- Email: [email protected]
Pharmacological Responses of Rabbit Urinary Bladder after Subtotal Cystectomy
0022-534 7/89 /1422-0409$02.00/0 THE JOURNAL OF UROLOGY Copyright© 1989 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Vol. 142, August
Printed in U.S.A.
PHARMACOLOGICAL RESPONSES OF RABBIT URINARY BLADDER AFTER SUBTOTAL CYSTECTOMY ALEX TONG-LONG LIN, KUMIKO KATO, FREDERICK MONSON, ALAN J. WEIN ROBERT M. LEVIN*
AND
From the Division of Urology, University of Pennsylvania School of Medicine and the Philadelphia Veterans Administration Medical Center, Philadelphia, and Division of Urology, Taiwan Veterans General Hospital, Taipei, Taiwan, Republic of China
ABSTRACT
Partial cystectomy is used clinically in specific circumstances. There have been some reports indicating that the bladder following subtotal cystectomy can regenerate to a certain degree_ The present study investigates the physiology and pharmacology of bladder regeneration at eight weeks after resection of a major part of the bladder body in rabbits. The following studies were performed on control and cystectomy bladders: 1) in vivo cystometry (prior to and four weeks after the operation, and before the sacrifice at eight weeks); 2) sequential muscle strip study (the strips were obtained by dividing bladder transversely into upper body, lower body, mid-bladder, upper base and lower base); and 3) in vitro whole bladder studies. The results can be summarized as follows: 1) grossly there was no regeneration of the bladder body; the cystometric capacity was increased slowly after the operation primarily due to increased size and capacity of the bladder base. 2) The contractile response of the subtotal cystectomized bladder body to field stimulation and bethanechol stimulation was less than the response of normal bladder body. 3) The contractile response to epinephrine showed that the ratio of alpha/beta adrenergic response was much greater in the cystectomy bladder than in the normal bladder. 4) In the whole bladder study, the intravesical pressure response to field stimulation was about the same in both groups, the response to bethanechol was less for cystectomized bladder, and the response to methoxamine was greater for the cystectomized bladder. 5) The ability of cystectomy bladder to empty in response to both field stimulation and bethanechol was impaired whereas the control bladder fully emptied to both field stimulation and bethanechol. In conclusion, over the eight week period following subtotal cystectomy the capacity increased via distention (and hypertrophy) of the bladder base area as opposed to regeneration of the bladder body. (J. Ural., 142: 409-412, 1989) Functionally, the urinary bladder can be divided into two parts: bladder body and base. The bladder body contains a greater density of muscarinic cholinergic receptors and responds to parasympathetic stimulation with a significantly greater contraction than does the bladder base. The bladder base contains a greater density of alpha adrenergic receptors than the bladder body, whereas the bladder body contains a greater density of beta adrenergic receptors. The bladder base responds to adrenergic stimulation with a contraction whereas the body relaxes. 1- 3 There is evidence in the literature suggesting that following subtotal cystectomy, the bladder can undergo regeneration. The present study was designed to determine the alterations in the pharmacological response of the urinary bladder following partial cystectomy.4-6 MATERIALS AND METHODS
Surgical technique. New Zealand rabbits with 4.5 to 5.0 kg. body weight were used for the experiment. Each rabbit was anesthetized with ketamine/xylazine (25 mg. ketamine, 10 mg. xylazine/kg.). The urinary bladder was exposed through a midline lower abdominal incision. The majority of the bladder body was excised three mm. above the ureterovesical junction. The Accepted for publication February 20, 1989. *Requests for reprints: Division of Urology, 5-Silverstein, Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104. Supported by grants from the Veterans Administration, NIH grants #R0-1-DK-2-6508, #R0-1-DK 33559, P-50-DK 39257 and the McCabe Fund.
remaining bladder base was closed in two layers with continuous 4-0 chromic sutures. Both ends of the suture line were marked with nonabsorbable black silk sutures for later identification. The bladder was returned to its natural position and the abdomen was closed with 2-0 chromic catgut. The skin was approximated with 3-0 silk continuous sutures. A French 8 Foley catheter was inserted into the bladder through the urethra and secured on the prepuce. The catheter was removed three days after the operation. In vivo cystometry. In vivo cystometry was done prior to surgery, four weeks after the operation and prior to sacrifice at eight weeks as follows: under anesthesia the bladder was catheterized with a French 8 catheter which was connected to a three-way stopcock with one end connected to a 60 ml. syringe mounted on a Harvard infusion pump (3.5 ml. normal saline/ min.). Intravesical pressure was continuously monitored via a Statham pressure transducer and recorded on a Grass polygraph. Immediately following the cystometry performed at eight weeks following surgery, the urinary bladder was removed for in vitro studies and the rabbit euthanized with T-61, iv. Muscle bath studies. Urinary bladders of four subtotal cystectomized and four normal rabbits were dissected free of fat and connective tissue. The bladder was separated the the level of the ureters into bladder body and base. Two sections of bladder body were used: upper and lower body. It should be remembered that in the cystectomized bladder, there was little dome present and although the size of all strips of body were similar, there was a very significantly greater amount of tissue to choose from in the control. The mid bladder represented the narrow section containing the ureteral orifices. Two sections of bladder base
409
410
LIN AND ASSOCIATES
were utilized: upper and lower. As opposite from the body, there was little tissue available from the control bladders whereas the bases of the cystectomized bladder were considerably larger. However, all strips of base were of equal size. Each segment was mounted in a 30 ml. bath chamber containing Tyrode'ssolution with following composition: 125 mM NaCl, 2.7 mM KCl, 0.4 mM NaHPO., 1.8 mM CaCl 2, 0.9 mM MgCl2, 23.8 mM NaHC0 3 and 0.2% glucose. The bath was equilibrated with 95% 02 + 5% CO2 and maintained at 37C. One gm. tension was applied on the strips and the experiment was performed the strips equilibrated for an additional 45 minutes. The contractile response to field stimulation or pharmacological stimulation was monitored via a FT 003 force transducer and was recorded on a 6-channel Grass polygraph. In vitro whole bladder studies. Urinary bladders of four subtotal cystectomized and four normal rabbits were dissected free of fat and the ureters tied. The urethra was cannulated on a saline-filled electrode tipped catheter. The preparation was mounted as an intact organ in an isolated muscle bath containing 300 ml. Tyrodes' solution. The catheter was connected via a three-way stopcock to a syringe mounted on a Harvard infusion pump, a saline bag mounted on an electronic balance, and a 100 ml. burette. lntravesical pressure is monitored by a Statham pressure transducer and recorded on a Grass Polygraph. 7 • 8 In vitro cystometry, and the ability of the bladder to both generate pressure and empty in response to field stimulation and pharmacological stimulation, was determined. For these studies, an intravesical volume of ½ capacity was used, emptying is given as a percent of maximum emptying and is thus normalized for the different volumes utilized. RESULTS
Eight weeks after subtotal cystectomy, the bladders were smaller than normal. The ureteral insertions were high on the bladder with no significant identifiable regenerated part above the marking black silk sutures. Figure 1 shows the in vivo cystometric capacity of normal bladder and after subtotal cystectomy. For normal male rabbits with 4.5 to 5.0 kg. body weight, the cystometric capacity is 72.3 ± 4.7 ml. (n = 11). Immediately after subtotal cystectomy, the estimated capacity was about five to 10 ml.; at four weeks after operation, the capacity was increased to 18.1 ± 1.5 ml.; at eight weeks, the capacity was increased to 31.9 ± 4.1 ml. Compliance (as related to the slope of the plateau phase of the cystometrogram) of cystectomized bladder was relatively normal. Figure 2 displays the contractile response to field stimulation (80 volts, 32 hz, one msec). The response was significantly reduced in the body of cystectomized bladder with no differences noted in mid bladder and base sections. The response of
CYSTOM ETRIC CAPACITY ,,...._ 80
E
'--"
~
60
O,_
40
D CONTROL !"22!1 IMMEDIATELY AFTER CYSTECTOMY &'S.'3 4 WEEKS AFTER CYSTECTOMY [Z] 8 WEEKS AFTER CYSTECTOMY
field stimulation was reduced by over 90% by preincubating with tetrodotoxin (one µM). Similarly, the response to both bethanechol (500 µM) and ATP (one mM) were reduced in the bladder body sections, but not in the mid-bladder or base regions (figs. 3 and 4). The contractile response to epinephrine (one mM) is displayed in figure 5. Epinephrine stimulates both adrenergic alpha receptors (contraction) and beta receptors (relaxation). The net effects depends on the relative density of alpha and beta receptors. As demonstrated previously, in the normal bladder epinephrine relaxed the body portions and contracted the mid-bladder and base sections. 1 In the cystectomized bladder, epinephrine relaxed the upper body and contracted all other segments. The mid-bladder and upper base responded with a significantly greater contraction than did these sections of the normal bladder. Isoproterenol (10 µM) relaxed all portions of both the cystectomized bladder and normal bladder to the same extent (figure 6). Figure 7 displays the pressure response of the cystectomized bladder and normal bladder to field stimulation, bethanechol, and epinephrine. The response to field stimulation was almost identical between normal and cystectomized bladder. The pressure response of the normal bladder to bethanechol was signif-
RESPONSE TO FIELD STIMULATION w
CJ CONTROL ~ CYSTECTOMY
6
CJ)
6
5
O,_
~'24
*
0::: -~
w ~ 3 _J f-i== E u o>2 <(._..,,
*
0:::
f-
z u
0
LOWER BASE FIG. 2. Response of sequential muscle strips to field stimulation (80 volts, 32 hz, 1 msec). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
RESPONSE TO BETHANECHOL CJ CONTROL ~ CYSTECTOMY
6
*
u <( <(
u
*
_J
<( u 20 CJ)
w
>
0
FIG. 1. Cystometric capacity after subtotal cystectomy. Each bar is mean ± S.E.M. of 8 animals. Data for immediately after cystectomy is estimated value.
UPPER BODY
LOWER MID UPPER BODY BLADDER BASE
LOWER BASE
FIG. 3. Response of sequential muscle strips to bethanechol (500 uM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
PHARMACOLOGY AND SUBTOTAL CYSTECTOMY
RESPOI\ISE ~
(f)
z
0
Q_
(/)
-
WC O::'. .2 C QJ
_J f-
CONTROL CYSTECTOMY
1.6
*
*
L2
(f)
,.._
W
C
0::: -~ C
~ ~ i== E 0,6
i== E
0~
U en 0.8
* *
0::: 1-
O'.'.
z
0.4
z u
(/)
0 D....
<( '--'
1-
CJ CONTROL ~ CYSTECTOi\~Y
w
z
2.0
(/l
w
RESPONSE TO ISOPROTERENOL
ATP D
2.4
w
411
0
0
0
O.O UPPER BODY
LOWER MID UPPER BODY BLADDER BASE
0. 2 UPPER BODY
LOWER BASE
FIG. 4. Response of sequential muscle strips to ATP (2 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
LOWER
MID UPPER BODY BLADDER BASE
LOWER BASE
FIG. 6. Response of sequential muscle strips to isoproterenol (10 µm.). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
RESPONSE TO EPINEPHRINE D ~
w
(f)
CONTROL CYSTECTOMY
z
w
::i (/)
w oc---
C 0
0::: 'iii
w _J
D.... 0
N
C Cl)
<(
f-
u
en '..J
7.5
E ll
5.0
(I)'--'
w
D:'.
~
I-
z
2.5
O'.'.
f---
0
u
CYSTECTOMY
_JI
i== E
u
~
10.0
(/)
,,_..
CJ CONTROL
12.5]
w
0:::
0
(1_ (/)
PRESSURE RESPONSE OF WHOLE BLADDER
0.0-'--------------------UPPER LOWER MID UPPER LOWER BODY BLADDER BASE BODY BASE
Fm. 5. Response of sequential muscle strips to epinephrine (1 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
icantly greater than the response of the cystectornized bladder. However, the response of the cystectomized bladder to epinephrine was significantly greater than the response of the normal bladder. The response to methoxamine was identical to the response observed for epinephrine (data not shown). The cystectomized bladder emptied a significantly smaller volume than the normal bladder in response to bethanechol and field stimulation. However, epinephrine (and methoxamine) stimulated a greater emptying in the cystectomized bladder than in the normal bladder (figure 8).
z
0.0
F. S.
BETH
EPI
FIG. 7. Pressure response of in vitro whole bladder to field stimulation (80 volts, 32 hz, 1 msec), bethanechol (500 µm.) and epinephrine (1 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
NG ABI
OF WHOI_E BLADDER CJ CONTROL ~ CYSTECTOMY
El 100 l i=
D...
CJ
75-
w DISCUSSION
Regeneration of urinary bladder after subtotal cystectomy is a controversial topic in urologic literatures.4· 6 • 9 - 14 In 1891 Schwarz resected most part of the urinary bladder in dogs and found that good capacity was regained later. 9 The same phenomenon was also observed later by others in rabbits and rats. Schiller performed subtotal cystectomy on rabbits and good size bladder was discovered later. 1° Kreschmer did subtotal cystectomy on rabbit and the cystogram showed normal size bladder as early as two months after the operation. 11 Liang carried out subtotal cystectomy on rats, which regained normal capacity of the bladder eight weeks after the operation. 6 Regeneration was claimed by these researchers as the mechanism for
~
3 0
50
*
>
~ 25
w
0 0:::
w
Q
0-'--l-...L.....l..>..>Cl..___
F.S
__,____t_il:l,,li__ _-=:=,._c,,.,.:'1__ _
BETH
EPI
FIG. 8. Emptying ability of in vitro whole bladder to field stimulation (80 volts, 32 hz, 1 msec), bethanechol (500 µm.) and epinephrine (1 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
412
LIN AND ASSOCIATES
regaining the capacity after massive resection of the urinary bladder. However, regeneration of detrusor muscle after subtotal cystectomy was questioned by others. Using autoradiography technique, Ross could not find evidence of regeneration of smooth muscle of bladder dome in dogs after partial cystectomy. 12 Goldstein suggested that it was stretching, not regeneration, that was responsible for the recovery of bladder capacity after subtotal cystectomy in rabbits. 13 Recently, Glasser et al. could not find regrowth of detrusor muscle in bladders subjected to total or subtotal cystectomy with insertion of silicone rubber prosthesis. 14 In the present study, eight weeks after excision of most part of the bladder body, no regeneration of bladder body was observed although the cystometric capacity increased substantially. Although the increased capacity of the bladder is via an increased capacity of the bladder base and midsection, it is not possible to tell whether the enlargement of the bladder base is due to simple dilatation or hypertrophy, which was suggested by Liang. 6 Further investigation is necessary to differentiate dilation from hypertrophy. Although this study didn't confirm the regeneration of the bladder body after massive resection by subtotal cystectomy, regeneration of the bladder body under other conditions is still possible. Love reported one case with ischemic vesical necrosis, the bladder regenerated and the bladder capacity became normal six months later. 15 The phenomenon of bladder regeneration had been applied to augment the capacity of contracted bladders. The diseased bladders were incised and materials, such as gelatin sponge or Japanese paper, were used as a guide allowing the bladder to regenerate and enlarge. Regeneration was identified with excellent results reported. 5 • 16 It is quite possible that under certain circumstances, some unknown mechanism will stimulate the smooth muscle of the bladder body to regenerate. Although the size of the bladder base was enlarged after subtotal cystectomy, its pharmacological properties were not changed. In the muscle strips study, the contractile response to cholinergic, adrenergic and purinergic stimulation was the same as the bladder base of the normal bladder. The removal of the bladder body would be expected to alter the ratio of autonomic receptors in the intact bladder. The normal bladder contains primarily muscarinic cholinergic and beta adrenergic receptors, whereas the cystectomized bladder would be expected to have primarily alpha-adrenergic receptors. This would explain the responses of the whole bladder to field and pharmacological stimulation. Field stimulation increases intravesical pressure primarily via the release of neurotransmitters (tetrodotoxin virtually eliminates the response). For the rabbit bladder, these would include acetylcholine (via muscarinic cholinergic receptors), norepinephrine (via alpha adrenergic receptors), and ATP (via purinergic receptors). It is difficult to separate field stimulation into these components because at present there are no specific purinergic antagonists. However, based on the pressure response to bethanechol, epinephrine, and methoxamine some conclusions can be theorized. Field stimulation produced similar pressure responses in both the normal and cystectomized bladder, whereas the response in the normal bladder was primarily related to muscarinic stimulation, the response in the cystectomized bladder may be related primarily to alpha adrenergic stimulation. This is supported by the demonstration that bethanechol (muscarinic agonist) stimulated a significantly greater pressure response in the normal bladder whereas epi-
nephrine (and methoxamine) stimulated a greater pressure response in the cystectomized bladder. In the emptying study, the in vitro cystectomized bladder could only evacuate 25 to 50% intravesical volume by various stimulation. This could be related in part to the relatively poor cholinergic response of the cystectomized bladder, the scarred tissue of the bladder dome, and/or morphologic damage associated with bladder base distention. It should be remembered that isometric pressure generation does not require a significant conformational change in the bladder, whereas bladder emptying requires both the generation of significant pressure and the conformational change which is required to empty the bladder. Bladder ischemia and outlet obstruction are two examples of pathological states which induce a significantly greater reduction in the ability of the bladder to empty than in the ability to generate isometric pressure. 17• 18 REFERENCES 1. Levin, R. M., Shofer, S. F. and Wein, A. J.: Cholinergic, adrenergic and purinergic response of sequential strips of rabbit urinary bladder. J. Pharmacol. Exp. Ther., 212: 536, 1980. 2. Ek, A., Alm, P., Andersson, K. E. and Persson, C. G. A.: Adrenergic and cholinergic nerves of the human urethra and urinary bladder. A histochemical study. Acta. Physiol. Scand., 99: 345, 1977. 3. El Badawi, A. and Schenk, E. A.: Dual innervation of the mammalian urinary bladder: A histochemical study of the distribution of cholinergic and adrenergic nerves. Am. J. Anat., 119: 405, 1966. 4. Baker, R., Kelly, T., Tehan, T., Putnam, C., Beaugard, C. and Beaugard, E. Subtotal cystectomy and total bladder regeneration in treatment of bladder cancer. JAMA, 168: 1178, 1958. 5. Taguchi, H., Ishizuka, E. and Saito, K. Cystoplasty by regeneration of the bladder. J. Urol., 118: 752, 1976. 6. Liang, D. S. and Gross, R. J. Regeneration of the bladder after subtotal cystectomy in rats. J. Urol., 89: 427, 1963. 7. Levin, R. M. and Wein, A. J.: Response of the in-vitro whole bladder (rabbit) preparation to autonomic agonists. J. Urol., 128: 1087, 1982. 8. Levin, R. M., Brendler, K. and Wein, A. J.: Comparative pharmacolological response of an in-vitro whole bladder preparation (rabbit) with the response of isolated smooth muscle strips. J. Urol., 30: 377, 1983. 9. Schwarz, R.: Sulla rigenerazione della vescica orinaria. Riv. Ven. Sci. Med., 20: 508, 1891. 10. Schiller, H.: Regeneration of resected urinary bladder in rabbits. Surg. Gynec. Obst., 36: 24, 1923. 11. Kretchmer, H. L. and Barber, K. E.: Regeneration of the bladder following resection. JAMA, 90: 355, 1928. 12. Ross, G., Thompson, I. M., Bynum, W. R. and Thomson, E. P.: The role of smooth muscle regeneration in urinary tract repair. J. Urol., 95: 541, 1966. 13. Goldstein, A. M. B., Gualtieri, V. and Getzoff, P. L.: Expansion mechanisms of the partially cystectomized bladder: an experimental study in rabbits. J. Urol., 104: 413, 1970. 14. Gasser, T. C., Larsen, E. H., England, D. M., Graversen, P. H., Dorflinger, T., Madsen, P. 0. and Bruskewitz, R. C.: Urinary bladder reformation: regeneration or dilatation? N eurourol. U rodyn., 6: 129, 1987. 15. Love, A. and Notley, R. G.: Vesical necrosis. J. R. Soc. Med., 71: 895, 1978. 16. Orikasa, S. and Tsuji, I.: Enlargement of contracted bladder by use of gelatin sponge bladder. J. Urol., 104: 107, 1970. 17. Lin, T. L., Wein, A. J., Gill, H. S. and Levin, R. M.: Functional effect of chronic ischemia on the rabbit urinary bladder. Neurourol. Urodyn, 7: 1, 1988. 18. Kato, K., Wein, A. J., Kitada, S., Haugaard, N. and Levin, R. M.: The functional effect of mild outlet obstruction on the rabbit urinary bladder. J. Urol. (in press)
Vol. 142, August
Printed in U.S.A.
PHARMACOLOGICAL RESPONSES OF RABBIT URINARY BLADDER AFTER SUBTOTAL CYSTECTOMY ALEX TONG-LONG LIN, KUMIKO KATO, FREDERICK MONSON, ALAN J. WEIN ROBERT M. LEVIN*
AND
From the Division of Urology, University of Pennsylvania School of Medicine and the Philadelphia Veterans Administration Medical Center, Philadelphia, and Division of Urology, Taiwan Veterans General Hospital, Taipei, Taiwan, Republic of China
ABSTRACT
Partial cystectomy is used clinically in specific circumstances. There have been some reports indicating that the bladder following subtotal cystectomy can regenerate to a certain degree_ The present study investigates the physiology and pharmacology of bladder regeneration at eight weeks after resection of a major part of the bladder body in rabbits. The following studies were performed on control and cystectomy bladders: 1) in vivo cystometry (prior to and four weeks after the operation, and before the sacrifice at eight weeks); 2) sequential muscle strip study (the strips were obtained by dividing bladder transversely into upper body, lower body, mid-bladder, upper base and lower base); and 3) in vitro whole bladder studies. The results can be summarized as follows: 1) grossly there was no regeneration of the bladder body; the cystometric capacity was increased slowly after the operation primarily due to increased size and capacity of the bladder base. 2) The contractile response of the subtotal cystectomized bladder body to field stimulation and bethanechol stimulation was less than the response of normal bladder body. 3) The contractile response to epinephrine showed that the ratio of alpha/beta adrenergic response was much greater in the cystectomy bladder than in the normal bladder. 4) In the whole bladder study, the intravesical pressure response to field stimulation was about the same in both groups, the response to bethanechol was less for cystectomized bladder, and the response to methoxamine was greater for the cystectomized bladder. 5) The ability of cystectomy bladder to empty in response to both field stimulation and bethanechol was impaired whereas the control bladder fully emptied to both field stimulation and bethanechol. In conclusion, over the eight week period following subtotal cystectomy the capacity increased via distention (and hypertrophy) of the bladder base area as opposed to regeneration of the bladder body. (J. Ural., 142: 409-412, 1989) Functionally, the urinary bladder can be divided into two parts: bladder body and base. The bladder body contains a greater density of muscarinic cholinergic receptors and responds to parasympathetic stimulation with a significantly greater contraction than does the bladder base. The bladder base contains a greater density of alpha adrenergic receptors than the bladder body, whereas the bladder body contains a greater density of beta adrenergic receptors. The bladder base responds to adrenergic stimulation with a contraction whereas the body relaxes. 1- 3 There is evidence in the literature suggesting that following subtotal cystectomy, the bladder can undergo regeneration. The present study was designed to determine the alterations in the pharmacological response of the urinary bladder following partial cystectomy.4-6 MATERIALS AND METHODS
Surgical technique. New Zealand rabbits with 4.5 to 5.0 kg. body weight were used for the experiment. Each rabbit was anesthetized with ketamine/xylazine (25 mg. ketamine, 10 mg. xylazine/kg.). The urinary bladder was exposed through a midline lower abdominal incision. The majority of the bladder body was excised three mm. above the ureterovesical junction. The Accepted for publication February 20, 1989. *Requests for reprints: Division of Urology, 5-Silverstein, Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104. Supported by grants from the Veterans Administration, NIH grants #R0-1-DK-2-6508, #R0-1-DK 33559, P-50-DK 39257 and the McCabe Fund.
remaining bladder base was closed in two layers with continuous 4-0 chromic sutures. Both ends of the suture line were marked with nonabsorbable black silk sutures for later identification. The bladder was returned to its natural position and the abdomen was closed with 2-0 chromic catgut. The skin was approximated with 3-0 silk continuous sutures. A French 8 Foley catheter was inserted into the bladder through the urethra and secured on the prepuce. The catheter was removed three days after the operation. In vivo cystometry. In vivo cystometry was done prior to surgery, four weeks after the operation and prior to sacrifice at eight weeks as follows: under anesthesia the bladder was catheterized with a French 8 catheter which was connected to a three-way stopcock with one end connected to a 60 ml. syringe mounted on a Harvard infusion pump (3.5 ml. normal saline/ min.). Intravesical pressure was continuously monitored via a Statham pressure transducer and recorded on a Grass polygraph. Immediately following the cystometry performed at eight weeks following surgery, the urinary bladder was removed for in vitro studies and the rabbit euthanized with T-61, iv. Muscle bath studies. Urinary bladders of four subtotal cystectomized and four normal rabbits were dissected free of fat and connective tissue. The bladder was separated the the level of the ureters into bladder body and base. Two sections of bladder body were used: upper and lower body. It should be remembered that in the cystectomized bladder, there was little dome present and although the size of all strips of body were similar, there was a very significantly greater amount of tissue to choose from in the control. The mid bladder represented the narrow section containing the ureteral orifices. Two sections of bladder base
409
410
LIN AND ASSOCIATES
were utilized: upper and lower. As opposite from the body, there was little tissue available from the control bladders whereas the bases of the cystectomized bladder were considerably larger. However, all strips of base were of equal size. Each segment was mounted in a 30 ml. bath chamber containing Tyrode'ssolution with following composition: 125 mM NaCl, 2.7 mM KCl, 0.4 mM NaHPO., 1.8 mM CaCl 2, 0.9 mM MgCl2, 23.8 mM NaHC0 3 and 0.2% glucose. The bath was equilibrated with 95% 02 + 5% CO2 and maintained at 37C. One gm. tension was applied on the strips and the experiment was performed the strips equilibrated for an additional 45 minutes. The contractile response to field stimulation or pharmacological stimulation was monitored via a FT 003 force transducer and was recorded on a 6-channel Grass polygraph. In vitro whole bladder studies. Urinary bladders of four subtotal cystectomized and four normal rabbits were dissected free of fat and the ureters tied. The urethra was cannulated on a saline-filled electrode tipped catheter. The preparation was mounted as an intact organ in an isolated muscle bath containing 300 ml. Tyrodes' solution. The catheter was connected via a three-way stopcock to a syringe mounted on a Harvard infusion pump, a saline bag mounted on an electronic balance, and a 100 ml. burette. lntravesical pressure is monitored by a Statham pressure transducer and recorded on a Grass Polygraph. 7 • 8 In vitro cystometry, and the ability of the bladder to both generate pressure and empty in response to field stimulation and pharmacological stimulation, was determined. For these studies, an intravesical volume of ½ capacity was used, emptying is given as a percent of maximum emptying and is thus normalized for the different volumes utilized. RESULTS
Eight weeks after subtotal cystectomy, the bladders were smaller than normal. The ureteral insertions were high on the bladder with no significant identifiable regenerated part above the marking black silk sutures. Figure 1 shows the in vivo cystometric capacity of normal bladder and after subtotal cystectomy. For normal male rabbits with 4.5 to 5.0 kg. body weight, the cystometric capacity is 72.3 ± 4.7 ml. (n = 11). Immediately after subtotal cystectomy, the estimated capacity was about five to 10 ml.; at four weeks after operation, the capacity was increased to 18.1 ± 1.5 ml.; at eight weeks, the capacity was increased to 31.9 ± 4.1 ml. Compliance (as related to the slope of the plateau phase of the cystometrogram) of cystectomized bladder was relatively normal. Figure 2 displays the contractile response to field stimulation (80 volts, 32 hz, one msec). The response was significantly reduced in the body of cystectomized bladder with no differences noted in mid bladder and base sections. The response of
CYSTOM ETRIC CAPACITY ,,...._ 80
E
'--"
~
60
O,_
40
D CONTROL !"22!1 IMMEDIATELY AFTER CYSTECTOMY &'S.'3 4 WEEKS AFTER CYSTECTOMY [Z] 8 WEEKS AFTER CYSTECTOMY
field stimulation was reduced by over 90% by preincubating with tetrodotoxin (one µM). Similarly, the response to both bethanechol (500 µM) and ATP (one mM) were reduced in the bladder body sections, but not in the mid-bladder or base regions (figs. 3 and 4). The contractile response to epinephrine (one mM) is displayed in figure 5. Epinephrine stimulates both adrenergic alpha receptors (contraction) and beta receptors (relaxation). The net effects depends on the relative density of alpha and beta receptors. As demonstrated previously, in the normal bladder epinephrine relaxed the body portions and contracted the mid-bladder and base sections. 1 In the cystectomized bladder, epinephrine relaxed the upper body and contracted all other segments. The mid-bladder and upper base responded with a significantly greater contraction than did these sections of the normal bladder. Isoproterenol (10 µM) relaxed all portions of both the cystectomized bladder and normal bladder to the same extent (figure 6). Figure 7 displays the pressure response of the cystectomized bladder and normal bladder to field stimulation, bethanechol, and epinephrine. The response to field stimulation was almost identical between normal and cystectomized bladder. The pressure response of the normal bladder to bethanechol was signif-
RESPONSE TO FIELD STIMULATION w
CJ CONTROL ~ CYSTECTOMY
6
CJ)
6
5
O,_
~'24
*
0::: -~
w ~ 3 _J f-i== E u o>2 <(._..,,
*
0:::
f-
z u
0
LOWER BASE FIG. 2. Response of sequential muscle strips to field stimulation (80 volts, 32 hz, 1 msec). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
RESPONSE TO BETHANECHOL CJ CONTROL ~ CYSTECTOMY
6
*
u <( <(
u
*
_J
<( u 20 CJ)
w
>
0
FIG. 1. Cystometric capacity after subtotal cystectomy. Each bar is mean ± S.E.M. of 8 animals. Data for immediately after cystectomy is estimated value.
UPPER BODY
LOWER MID UPPER BODY BLADDER BASE
LOWER BASE
FIG. 3. Response of sequential muscle strips to bethanechol (500 uM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
PHARMACOLOGY AND SUBTOTAL CYSTECTOMY
RESPOI\ISE ~
(f)
z
0
Q_
(/)
-
WC O::'. .2 C QJ
_J f-
CONTROL CYSTECTOMY
1.6
*
*
L2
(f)
,.._
W
C
0::: -~ C
~ ~ i== E 0,6
i== E
0~
U en 0.8
* *
0::: 1-
O'.'.
z
0.4
z u
(/)
0 D....
<( '--'
1-
CJ CONTROL ~ CYSTECTOi\~Y
w
z
2.0
(/l
w
RESPONSE TO ISOPROTERENOL
ATP D
2.4
w
411
0
0
0
O.O UPPER BODY
LOWER MID UPPER BODY BLADDER BASE
0. 2 UPPER BODY
LOWER BASE
FIG. 4. Response of sequential muscle strips to ATP (2 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
LOWER
MID UPPER BODY BLADDER BASE
LOWER BASE
FIG. 6. Response of sequential muscle strips to isoproterenol (10 µm.). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
RESPONSE TO EPINEPHRINE D ~
w
(f)
CONTROL CYSTECTOMY
z
w
::i (/)
w oc---
C 0
0::: 'iii
w _J
D.... 0
N
C Cl)
<(
f-
u
en '..J
7.5
E ll
5.0
(I)'--'
w
D:'.
~
I-
z
2.5
O'.'.
f---
0
u
CYSTECTOMY
_JI
i== E
u
~
10.0
(/)
,,_..
CJ CONTROL
12.5]
w
0:::
0
(1_ (/)
PRESSURE RESPONSE OF WHOLE BLADDER
0.0-'--------------------UPPER LOWER MID UPPER LOWER BODY BLADDER BASE BODY BASE
Fm. 5. Response of sequential muscle strips to epinephrine (1 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
icantly greater than the response of the cystectornized bladder. However, the response of the cystectomized bladder to epinephrine was significantly greater than the response of the normal bladder. The response to methoxamine was identical to the response observed for epinephrine (data not shown). The cystectomized bladder emptied a significantly smaller volume than the normal bladder in response to bethanechol and field stimulation. However, epinephrine (and methoxamine) stimulated a greater emptying in the cystectomized bladder than in the normal bladder (figure 8).
z
0.0
F. S.
BETH
EPI
FIG. 7. Pressure response of in vitro whole bladder to field stimulation (80 volts, 32 hz, 1 msec), bethanechol (500 µm.) and epinephrine (1 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
NG ABI
OF WHOI_E BLADDER CJ CONTROL ~ CYSTECTOMY
El 100 l i=
D...
CJ
75-
w DISCUSSION
Regeneration of urinary bladder after subtotal cystectomy is a controversial topic in urologic literatures.4· 6 • 9 - 14 In 1891 Schwarz resected most part of the urinary bladder in dogs and found that good capacity was regained later. 9 The same phenomenon was also observed later by others in rabbits and rats. Schiller performed subtotal cystectomy on rabbits and good size bladder was discovered later. 1° Kreschmer did subtotal cystectomy on rabbit and the cystogram showed normal size bladder as early as two months after the operation. 11 Liang carried out subtotal cystectomy on rats, which regained normal capacity of the bladder eight weeks after the operation. 6 Regeneration was claimed by these researchers as the mechanism for
~
3 0
50
*
>
~ 25
w
0 0:::
w
Q
0-'--l-...L.....l..>..>Cl..___
F.S
__,____t_il:l,,li__ _-=:=,._c,,.,.:'1__ _
BETH
EPI
FIG. 8. Emptying ability of in vitro whole bladder to field stimulation (80 volts, 32 hz, 1 msec), bethanechol (500 µm.) and epinephrine (1 mM). Each bar is mean ± S.E.M. of results from 4 animals. * indicates that value is statistically different from control (p < 0.05).
412
LIN AND ASSOCIATES
regaining the capacity after massive resection of the urinary bladder. However, regeneration of detrusor muscle after subtotal cystectomy was questioned by others. Using autoradiography technique, Ross could not find evidence of regeneration of smooth muscle of bladder dome in dogs after partial cystectomy. 12 Goldstein suggested that it was stretching, not regeneration, that was responsible for the recovery of bladder capacity after subtotal cystectomy in rabbits. 13 Recently, Glasser et al. could not find regrowth of detrusor muscle in bladders subjected to total or subtotal cystectomy with insertion of silicone rubber prosthesis. 14 In the present study, eight weeks after excision of most part of the bladder body, no regeneration of bladder body was observed although the cystometric capacity increased substantially. Although the increased capacity of the bladder is via an increased capacity of the bladder base and midsection, it is not possible to tell whether the enlargement of the bladder base is due to simple dilatation or hypertrophy, which was suggested by Liang. 6 Further investigation is necessary to differentiate dilation from hypertrophy. Although this study didn't confirm the regeneration of the bladder body after massive resection by subtotal cystectomy, regeneration of the bladder body under other conditions is still possible. Love reported one case with ischemic vesical necrosis, the bladder regenerated and the bladder capacity became normal six months later. 15 The phenomenon of bladder regeneration had been applied to augment the capacity of contracted bladders. The diseased bladders were incised and materials, such as gelatin sponge or Japanese paper, were used as a guide allowing the bladder to regenerate and enlarge. Regeneration was identified with excellent results reported. 5 • 16 It is quite possible that under certain circumstances, some unknown mechanism will stimulate the smooth muscle of the bladder body to regenerate. Although the size of the bladder base was enlarged after subtotal cystectomy, its pharmacological properties were not changed. In the muscle strips study, the contractile response to cholinergic, adrenergic and purinergic stimulation was the same as the bladder base of the normal bladder. The removal of the bladder body would be expected to alter the ratio of autonomic receptors in the intact bladder. The normal bladder contains primarily muscarinic cholinergic and beta adrenergic receptors, whereas the cystectomized bladder would be expected to have primarily alpha-adrenergic receptors. This would explain the responses of the whole bladder to field and pharmacological stimulation. Field stimulation increases intravesical pressure primarily via the release of neurotransmitters (tetrodotoxin virtually eliminates the response). For the rabbit bladder, these would include acetylcholine (via muscarinic cholinergic receptors), norepinephrine (via alpha adrenergic receptors), and ATP (via purinergic receptors). It is difficult to separate field stimulation into these components because at present there are no specific purinergic antagonists. However, based on the pressure response to bethanechol, epinephrine, and methoxamine some conclusions can be theorized. Field stimulation produced similar pressure responses in both the normal and cystectomized bladder, whereas the response in the normal bladder was primarily related to muscarinic stimulation, the response in the cystectomized bladder may be related primarily to alpha adrenergic stimulation. This is supported by the demonstration that bethanechol (muscarinic agonist) stimulated a significantly greater pressure response in the normal bladder whereas epi-
nephrine (and methoxamine) stimulated a greater pressure response in the cystectomized bladder. In the emptying study, the in vitro cystectomized bladder could only evacuate 25 to 50% intravesical volume by various stimulation. This could be related in part to the relatively poor cholinergic response of the cystectomized bladder, the scarred tissue of the bladder dome, and/or morphologic damage associated with bladder base distention. It should be remembered that isometric pressure generation does not require a significant conformational change in the bladder, whereas bladder emptying requires both the generation of significant pressure and the conformational change which is required to empty the bladder. Bladder ischemia and outlet obstruction are two examples of pathological states which induce a significantly greater reduction in the ability of the bladder to empty than in the ability to generate isometric pressure. 17• 18 REFERENCES 1. Levin, R. M., Shofer, S. F. and Wein, A. J.: Cholinergic, adrenergic and purinergic response of sequential strips of rabbit urinary bladder. J. Pharmacol. Exp. Ther., 212: 536, 1980. 2. Ek, A., Alm, P., Andersson, K. E. and Persson, C. G. A.: Adrenergic and cholinergic nerves of the human urethra and urinary bladder. A histochemical study. Acta. Physiol. Scand., 99: 345, 1977. 3. El Badawi, A. and Schenk, E. A.: Dual innervation of the mammalian urinary bladder: A histochemical study of the distribution of cholinergic and adrenergic nerves. Am. J. Anat., 119: 405, 1966. 4. Baker, R., Kelly, T., Tehan, T., Putnam, C., Beaugard, C. and Beaugard, E. Subtotal cystectomy and total bladder regeneration in treatment of bladder cancer. JAMA, 168: 1178, 1958. 5. Taguchi, H., Ishizuka, E. and Saito, K. Cystoplasty by regeneration of the bladder. J. Urol., 118: 752, 1976. 6. Liang, D. S. and Gross, R. J. Regeneration of the bladder after subtotal cystectomy in rats. J. Urol., 89: 427, 1963. 7. Levin, R. M. and Wein, A. J.: Response of the in-vitro whole bladder (rabbit) preparation to autonomic agonists. J. Urol., 128: 1087, 1982. 8. Levin, R. M., Brendler, K. and Wein, A. J.: Comparative pharmacolological response of an in-vitro whole bladder preparation (rabbit) with the response of isolated smooth muscle strips. J. Urol., 30: 377, 1983. 9. Schwarz, R.: Sulla rigenerazione della vescica orinaria. Riv. Ven. Sci. Med., 20: 508, 1891. 10. Schiller, H.: Regeneration of resected urinary bladder in rabbits. Surg. Gynec. Obst., 36: 24, 1923. 11. Kretchmer, H. L. and Barber, K. E.: Regeneration of the bladder following resection. JAMA, 90: 355, 1928. 12. Ross, G., Thompson, I. M., Bynum, W. R. and Thomson, E. P.: The role of smooth muscle regeneration in urinary tract repair. J. Urol., 95: 541, 1966. 13. Goldstein, A. M. B., Gualtieri, V. and Getzoff, P. L.: Expansion mechanisms of the partially cystectomized bladder: an experimental study in rabbits. J. Urol., 104: 413, 1970. 14. Gasser, T. C., Larsen, E. H., England, D. M., Graversen, P. H., Dorflinger, T., Madsen, P. 0. and Bruskewitz, R. C.: Urinary bladder reformation: regeneration or dilatation? N eurourol. U rodyn., 6: 129, 1987. 15. Love, A. and Notley, R. G.: Vesical necrosis. J. R. Soc. Med., 71: 895, 1978. 16. Orikasa, S. and Tsuji, I.: Enlargement of contracted bladder by use of gelatin sponge bladder. J. Urol., 104: 107, 1970. 17. Lin, T. L., Wein, A. J., Gill, H. S. and Levin, R. M.: Functional effect of chronic ischemia on the rabbit urinary bladder. Neurourol. Urodyn, 7: 1, 1988. 18. Kato, K., Wein, A. J., Kitada, S., Haugaard, N. and Levin, R. M.: The functional effect of mild outlet obstruction on the rabbit urinary bladder. J. Urol. (in press)