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Structural and vascular response of normal and obstructed rabbit whole bladders to distension
BASIC SCIENCE
STRUCTURAL AND VASCULAR RESPONSE OF NORMAL AND OBSTRUCTED RABBIT WHOLE BLADDERS TO DISTENSION SEIJI MATSUMOTO, PAUL CHICHESTER, BARRY A. KOGAN,
AND
ROBERT M. LEVIN
ABSTRACT Objectives. To investigate the structural and morphologic effect of distension after partial outlet obstruction in rabbits. Methods. Thirty male New Zealand white rabbits were separated into two groups: control (sham) and partial outlet obstruction (3 weeks). Three rabbits from each group were distended to 5%, 25%, 50%, 100%, and 125% of capacity. Each bladder was fixed at the volume in buffered formalin for 6 to 8 hours. Sections of dorsal and ventral bladder were blocked, and cross sections were evaluated. Quantitative morphometry was performed, and CD31 immunohistochemistry was used to characterize the vascularity. Results. Partial outlet obstruction resulted in increased bladder weight and capacity and increased thickness of the mucosa, submucosa, detrusor, and serosa. In the control bladder, the greatest thinning was seen between 5% and 25% capacity, and in the obstructed group, the greatest thinning occurred between 25% and 50%. The level of vascular collapse was significantly greater for the control bladders than for the obstructed bladders at all levels of distension. Finally, the obstructed bladders showed a significantly greater level of vascularity in the submucosa than the control bladders. Conclusions. Normal bladder distension resulted in significant morphologic changes when the bladder was distended to 25% of capacity but changed relatively little between 25% and 125%. However, distension of the obstructed bladder resulted in significant morphologic changes when the bladder was distended from 25% to 50% of capacity but changed relatively little between 50% and 125%. UROLOGY 62: 1129–1133, 2003. © 2003 Elsevier Inc.
T
he urinary bladder can be filled close to its maximal capacity without a large increase in intravesical pressure. It has been clearly demonstrated that one of the major responses to obstruction in both men secondary to benign prostatic hyperplasia and in animals secondary to partial outlet obstruction is a thickening of the bladder wall and a decrease in compliance.1–3 Although urinary bladder distension and overdistension have been the subject of numerous studies,4 –14 the effects of bladder distension with or without bladder outlet obstruction on structure and vascularity have not been thoroughly investigated. In This work was supported by grants from the Veterans Affairs Medical Center and NIH grant NIH RO-1-DK53965. From the Division of Urology, Albany College of Medicine; Stratton Veterans Affairs Medical Center, Albany, New York; Department of Urology, Kinki University School of Medicine, Osaka, Japan; and Division of Basic and Pharmaceutical Sciences, Albany College of Pharmacy, Albany, New York Reprint requests: Robert M. Levin, Ph.D., Albany College of Pharmacy, 106 New Scotland Avenue, Albany, NY 12208 Submitted: March 12, 2003, accepted (with revisions): June 19, 2003 © 2003 ELSEVIER INC. ALL RIGHTS RESERVED
previous studies, we observed that the thickness of the ventral and dorsal sections of the obstructed rabbit bladder were very different. Thus, the objective of the current study was to compare and correlate the effects of distension on the ventral and dorsal sections of both control and obstructed rabbit bladder morphology. The rabbit has been used extensively for the study of urinary bladder function and dysfunction.2,3,15,16 The adult rabbit has a comparatively large bladder that can be evaluated using standard cystometry techniques. The in vitro whole rabbit bladder model has been used effectively to evaluate the effect of filling on bladder function.17–19 In these early studies, no attempt was made to correlate changes in function with changes in structure.17,19 MATERIAL AND METHODS EXPERIMENTAL PROTOCOL A total of 30 mature male New Zealand white rabbits weighing 3 to 4 kg were separated into two groups of 15 control (sham) and 15 obstructed rabbits. 0090-4295/03/$30.00 doi:10.1016/S0090-4295(03)00686-1 1129
SURGICAL CREATION OF BLADDER OUTLET PARTIAL OBSTRUCTION Each rabbit was anesthetized with sodium pentobarbital (25 mg/kg intravenously). An 8F Foley catheter was inserted into the bladder and the urine drained. The bladder and urethrovesical junction were exposed through a midline incision. Placing a 2-0 silk ligature loosely around the catheterized proximal urethra created partial bladder outlet obstruction. The catheter was then removed. The bladder was returned, and the wound was closed. A dose of 1 mg/kg gentamicin and 0.3 mg/kg buprenorphine hydrochloride was given intramuscularly on postoperative days 1 and 2. Sham operations consisted of anesthesia, a midline incision, and cleaning the bladder neck region. No ligature was tied.
IN VIVO CYSTOMETRY At 3 weeks after surgery, cystometry was performed. With the animal in the supine position, an 8F catheter was inserted into the bladder and the volume of urine measured. The bladder was filled with saline at 1.42 mL/min until either micturition contraction or overflow incontinence occurred. The bladder capacity was determined. The bladder was then emptied and filled with buffered formalin to one of the following volumes: 5%, 25%, 50%, 100%, and 125% of capacity. Three control and three obstructed bladders were filled to each volume. The bladders were excised en bloc and placed in buffered formalin for 6 to 8 hours. After fixation, full-thickness sections of the ventral and dorsal bladder body were cut and processed for both light microscopy and immunohistochemistry.
MICROSCOPY After fixation, a 2-cm square sample was cut from the dorsal and ventral sides of the bladder body and processed. The samples were then cut into strips and embedded on edge in paraffin to get a cross-sectional view when sectioned. Tissue blocks were sectioned at 5 m mounted on glass slides, deparaffinized, rehydrated, and stained with hematoxylin-eosin. The effect of distension on bladder morphology was evaluated by measuring the bladder wall thickness using quantitative image analysis with Image Pro Plus software (Media Cybernetics, Silver Spring, Md); 40⫻ image magnification was used for all determinations, and all parameters were measured in 10 different locations on each sample cross-section and an average taken.
IMMUNOSTAINING Tissue sections were stained for vascular endothelium with a mouse monoclonal antibody to CD31 on a Ventana ES automated immunostainer (Ventana Medical Systems, Tucson, Ariz) using a biotin avidin-conjugated horseradish peroxidase amplification method with diaminobenzidine chromogen detection followed by a light hematoxylin counterstain. After immunostaining, all slides were dehydrated through graded alcohols and two changes of xylene and then mounted with Permount mounting medium.
STATISTICAL ANALYSIS
The data are expressed as the mean ⫾ the standard error of mean (SEM). Comparisons among groups were made using analysis of variance followed by the Newman-Keuls test and paired Student t test for individual differences. Statistical significance required a P value of less than 0.05. The Stratton Veterans Affairs Medical Center Institutional Animal Care and Use Committee reviewed and approved these studies. 1130
FIGURE 1. Effect of distension on bladder wall thickness. Values presented as the mean ⫾ SEM of 3 rabbits per group. *Significantly different from 5% (P ⬍0.05). †Significantly different from dorsal (P ⬍0.05).
RESULTS Three weeks of partial bladder outlet obstruction resulted in a significant increase in bladder weight (2.24 ⫾ 0.09 versus 11.77 ⫾ 1.06 mg) and bladder capacity (60 ⫾ 4.01 versus 133.01 ⫾ 27.90 mL). MICROSCOPY Histologic evaluation of the control bladders at all levels of distension showed no significant differences between the dorsal and ventral sides. Thus, in all figures, we present the data for the control bladder as the mean of the ventral and dorsal sections. Histologic evaluations of the obstructed bladders showed significant differences between the ventral and dorsal sections and thus were evaluated separately. At 5% capacity (⬃empty), the thickness of the dorsal side of the obstructed bladder was similar to the thickness of the control bladder; however, the ventral side was significantly thicker than both the dorsal side and the control bladder (Fig. 1). Compared with the nondistended bladders, distending the bladders to 25% resulted in significant thinning of the control bladder, but not for either the dorsal or ventral walls of the obstructed bladder. Compared with the bladders distended to 25% of capacity, distending the bladders to 50% of capacity resulted in no further thinning of the control bladder, but a significant thinning of the ventral wall of the obstructed bladder. Similarly, compared with bladders distended to 50% of capacity, distending the bladders to 100% of capacity resulted in significant thinning of the dorsal wall of the obstructed bladder. No statistically significant differences were found between bladders distended to 100% and those distended to 125% (Fig. 1). UROLOGY 62 (6), 2003
FIGURE 2. Representative example of transverse sections of normal and obstructed bladder walls distended to 5%, 25%, and 125% capacity. Hematoxylin-eosin stain, original magnification ⫻40. (A) Control (distended to 5%, 25%, and 125% capacity), (B) obstructed dorsal wall (distended to 25%, 50%, and 125% capacity), and (C) obstructed ventral wall (distended to 25%, 50%, and 125% capacity).
Figure 2A shows representative cross-sections of control bladders at 5%, 25%, and 125% capacity. Figure 2B,C (ventral and dorsal, respectively) show representative cross-sections of obstructed bladders at 25%, 50%, and 125% capacity. For the obstructed bladder, no statistically significant differences were found between 5% and 25% distension (representative sections of obstructed bladder at 5% distension not shown because of space considerations). No statistically significant differences were found in the cross-sectional areas between the venUROLOGY 62 (6), 2003
FIGURE 3. Representative example of transverse sections of control bladder wall. Vascular endothelium immunostained with anti-CD31 antibodies. (A) Control bladder distended to 5% (⫻40) of capacity and (B) control bladder distended to 25% of capacity.
tral and dorsal obstructed bladders for either the mucosa or detrusor. The cross-sectional area of the ventral serosa was significantly greater than that of the dorsal obstructed bladder for all levels of distension. However, although distension significantly thinned the ventral serosa, the cross-sectional area of the obstructed dorsal section did not significantly change with distension. For both control and obstructed bladders, the mucosa at 5% capacity was multilayered with folds. On distension, it thinned and stretched and appeared as a single layer with a flat surface. For both control and obstructed bladders, the smooth muscle bundles within the detrusor that were large and the most prominent portion of the wall at 5% capacity stretched and thinned and were reduced to longitudinal strands by 25% capacity for control 1131
bladders, but not until 125% capacity for obstructed bladders. Partial outlet obstruction resulted in a marked thickening and repositioning of the serosal layer. In the control, it is a thin, one-cell-thick, connective tissue sheath around the bladder. In obstructed bladders, the serosa was significantly thicker on the ventral side compared with the dorsal side. The histologic changes at the serosa for both the dorsal and the ventral obstructed sides were similar in that both showed an increase in connective tissue, fibroblasts, and microvessels. However, the marked difference was that on the ventral side all of the serosal growth was extrinsic to the detrusor, and on the dorsal side, almost one half of this additional growth of connective tissue was within the detrusor. On distension, the serosa on the ventral surface thinned to a significantly greater extent than the serosa on the dorsal surface. IMMUNOSTAINING Figure 3 displays a cross-section of a control bladder at 5% and 25% capacity showing blood vessels immunostained for vascular endothelium with CD31 antibodies. At 5% capacity, all vessels, arteries, and veins were open. At 25% capacity, virtually all veins showed significant compression and most appeared collapsed. The arteries and arterioles remained open at 25% capacity. With increasing volumes, the vessels showed additional compression. With regard to the obstructed bladder, little change was noted in the structure between 5% and 25% in the obstructed bladders. All vessels remained fully opened. As the bladder distended beyond 25%, some collapsing of veins occurred although many of them remained open. Qualitatively, the vessels of the dorsal side were affected to a greater extent than the vessels of the ventral side. Thus, a marked difference was found between the control and obstructed bladders with regard to the effect of distension on vessel morphology. COMMENT In experimental animals, partial outlet obstruction stimulates a rapid increase in bladder mass and bladder capacity. In addition, a direct correlation appears to exist between the severity of decompensation and the magnitude of the increase in bladder mass.2,3,16,20 In vivo studies have shown that overdistension can result in edema within the bladder wall.5,7,9 Because edema takes some time to occur after overdistension and in our studies, the bladders were fixed during and immediately after distension, there was no time for edema to occur and thus edema was not a factor in our observations. 1132
In the current study, we directly compared the structural responses of control and obstructed bladders to distension using the isolated whole bladder model. Kojima et al.21 demonstrated that cadaveric human bladders revealed no statistically significant differences between the thickness of the anterior or lateral bladder walls, trigone, or dome. Our data on the control rabbit bladder likewise showed no significant structural differences between the ventral and dorsal wall. However, statistically significant differences were noted for the obstructed bladder, primarily owing to the increased thickness of the serosa on the ventral side. Serosal thickness was not affected by sham surgery. Oelke et al.22 showed detrusor wall thickness during bladder filling in humans using ultrasound devices. Their results showed that the detrusor wall thickness decreased continuously while the bladder filled to 50% of its capacity and then remained constant until 100%. In our study, the rabbit bladder wall thinned up to 25% capacity and then remained constant. The difference was most likely due to the thinner wall of the rabbit bladder compared with that of the human bladder. In a recent study, Schroder et al.23 reported on the effect of distension on the expansion of control and obstructed rabbit bladders and on the regional contractile responses. These studies demonstrated that although the control bladders expanded evenly during filling, the obstructed bladders did not.23 Our current studies showing uneven growth of the ventral and dorsal sides of the bladder can explain the functional observations of Schroder et al. Similarly, Schroder et al.23 demonstrated that the contractile responses of the control dorsal and ventral strips were similar, although after obstruction significant differences existed. These differences would be directly related to the different structural responses of the dorsal and ventral sides of the bladder to obstruction. The increase in bladder mass is mediated primarily through urothelial and fibroblast hyperplasia and smooth muscle hypertrophy.24 The development of serosal thickening is a characteristic response of the rabbit bladder to obstruction.25 The origin of the connective tissue is from serosal submesothelial mesenchymal cells. They proliferate in early obstruction, contribute to the dorsal side serosal thickening, and also differentiate into myofibroblasts.24 However, these myofibroblasts do not differentiate into smooth muscle cells in the ventral serosa. The asymmetric appearance of these cells explains the uneven distension of the obstructed bladders. The significantly increased thickening of the ventral side of the bladder compared with the dorsal side may relate to the position of the ventral side (ie, the ventral side has greater stress because of gravity than the dorsal UROLOGY 62 (6), 2003
side). It may be that the increased thickness is a response to the greater forces placed on this side by the increased volume of urine the obstructed bladder contains. The results of human and animal experiments have shown that blood flow decreases with distension.11,12,14 In patients with poorly compliant bladders, distension resulted in decreased bladder wall blood flow to a significantly greater extent than it did in patients with normal compliance. Thus, bladder distension can lead to bladder wall ischemia that, ultimately, may result in reduced bladder contractility, increased residual volume, and bladder dysfunction. We have previously demonstrated that outlet obstruction results in angiogenesis in the rapidly growing bladder26 and increased vascularization, so that in compensated function vascular density is not significantly different from control bladders.27,28 In the current study, vascular compression during distension was significantly greater for the controls than for the obstructed bladders. This may be a compensatory factor that will allow the obstructed bladder to maintain blood flow in the presence of overdistension, which might have less effect on blood flow in the obstructed than in the control. CONCLUSIONS The response of the rabbit bladder to obstruction is uneven. The morphologic responses of the dorsal and ventral sides of the bladder were significantly different from each other, with the ventral side showing significantly greater serosal thickness than the dorsal. REFERENCES 1. Zderic SA, Levin RM, and Wein AJ: Voiding function and dysfunction: A—relevant anatomy, physiology, and pharmacology, and molecular biology, in Gillenwater JY, Grayhack JT, Howards SS, et al (Eds): Adult and Pediatric Urology, 3rd ed. Chicago, Mosby Year Book Medical, 1996, pp 1159 –1219. 2. Gosling JA, Kung LS, Dixon JS, et al: Correlation between the structure and function of the urinary bladder following partial outlet obstruction. J Urol 163: 1349 –1356, 2000. 3. Levin RM, Haugaard N, O’Connor L, et al: Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol Urodyn 19: 609 –629, 2000. 4. Dunn M: A study of the bladder blood flow during distension in rabbits. Br J Urol 47: 67–72, 1975. 5. Gosling JA, Dixon JS, and Dunn M: The structure of the rabbit urinary bladder after experimental distension. Invest Urol 14: 386 –389, 1977. 6. Kang J, Wein AJ, and Levin RM: Bladder functional recovery following acute overdistension. Neurourol Urodyn 11: 253–261, 1992. 7. Monson FC, Wein AJ, Eika B, et al: Stimulation of the proliferation of rabbit bladder urothelium by partial outlet obstruction and acute overdistension. Neurourol Urodyn 13: 51–62, 1994. UROLOGY 62 (6), 2003
8. Tammela T, Lasanen L, and Waris T: Effect of distension on adrenergic innervation of the rat urinary bladder. Urol Res 18: 345–348, 1990. 9. Tammela T, Autio-Harmainen H, Lukkarinen O, et al: Effect of distension on function and nervous ultrastructure in the canine urinary bladder. Urol Int 42: 265–270, 1987. 10. Tammela TL, Levin RM, Monson FC, et al: The influence of acute overdistension on rat bladder function and DNA synthesis. J Urol 150: 1533–1539, 1993. 11. Nemeth CJ, Khan RM, Kirchner P, et al: Change in canine bladder perfusion with distension. Invest Urol 15: 149 –150, 1977. 12. Finkbeiner A, and Lapides J: Effect of distension on blood flow in dog urinary bladder. Invest Urol 12: 210 –212, 1974. 13. Weaver LC: Organization of sympathetic responses to distension of urinary bladder. Am J Physiol 248: R236 –R240, 1985. 14. Brading AF, Greenland JE, and Milis IW: Blood supply to the bladder during filling. Scand J Urol Nephrol 201: 25–31, 1999. 15. Levin RM, Monson FC, Longhurst PA, et al: The rabbit as a model of urinary bladder function. Neurourol Urodyn 13: 119 –136, 1994. 16. Levin RM, Brading AF, Mills IW, et al: Experimental models of bladder obstruction, in Lepor H (Ed): Prostatic Disease. Philadelphia, WB Saunders, 2000, pp 169 –196. 17. Levin RM, and Wein AJ: Response of the in-vitro whole bladder (rabbit) preparation to autonomic agonists. J Urol 128: 1087–1090, 1982. 18. Levin RM, Brendler K, and Wein AJ: Comparative pharmacological response of an in-vitro whole bladder preparation (rabbit) with the response of isolated smooth muscle strips. J Urol 30: 377–381, 1983. 19. Kaplan SA, Blaivas JG, Brown WC, et al: Parameters of detrusor contractility. I. The effects of electrical stimulation, hysteresis and bladder volume in an in-vitro whole rabbit bladder model. Neurourol Urodyn 10: 53–60, 1991. 20. Kato K, Wein AJ, Longhurst PA, et al: The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J Urol 143: 600 –606, 1990. 21. Kojima M, Inui E, Ochiai A, et al: Ultrasonic estimation of bladder weight as a measure of bladder hypertrophy in men with intravesical obstruction: a preliminary report. Urology 47: 942–947, 1996. 22. Oelke M, Hofner K, Wiese B, et al: Increase in detrusor wall thickness indicates bladder outlet obstruction (BOO) in men. World J Urol 19: 443–452, 2002. 23. Schroder A, Uvelius B, Capello SA, et al: Regional differences in bladder enlargement and in vitro contractility after outlet obstruction in the rabbit. J Urol 168: 1240 –1246, 2002. 24. Levin RM, Monson FC, Haugaard N, et al: Genetic and cellular characteristics of bladder outlet obstruction. Urol Clin North Am 22: 263–283, 1995. 25. Monson FC, Goldschmidt MH, Zderic SA, et al: Use of a undescribed elastic lamina of serosa to characterize connective tissue hypertrophy of rabbit bladder wall following partial outlet obstruction. Neurourol Urodyn 7: 385–396, 1988. 26. Ghafar MA, Anastasiadis AG, Olsson LE, et al: Hypoxia and an angiogenic response in the partially obstructed rat bladder. Lab Invest 82: 903–909, 2002. 27. Chichester P, Lieb J, Levin SS, et al: Vascular response of the rabbit bladder to short term partial outlet obstruction. Mol Cell Biochem 208: 19 –26, 2000. 28. Chichester P, Schroder A, Horan P, et al: Vascular response of the rabbit bladder to chronic partial outlet obstruction. Mol Cell Biochem 226: 1–8, 2001. 1133
STRUCTURAL AND VASCULAR RESPONSE OF NORMAL AND OBSTRUCTED RABBIT WHOLE BLADDERS TO DISTENSION SEIJI MATSUMOTO, PAUL CHICHESTER, BARRY A. KOGAN,
AND
ROBERT M. LEVIN
ABSTRACT Objectives. To investigate the structural and morphologic effect of distension after partial outlet obstruction in rabbits. Methods. Thirty male New Zealand white rabbits were separated into two groups: control (sham) and partial outlet obstruction (3 weeks). Three rabbits from each group were distended to 5%, 25%, 50%, 100%, and 125% of capacity. Each bladder was fixed at the volume in buffered formalin for 6 to 8 hours. Sections of dorsal and ventral bladder were blocked, and cross sections were evaluated. Quantitative morphometry was performed, and CD31 immunohistochemistry was used to characterize the vascularity. Results. Partial outlet obstruction resulted in increased bladder weight and capacity and increased thickness of the mucosa, submucosa, detrusor, and serosa. In the control bladder, the greatest thinning was seen between 5% and 25% capacity, and in the obstructed group, the greatest thinning occurred between 25% and 50%. The level of vascular collapse was significantly greater for the control bladders than for the obstructed bladders at all levels of distension. Finally, the obstructed bladders showed a significantly greater level of vascularity in the submucosa than the control bladders. Conclusions. Normal bladder distension resulted in significant morphologic changes when the bladder was distended to 25% of capacity but changed relatively little between 25% and 125%. However, distension of the obstructed bladder resulted in significant morphologic changes when the bladder was distended from 25% to 50% of capacity but changed relatively little between 50% and 125%. UROLOGY 62: 1129–1133, 2003. © 2003 Elsevier Inc.
T
he urinary bladder can be filled close to its maximal capacity without a large increase in intravesical pressure. It has been clearly demonstrated that one of the major responses to obstruction in both men secondary to benign prostatic hyperplasia and in animals secondary to partial outlet obstruction is a thickening of the bladder wall and a decrease in compliance.1–3 Although urinary bladder distension and overdistension have been the subject of numerous studies,4 –14 the effects of bladder distension with or without bladder outlet obstruction on structure and vascularity have not been thoroughly investigated. In This work was supported by grants from the Veterans Affairs Medical Center and NIH grant NIH RO-1-DK53965. From the Division of Urology, Albany College of Medicine; Stratton Veterans Affairs Medical Center, Albany, New York; Department of Urology, Kinki University School of Medicine, Osaka, Japan; and Division of Basic and Pharmaceutical Sciences, Albany College of Pharmacy, Albany, New York Reprint requests: Robert M. Levin, Ph.D., Albany College of Pharmacy, 106 New Scotland Avenue, Albany, NY 12208 Submitted: March 12, 2003, accepted (with revisions): June 19, 2003 © 2003 ELSEVIER INC. ALL RIGHTS RESERVED
previous studies, we observed that the thickness of the ventral and dorsal sections of the obstructed rabbit bladder were very different. Thus, the objective of the current study was to compare and correlate the effects of distension on the ventral and dorsal sections of both control and obstructed rabbit bladder morphology. The rabbit has been used extensively for the study of urinary bladder function and dysfunction.2,3,15,16 The adult rabbit has a comparatively large bladder that can be evaluated using standard cystometry techniques. The in vitro whole rabbit bladder model has been used effectively to evaluate the effect of filling on bladder function.17–19 In these early studies, no attempt was made to correlate changes in function with changes in structure.17,19 MATERIAL AND METHODS EXPERIMENTAL PROTOCOL A total of 30 mature male New Zealand white rabbits weighing 3 to 4 kg were separated into two groups of 15 control (sham) and 15 obstructed rabbits. 0090-4295/03/$30.00 doi:10.1016/S0090-4295(03)00686-1 1129
SURGICAL CREATION OF BLADDER OUTLET PARTIAL OBSTRUCTION Each rabbit was anesthetized with sodium pentobarbital (25 mg/kg intravenously). An 8F Foley catheter was inserted into the bladder and the urine drained. The bladder and urethrovesical junction were exposed through a midline incision. Placing a 2-0 silk ligature loosely around the catheterized proximal urethra created partial bladder outlet obstruction. The catheter was then removed. The bladder was returned, and the wound was closed. A dose of 1 mg/kg gentamicin and 0.3 mg/kg buprenorphine hydrochloride was given intramuscularly on postoperative days 1 and 2. Sham operations consisted of anesthesia, a midline incision, and cleaning the bladder neck region. No ligature was tied.
IN VIVO CYSTOMETRY At 3 weeks after surgery, cystometry was performed. With the animal in the supine position, an 8F catheter was inserted into the bladder and the volume of urine measured. The bladder was filled with saline at 1.42 mL/min until either micturition contraction or overflow incontinence occurred. The bladder capacity was determined. The bladder was then emptied and filled with buffered formalin to one of the following volumes: 5%, 25%, 50%, 100%, and 125% of capacity. Three control and three obstructed bladders were filled to each volume. The bladders were excised en bloc and placed in buffered formalin for 6 to 8 hours. After fixation, full-thickness sections of the ventral and dorsal bladder body were cut and processed for both light microscopy and immunohistochemistry.
MICROSCOPY After fixation, a 2-cm square sample was cut from the dorsal and ventral sides of the bladder body and processed. The samples were then cut into strips and embedded on edge in paraffin to get a cross-sectional view when sectioned. Tissue blocks were sectioned at 5 m mounted on glass slides, deparaffinized, rehydrated, and stained with hematoxylin-eosin. The effect of distension on bladder morphology was evaluated by measuring the bladder wall thickness using quantitative image analysis with Image Pro Plus software (Media Cybernetics, Silver Spring, Md); 40⫻ image magnification was used for all determinations, and all parameters were measured in 10 different locations on each sample cross-section and an average taken.
IMMUNOSTAINING Tissue sections were stained for vascular endothelium with a mouse monoclonal antibody to CD31 on a Ventana ES automated immunostainer (Ventana Medical Systems, Tucson, Ariz) using a biotin avidin-conjugated horseradish peroxidase amplification method with diaminobenzidine chromogen detection followed by a light hematoxylin counterstain. After immunostaining, all slides were dehydrated through graded alcohols and two changes of xylene and then mounted with Permount mounting medium.
STATISTICAL ANALYSIS
The data are expressed as the mean ⫾ the standard error of mean (SEM). Comparisons among groups were made using analysis of variance followed by the Newman-Keuls test and paired Student t test for individual differences. Statistical significance required a P value of less than 0.05. The Stratton Veterans Affairs Medical Center Institutional Animal Care and Use Committee reviewed and approved these studies. 1130
FIGURE 1. Effect of distension on bladder wall thickness. Values presented as the mean ⫾ SEM of 3 rabbits per group. *Significantly different from 5% (P ⬍0.05). †Significantly different from dorsal (P ⬍0.05).
RESULTS Three weeks of partial bladder outlet obstruction resulted in a significant increase in bladder weight (2.24 ⫾ 0.09 versus 11.77 ⫾ 1.06 mg) and bladder capacity (60 ⫾ 4.01 versus 133.01 ⫾ 27.90 mL). MICROSCOPY Histologic evaluation of the control bladders at all levels of distension showed no significant differences between the dorsal and ventral sides. Thus, in all figures, we present the data for the control bladder as the mean of the ventral and dorsal sections. Histologic evaluations of the obstructed bladders showed significant differences between the ventral and dorsal sections and thus were evaluated separately. At 5% capacity (⬃empty), the thickness of the dorsal side of the obstructed bladder was similar to the thickness of the control bladder; however, the ventral side was significantly thicker than both the dorsal side and the control bladder (Fig. 1). Compared with the nondistended bladders, distending the bladders to 25% resulted in significant thinning of the control bladder, but not for either the dorsal or ventral walls of the obstructed bladder. Compared with the bladders distended to 25% of capacity, distending the bladders to 50% of capacity resulted in no further thinning of the control bladder, but a significant thinning of the ventral wall of the obstructed bladder. Similarly, compared with bladders distended to 50% of capacity, distending the bladders to 100% of capacity resulted in significant thinning of the dorsal wall of the obstructed bladder. No statistically significant differences were found between bladders distended to 100% and those distended to 125% (Fig. 1). UROLOGY 62 (6), 2003
FIGURE 2. Representative example of transverse sections of normal and obstructed bladder walls distended to 5%, 25%, and 125% capacity. Hematoxylin-eosin stain, original magnification ⫻40. (A) Control (distended to 5%, 25%, and 125% capacity), (B) obstructed dorsal wall (distended to 25%, 50%, and 125% capacity), and (C) obstructed ventral wall (distended to 25%, 50%, and 125% capacity).
Figure 2A shows representative cross-sections of control bladders at 5%, 25%, and 125% capacity. Figure 2B,C (ventral and dorsal, respectively) show representative cross-sections of obstructed bladders at 25%, 50%, and 125% capacity. For the obstructed bladder, no statistically significant differences were found between 5% and 25% distension (representative sections of obstructed bladder at 5% distension not shown because of space considerations). No statistically significant differences were found in the cross-sectional areas between the venUROLOGY 62 (6), 2003
FIGURE 3. Representative example of transverse sections of control bladder wall. Vascular endothelium immunostained with anti-CD31 antibodies. (A) Control bladder distended to 5% (⫻40) of capacity and (B) control bladder distended to 25% of capacity.
tral and dorsal obstructed bladders for either the mucosa or detrusor. The cross-sectional area of the ventral serosa was significantly greater than that of the dorsal obstructed bladder for all levels of distension. However, although distension significantly thinned the ventral serosa, the cross-sectional area of the obstructed dorsal section did not significantly change with distension. For both control and obstructed bladders, the mucosa at 5% capacity was multilayered with folds. On distension, it thinned and stretched and appeared as a single layer with a flat surface. For both control and obstructed bladders, the smooth muscle bundles within the detrusor that were large and the most prominent portion of the wall at 5% capacity stretched and thinned and were reduced to longitudinal strands by 25% capacity for control 1131
bladders, but not until 125% capacity for obstructed bladders. Partial outlet obstruction resulted in a marked thickening and repositioning of the serosal layer. In the control, it is a thin, one-cell-thick, connective tissue sheath around the bladder. In obstructed bladders, the serosa was significantly thicker on the ventral side compared with the dorsal side. The histologic changes at the serosa for both the dorsal and the ventral obstructed sides were similar in that both showed an increase in connective tissue, fibroblasts, and microvessels. However, the marked difference was that on the ventral side all of the serosal growth was extrinsic to the detrusor, and on the dorsal side, almost one half of this additional growth of connective tissue was within the detrusor. On distension, the serosa on the ventral surface thinned to a significantly greater extent than the serosa on the dorsal surface. IMMUNOSTAINING Figure 3 displays a cross-section of a control bladder at 5% and 25% capacity showing blood vessels immunostained for vascular endothelium with CD31 antibodies. At 5% capacity, all vessels, arteries, and veins were open. At 25% capacity, virtually all veins showed significant compression and most appeared collapsed. The arteries and arterioles remained open at 25% capacity. With increasing volumes, the vessels showed additional compression. With regard to the obstructed bladder, little change was noted in the structure between 5% and 25% in the obstructed bladders. All vessels remained fully opened. As the bladder distended beyond 25%, some collapsing of veins occurred although many of them remained open. Qualitatively, the vessels of the dorsal side were affected to a greater extent than the vessels of the ventral side. Thus, a marked difference was found between the control and obstructed bladders with regard to the effect of distension on vessel morphology. COMMENT In experimental animals, partial outlet obstruction stimulates a rapid increase in bladder mass and bladder capacity. In addition, a direct correlation appears to exist between the severity of decompensation and the magnitude of the increase in bladder mass.2,3,16,20 In vivo studies have shown that overdistension can result in edema within the bladder wall.5,7,9 Because edema takes some time to occur after overdistension and in our studies, the bladders were fixed during and immediately after distension, there was no time for edema to occur and thus edema was not a factor in our observations. 1132
In the current study, we directly compared the structural responses of control and obstructed bladders to distension using the isolated whole bladder model. Kojima et al.21 demonstrated that cadaveric human bladders revealed no statistically significant differences between the thickness of the anterior or lateral bladder walls, trigone, or dome. Our data on the control rabbit bladder likewise showed no significant structural differences between the ventral and dorsal wall. However, statistically significant differences were noted for the obstructed bladder, primarily owing to the increased thickness of the serosa on the ventral side. Serosal thickness was not affected by sham surgery. Oelke et al.22 showed detrusor wall thickness during bladder filling in humans using ultrasound devices. Their results showed that the detrusor wall thickness decreased continuously while the bladder filled to 50% of its capacity and then remained constant until 100%. In our study, the rabbit bladder wall thinned up to 25% capacity and then remained constant. The difference was most likely due to the thinner wall of the rabbit bladder compared with that of the human bladder. In a recent study, Schroder et al.23 reported on the effect of distension on the expansion of control and obstructed rabbit bladders and on the regional contractile responses. These studies demonstrated that although the control bladders expanded evenly during filling, the obstructed bladders did not.23 Our current studies showing uneven growth of the ventral and dorsal sides of the bladder can explain the functional observations of Schroder et al. Similarly, Schroder et al.23 demonstrated that the contractile responses of the control dorsal and ventral strips were similar, although after obstruction significant differences existed. These differences would be directly related to the different structural responses of the dorsal and ventral sides of the bladder to obstruction. The increase in bladder mass is mediated primarily through urothelial and fibroblast hyperplasia and smooth muscle hypertrophy.24 The development of serosal thickening is a characteristic response of the rabbit bladder to obstruction.25 The origin of the connective tissue is from serosal submesothelial mesenchymal cells. They proliferate in early obstruction, contribute to the dorsal side serosal thickening, and also differentiate into myofibroblasts.24 However, these myofibroblasts do not differentiate into smooth muscle cells in the ventral serosa. The asymmetric appearance of these cells explains the uneven distension of the obstructed bladders. The significantly increased thickening of the ventral side of the bladder compared with the dorsal side may relate to the position of the ventral side (ie, the ventral side has greater stress because of gravity than the dorsal UROLOGY 62 (6), 2003
side). It may be that the increased thickness is a response to the greater forces placed on this side by the increased volume of urine the obstructed bladder contains. The results of human and animal experiments have shown that blood flow decreases with distension.11,12,14 In patients with poorly compliant bladders, distension resulted in decreased bladder wall blood flow to a significantly greater extent than it did in patients with normal compliance. Thus, bladder distension can lead to bladder wall ischemia that, ultimately, may result in reduced bladder contractility, increased residual volume, and bladder dysfunction. We have previously demonstrated that outlet obstruction results in angiogenesis in the rapidly growing bladder26 and increased vascularization, so that in compensated function vascular density is not significantly different from control bladders.27,28 In the current study, vascular compression during distension was significantly greater for the controls than for the obstructed bladders. This may be a compensatory factor that will allow the obstructed bladder to maintain blood flow in the presence of overdistension, which might have less effect on blood flow in the obstructed than in the control. CONCLUSIONS The response of the rabbit bladder to obstruction is uneven. The morphologic responses of the dorsal and ventral sides of the bladder were significantly different from each other, with the ventral side showing significantly greater serosal thickness than the dorsal. REFERENCES 1. Zderic SA, Levin RM, and Wein AJ: Voiding function and dysfunction: A—relevant anatomy, physiology, and pharmacology, and molecular biology, in Gillenwater JY, Grayhack JT, Howards SS, et al (Eds): Adult and Pediatric Urology, 3rd ed. Chicago, Mosby Year Book Medical, 1996, pp 1159 –1219. 2. Gosling JA, Kung LS, Dixon JS, et al: Correlation between the structure and function of the urinary bladder following partial outlet obstruction. J Urol 163: 1349 –1356, 2000. 3. Levin RM, Haugaard N, O’Connor L, et al: Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol Urodyn 19: 609 –629, 2000. 4. Dunn M: A study of the bladder blood flow during distension in rabbits. Br J Urol 47: 67–72, 1975. 5. Gosling JA, Dixon JS, and Dunn M: The structure of the rabbit urinary bladder after experimental distension. Invest Urol 14: 386 –389, 1977. 6. Kang J, Wein AJ, and Levin RM: Bladder functional recovery following acute overdistension. Neurourol Urodyn 11: 253–261, 1992. 7. Monson FC, Wein AJ, Eika B, et al: Stimulation of the proliferation of rabbit bladder urothelium by partial outlet obstruction and acute overdistension. Neurourol Urodyn 13: 51–62, 1994. UROLOGY 62 (6), 2003
8. Tammela T, Lasanen L, and Waris T: Effect of distension on adrenergic innervation of the rat urinary bladder. Urol Res 18: 345–348, 1990. 9. Tammela T, Autio-Harmainen H, Lukkarinen O, et al: Effect of distension on function and nervous ultrastructure in the canine urinary bladder. Urol Int 42: 265–270, 1987. 10. Tammela TL, Levin RM, Monson FC, et al: The influence of acute overdistension on rat bladder function and DNA synthesis. J Urol 150: 1533–1539, 1993. 11. Nemeth CJ, Khan RM, Kirchner P, et al: Change in canine bladder perfusion with distension. Invest Urol 15: 149 –150, 1977. 12. Finkbeiner A, and Lapides J: Effect of distension on blood flow in dog urinary bladder. Invest Urol 12: 210 –212, 1974. 13. Weaver LC: Organization of sympathetic responses to distension of urinary bladder. Am J Physiol 248: R236 –R240, 1985. 14. Brading AF, Greenland JE, and Milis IW: Blood supply to the bladder during filling. Scand J Urol Nephrol 201: 25–31, 1999. 15. Levin RM, Monson FC, Longhurst PA, et al: The rabbit as a model of urinary bladder function. Neurourol Urodyn 13: 119 –136, 1994. 16. Levin RM, Brading AF, Mills IW, et al: Experimental models of bladder obstruction, in Lepor H (Ed): Prostatic Disease. Philadelphia, WB Saunders, 2000, pp 169 –196. 17. Levin RM, and Wein AJ: Response of the in-vitro whole bladder (rabbit) preparation to autonomic agonists. J Urol 128: 1087–1090, 1982. 18. Levin RM, Brendler K, and Wein AJ: Comparative pharmacological response of an in-vitro whole bladder preparation (rabbit) with the response of isolated smooth muscle strips. J Urol 30: 377–381, 1983. 19. Kaplan SA, Blaivas JG, Brown WC, et al: Parameters of detrusor contractility. I. The effects of electrical stimulation, hysteresis and bladder volume in an in-vitro whole rabbit bladder model. Neurourol Urodyn 10: 53–60, 1991. 20. Kato K, Wein AJ, Longhurst PA, et al: The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J Urol 143: 600 –606, 1990. 21. Kojima M, Inui E, Ochiai A, et al: Ultrasonic estimation of bladder weight as a measure of bladder hypertrophy in men with intravesical obstruction: a preliminary report. Urology 47: 942–947, 1996. 22. Oelke M, Hofner K, Wiese B, et al: Increase in detrusor wall thickness indicates bladder outlet obstruction (BOO) in men. World J Urol 19: 443–452, 2002. 23. Schroder A, Uvelius B, Capello SA, et al: Regional differences in bladder enlargement and in vitro contractility after outlet obstruction in the rabbit. J Urol 168: 1240 –1246, 2002. 24. Levin RM, Monson FC, Haugaard N, et al: Genetic and cellular characteristics of bladder outlet obstruction. Urol Clin North Am 22: 263–283, 1995. 25. Monson FC, Goldschmidt MH, Zderic SA, et al: Use of a undescribed elastic lamina of serosa to characterize connective tissue hypertrophy of rabbit bladder wall following partial outlet obstruction. Neurourol Urodyn 7: 385–396, 1988. 26. Ghafar MA, Anastasiadis AG, Olsson LE, et al: Hypoxia and an angiogenic response in the partially obstructed rat bladder. Lab Invest 82: 903–909, 2002. 27. Chichester P, Lieb J, Levin SS, et al: Vascular response of the rabbit bladder to short term partial outlet obstruction. Mol Cell Biochem 208: 19 –26, 2000. 28. Chichester P, Schroder A, Horan P, et al: Vascular response of the rabbit bladder to chronic partial outlet obstruction. Mol Cell Biochem 226: 1–8, 2001. 1133