tracings of relaxant response to field stimulation of defunctionalized bladder pre-contracted with carbachol. A, biphasic responses. B, phasic relaxations.
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FIG. 6. Effect of urinary diversion on relaxant responses to field stimulation of carbachol pre-contracted fetal bladders. Bars represent mean ⫾ SEM of 5 or 6 bladders. Asterisk indicates significantly different from control fetal bladder response (p ⬍0.05).
FIG. 7. Effect of urinary diversion on contractile responses of fetal bladder to 1 mM. ATP, 10 M. carbachol and 120 mM. KCl. Bars represent mean ⫾ SEM of 5 or 6 bladders. Asterisk indicates significantly different from control fetal bladder response (p ⬍0.05).
Although changes in defunctionalized bladders have been reported,1, 2 the defunctionalized fetal bladder has not been well studied. Lipski et al reported the effects of defunctionalized bladders in a new urinary diversion model in infant rabbits.1 Their results also showed that defunctionalized bladders grew more slowly than normal, aged matched control bladders. Responses of control fetal bladders to neurogenic (field stimulation) and pharmacological stimulation (ATP and carbachol) were similar to those seen previously.12 These findings are in agreement with the results of in vivo studies in fetal sheep at 120 days of gestation that showed significant bladder contractions in response to pharmacological stimulation.5 Furthermore, there were no differences in control and defunctionalized fetal bladder responses to field stimulation or pharmacological stimulation with ATP, KCl, isoproterenol or nitroprusside after pre-contraction indicating that, although significant changes occur in detrusor structure in response to diversion, the muscle that remains is functional. A major pharmacological difference in the responses of the control bladder compared to the defunctionalized bladder was the reduced response to carbachol. The decreased response to carbachol would normally indicate a decreased response to direct muscarinic stimulation or decreased receptor density. However, the response to field stimulation was not decreased. Because cholinergic neurotransmission is a primary response to field stimulation, this finding would indicate that control and defunctionalized bladders have a similar cholinergic response, at least via neurogenic stimulation. Another possible explanation relates to the increased thickness of the strips. If the diffusion rate for carbachol were slower for defunctionalized bladder strips than for control strips, it could result in a decreased response to carbachol administration but not to field stimulation since the release of acetylcholine occurs within the synaptic cleft and there is no diffusion barrier. Further studies are required to identify the mechanism of this apparent disparity. Control and defunctionalized fetal sheep bladder strips pre-contracted with carbachol relaxed in response to field stimulation. These results agree with those of previous studies that consistently showed field stimulated relaxation of ovine and bovine fetal bladders.10, 12, 13 In each species this relaxation response to field stimulation is mediated by the neurogenic release of nitric oxide (NO) and this NO response is lost in neonates and adult animals. Interestingly the magnitude of the NO response was significantly greater in defunctionalized bladder strips than in control bladder strips. Thus, an increase in NO-neuronal release mechanisms is induced by defunctionalization. CONCLUSIONS
Although short-term urinary diversion in normal fetal sheep resulted in reduced bladder growth and marked connective tissue infiltration, there were few qualitative or quantitative changes in bladder contractile or relaxant responses. This study indicates that structural changes induced by diversion precede contractile dysfunctions. Nobuaki Ohto and Robert Edward Leggett provided technical assistance. FIG. 8. Effect of urinary diversion on relaxant responses to 20 M. isoproterenol and 100 M. nitroprusside of carbachol pre-contracted fetal bladders. Bars represents mean ⫾ SEM of 5 or 6 bladders.
becomes altered because normal cycling is a requirement for achieving normal bladder architecture. It would also appear that the structural changes in the bladder precede major contractile dysfunctions.
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8. Anderson, K. E.: Pharmacology of lower urinary tract smooth muscles and penile erectile tissue. Pharmacol Rev, 45: 253, 1993 9. Liard, A., Seguier-Lipszyc, E. and Mitrofanoff, P.: Temporary high diversion for posterior urethral valves. J Urol, 164: 145, 2000 10. Koo, H. P., Macarak, E. J., Duckett, J. W., Snyder, H. M., 3rd, Wein, A. J. and Levin, R. M.: Fetal bovine bladder: physiology and pharmacology. Adv Exp Med Biol, 385: 251, 1995 11. Chang, S. L., Chung, J. S., Yeung, M. K., Howard, P. S. and Macarak, E. J.: Roles of the lamina propria and the detrusor in tension transfer during bladder filling. Scand J Urol Neprol, suppl., 201: 38, 1999 12. Levin, R. M., Macarak, E., Howard, P., Horan, P. and Kogan, B. A.: The response of fetal sheep bladder tissue to partial outlet obstruction. J Urol, 166: 1156, 2001 13. Mevorach, R. A., Bogaert, G. A. and Kogan, B. A.: Role of nitric oxide in fetal lower urinary tract function. J Urol, 152: 510, 1994