Acute Biochemical and Functional Alterations in The Partially Obstructed Rabbit Urinary Bladder

0022-5347 /86/1364-1324$02.00/0 Vol. 136, December

THE JOURNAL OF UROLOGY

Copyright © 1986 by The Williams & Wilkins Co.

Printed in U.S.A.

ACUTE BIOCHEMICAL AND FUNCTIONAL ALTERATIONS IN THE PARTIALLY OBSTRUCTED RABBIT URINARY BLADDER S. BRUCE MALKOWICZ, ALAN J. WEIN, AHMAD ELBADAWI, KEITH VAN ARSDALEN, MICHAEL R. RUGGIERI AND ROBERT M. LEVIN* From the Division of Urology, University of Pennsylvania School of Medicine, the Philadelphia Veterans Administration Medical Center, Philadelphia, Pennsylvania, and the Department of Pathology, SUNY Health Science Center, Syracuse, New York

ABSTRACT

Rapid structural and functional alterations have been noted in several models of partial outlet obstruction. To better characterize the rapid progression of alterations, the partially obstructed urinary bladders of mature NZW male rabbits were studied at 1, 3, 5, 7 and 14 days of outlet obstruction with respect to muscarinic receptor density, DNA, RNA, lipid and hydroxyproline content. Functional characteristics were assessed by measuring the in vitro response of the whole bladder to cholinergic and field stimulation. Wet weight increased eight-fold by day 7, decreasing to four-fold at day 14. Receptor density decreased by 50% by day 1 and remained low throughout. Although DNA concentration varied only slightly from controls, RNA increased four-fold by day 7. Hydroxyproline concentration per mg. tissue decreased in the obstructed bladder, yet total hydroxyproline content of the obstructed bladder significantly increased. Total lipids increased significantly during day 3 through 7 and decreased by day 14. Cystometry revealed a large capacity low pressure system at day 1 which rapidly changed to a low compliance system of lesser volume by day 14. Bladder emptying was significantly impaired in all obstructed specimens. Additionally, electrical field stimulation was significantly less effective than cholinergic stimulation in effecting bladder emptying. The above findings suggest that rapid changes in biochemical parameters occur during the early stage of acute obstruction which may in part be secondary to metabolic or inflammatory alterations in the detrusor. It additionally suggests that the myogenic alterations in partial outlet obstruction are rapid and partially adaptive, while neurogenic alterations appear degenerative and display a lesser degree of short term adaptation. Bladder outlet obstruction is a condition which can produce significant alterations in detrusor structure and function. It is conventionally presumed that the sequential response to such stress is bladder hyperplasia and or hypertrophy followed by connective tissue replacement with functional decompensation. In many investigations employing models of subacute partial obstruction, morphologic changes have been rapid. 1- 7 These changes have been accompanied by poorly maintained intravesical pressure generation resulting in incomplete bladder emptying. It is thought that hypertrophy/hyperplasia may maintain peak force but does not provide the bladder with the ability to perform the additional work needed to void efficiently when obstructed. 8 These functional changes have been attributed to increased noncontractile elements of the bladder as well as inefficient incorporation of new muscle cells. 2•5 Additionally, changes in the properties of hypertrophic cells and decreased propagation of action potentials through detrusor muscle secondary to nexus disruption have been proposed. 9 Prior studies using a rabbit model of outlet obstruction have demonstrated dramatic changes in structure and function, including a ten-fold increase in bladder weight and a 75% reduction in bladder function. 6 These dramatic changes after one week of obstruction prompted further investigation regarding the initial alterations from the onset of obstruction. The purpose of this study was to investigate the sequential biochemical and in vitro functional Accepted for publication June 24, 1986. * Requests for reprints: Division of Urology, 3006 Ravdin Courtyard Bldg., Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA. Supported by grants from the Veterans Administration, NIH grants #R0-1-AM-2-6508-05, R0-1-AM-33559-01, AG 0620201 and the McCabe Fund.

response of the partially obstructed rabbit urinary bladder over the first two weeks of obstruction. This was accomplished by assaying muscarinic cholinergic receptor density, RNA, DNA, hydroxyproline and lipid content of bladder specimens at different stages of obstruction. Functional characteristics of this model were assessed by: 1) measuring in vitro cystometric characteristics and 2) determining in vitro response to pharmacologic (bethanechol) and electrical field stimulation. MATERIALS AND METHODS

Obstruction procedure. Mature male New Zealand White rabbits were sedated with an intramuscular injection (0.7 ml./ kg.) of a ketamine/xylazine mixture (29.2 mg./ml. ketamine, 8.3 mg./ml. xylazine). Surgical anesthesia was maintained with one ml. of 50 mg./ml. pentobarbital given over the course of surgery. Partial bladder outlet obstruction was created by securing a 2-0 silk ligature about the exposed, temporarily catheterized (8 F) urethra. After 1, 3, 5, 7 or 14 days the animals were anesthetized in the previously described manner. Each group consisted of between 12 and 16 animals. Each bladder was exposed through a midline incision, the ureters tied, and the bladder excised as low on the urethra as possible. The bladders were either used immediately for the whole-bladder studies, or the bladder was rapidly separated between base and body at the ureteral orifices and the bladder body separated into four strips and each strip frozen rapidly by immersion in liquid nitrogen. The frozen tissue was maintained under liquid nitrogen until utilized. Whole bladder preparation. 1• 11 The exposed bladder was emptied and the urethra cannulated with a saline-filled, electrodetipped catheter. The bladder was mounted in a 300 ml. isolated bath chamber containing Tyrode's solution equilibrated with 95% 02/5% CO2 and maintained at 37C.

1324

1325

RABBIT BLADDER OUTLET OBSTRUCTION

The catheter was connected by a three-way valve to either a saline-filled graduated burette which could be rapidly moved up or down, or a saline-filled bag resting on top of an electronic pan balance. The balance was mounted on a platform which could also be raised or lowered. Intravesical pressure was monitored with a Statham pressure transducer. With the valve open to the burette, the level of saline in the burette was set at the same level as the bladder base. At this point, the pressure was set to zero (volume= 0). After a 30 minute equilibrium period, the burette was raised in 10 ml. (6 cm.) increments. After each increment, the bladder was allowed to equilibrate as the saline entered (5 minutes). At equilibrium both the volume which entered the bladder and the pressure were recorded. By this method the volume-pressure relationship (cystometry) for the isolated bladder was determined. In order to determine if there is "tone" present in the urinary bladder, the isolated bladder is washed three times with calcium free tyrodes containing one mM EGTA at five minute intervals. At the end of this time, a second cystometrogram is generated. The difference between the cystometrogram in the presence and absence of calcium is a reflection of the "tone" present in the in vitro bladder. For pharmacological studies one of two systems was used: "Closed system": the bladder was filled to a specific volume by raising the burette as described above. After equilibration at the specific volume, the three-way valve was rotated so the bladder was "closed" to both the burette and the saline bag. The drug was then added to the bath in a cumulative manner at five minute intervals. In this system the volume of the bladder cannot change and thus the isometric (isovolumic) contraction was measured as a rise in intravesical pressure. "Expulsion system": the bladder was filled to a specific volume by raising the burette as previously described. After equilibration the burette valve was closed and the saline filled expulsion tube was connected to the system. The top of the saline reservoir bag was set equal to the saline level in the burette, thus maintaining the same pressure in the system. The bladder was opened to the saline bag so saline could move freely between the bladder and bag. Drugs were then added to the bath as previously described. In this system the bladder was allowed to respond to the drug with either an increase or decrease in volume. Thus the pressure in the static system was constant and the ability of the bladder to expel saline against a specific pressure could be determined. Changes in bladder volume were reflected in changes in the weight of the saline bag. The digital electronic balance was connected via an analog converter to a Grass polygraph so volume changes were recorded along with changes in intravesical pressure. For field stimulation, as previously mentioned, the bladder was catheterized on a specially designed electrode-tipped catheter. Two serially connected platinum electrodes were placed on either side of the catheterized bladder. The bladder was transmurally stimulated using biphasic pulses at 80 volts, one ms duration, 30 Hz. The signal was processed through a constant current power amplifier which was capable of maximally stimulating bladder contraction. '1H-QNB binding: muscarinic receptor determination. 12 The frozen tissue samples were rapidly weighed and homogenized at 200 mg./ml. in ice cold phosphate buffer (50 mM), pH 7.4, with a Brinkman Polytron homogenizer. The homogenate was centrifuged at 900 g for 10 minutes and the crude membrane pellet was discarded. The supernatant was then centrifuged at 30,000 g for 60 minutes. The pellet was resuspended in an equal volume of fresh buffer and used immediately for the binding assay. 250 µl. of membrane fraction was incubated with 3 H-QNB (quinuclidinyl benzilate) (0.10-20 nM) in the presence and absence of 10 µM atropine sulfate in a total volume of 350 µl. for 60 minutes at 25C. The reaction was stopped by the addition of four ml. of ice cold buffer and was rapidly filtered through a Whatman GFC glass fiber filter. The filter was washed with

three 10 mL portions of ice cold buffer. They were then placed in vials with five ml. of scintillation fluid. Radioactivity was determined by liquid scintillation spectrometry. Nonspecific binding was defined as 3 H-QNB binding in the presence of 10 µM atropine and accounted for 20% of total binding. Protein concentrations were determined by the method of Lowry. Tissue DNA and RNA and lipid concentration determinations. 13 Frozen bladder sections (250 mg.) were weighed and homogenized in 10 ml. of 10% trichloroacetic acid (TCA). One ml. of homogenate was neutralized and saved for protein assay. The remaining nine ml. was centrifuged at 10,000 g for 15 minutes, the supernatant was discarded and the pellet was washed with five ml. of TCA to remove acid soluble compounds. The washed pellet was resedimented by centrifugation. The pellet was extracted three times with 95% ethanol to remove lipids. This lipid-containing ethanol was filtered and dried under a vacuum at 50C and the total lipid content determined gravimetrically. The lipid-free pellet was suspended in 2.5 ml of 5% TCA and heated at 90C for 30 minutes with occasional stirring. The sediment was recovered by centrifugation at 12,000 g for 15 minutes. The sediment was extracted a second time with 2.5 ml. 5% TCA at 90C for 30 minutes and resedimented by centrifugation. The two supernates containing the nucleic acids were combined while the pellet was discarded. DNA assay. One ml. of supernate (in triplicate) was reacted with two ml. diphenylamine reagent and heated for 10 minutes in a boiling water bath. The blue color was read at 600 nM. Standard curves using purified DNA were run with each assay. RNA assay. 200 µl. of supernate (in triplicate) was reacted with 1.3 ml. of water and 1.5 ml. of Orcinol reagent and heated in a boiling water bath for 20 minutes. The color was 1·ead at 660 nM. Standard curves using purified RNA were run with each assay. Tissue collagen determination. 14 100 mg. of frozen tissue samples were homogenized in 5 ml. distilled water. One ml. was stored for protein assay. Four ml. of homogenate was mixed with four ml. of 12 M HCL. The mixture was then sonicated to insure total reaction. Two ml. aliquots were hydrolyzed under pressure (30 psi) at 250F for five hours. The hydrolysate was then dried at 55C under a vacuum. The dried hydrolyzed collagen was dissolved in four ml. buffer (33.25 gm. citric acid, 8 ml. glacial acetic acid, 80 gm. sodium acetate, 22.75 gm. sodium hydroxide, 200 ml. n-propanol in 1000 ml, pH 6.4). Two ml. aliquots of each sample were reacted with one ml. chloramine T reagent for 25 minutes and then one ml. of aldehyde perchloric acid reagent for 15 minutes at 60C. The color was read at 550 nM. Standard curves of hydroxyproline were run with each assay. Statistical significance was determined by analysis of variance. All values presented in the results represent the mean +/- standard error of the mean of six to ten independent determinations. A probability level less than 0.05 was required for statistical significance. All materials were of the highest purity available, purchased through regular commercial sources. RESULTS

At the time of sacrifice the bladders were visually inspected and weighed. The wet weight of the specimens is displayed in figure 1. A progressive eight-fold increase in wet weight was noted between day one and day seven. The most rapid progression in weight gain was seen between day three and day five. A significant decrease in weight to four times that of controls was observed at day fourteen. The percentage of protein (per wet weight) was 10.58 +/- 1.6% for both control and obstructed tissue and remained constant throughout the study demonstrating that the increase in bladder mass was not solely due to edema. On gross inspection the bladders obstructed for one day displayed an increased intravesical capacity and--'an attenuated

1326

MALKOWICZ AND ASSOCIATES

vesical wall. By three days gross concentric thickening was noted, which progressed over the next several days. At fourteen days the specimens were larger than the controls and had a thicker vesical wall. Biochemical parameters. The concentrations of a variety of intracellular components are presented in table 1. Muscarinic cholinergic receptors. Practically no change was noted in our estimated receptor number over the first three days of obstruction, yet by one week the estimated number of receptors per bladder had more than doubled (six pmoles vs. 13 pmoles) and remained at this level at 14 days. Receptor density measured in fmoles per mg. protein dropped dramatically over the first week to almost 25% (10 vs. 38 fmoles per mg. protein) of control values, but recovered to approximately 50% of control values (21 fmoles/mg. protein) by 14 days. There was no change in the Kd of QNB binding within the various groups; the average Kd was .36 +/- .11 nM. Nucleic acid concentration. A 50% increase in DNA concentration over control values was noted at 1 and 3 days (10 vs. 15 µg./mg. protein), yet this figure returned to control values by day seven and remained so at day 14. RNA concentration increased dramatically by day 3 and day 7. It subsequently returned to control values by day 14. Lipid content. Lipid concentration was significantly increased at one day of obstruction (28 mg. vs. 47 mg. per gram tissue) and was markedly increased by day three (87 mg./gm. tissue) and seven (55 mg./gm. tissue). By day 14, lipids had decreased to about 50% of control values (15.6 mg./gm. tissue). Hydroxyproline concentration. The hydroxyproline content

Effect of Obstruction on Bladder Weight

of the bladder increased progressively through day 7. Although the hydroxyproline content at day 14 was lower than at day 7, it was still significantly above the control value. The hydroxyproline concentration (per mg. of tissue) decreased in the obstructed bladders. Functional parameters. In vitro cystometrograms for control and obstructed bladders are shown in figure 2. Each group contained six to eight individual animals. After one day of obstruction, a long flat filling pattern was noted. By day 7 a steeper filling limb was seen with a right shift of the terminal limb with respect to controls. At fourteen days a marked increase in slope of the filling limb was evident and a left shift of the terminal limb was observed. Data for days 3 and 5 displayed a stepwise progression from one to seven days but was omitted for sake of clarity. Figure 3A-C presents the effect of EGTA on the in vitro cystometrogram of control, one week, and two week obstructed animals. The nonobstructed bladders responded to EGTA with a significant shift to the right. During the plateau phase of the EGTA curve, the intravesical pressure for a specific volume was approximately 50% of the control pressure. There was no difference in the bladder capacity or the maximum pressure. The one-week obstructed bladder showed a similar response to EGTA. EGTA shifted the curve to the right. Although the plateau pressure was significantly reduced, no change in either bladder capacity or maximum pressure was noted. EGTA had no effect on the cystometry of the fourteen day obstructed bladder. Figure 4 displays the peak intravesical pressure response of the specimens to bethanechol or field stimulation. It should be noted that the response to either stimulus was most depressed after the initial 24 hours of obstruction. The pressure response to field stimulation was significantly reduced throughout the

12.0

EFFECT OF OBSTRUCTION ON IN VITRO CYSTOMETRIC CURVES 10.0

-;;; E !:)

0

8.0

E s

Cl

·;;;

s:

6.0

~

oi

:,

,::, ,::,

.,

ii'i

60

£

:i::

50

40

(/) (/)

4.0

(!)

2.0

a:

30


20

(!)

> ~

£

10

Days of Obstruction

20

FIG. 1. Wet weight of control and obstructed bladders. Histograms are presented for control, 1, 3, 5, 7 and 14 days of obstruction. Asterisk denotes difference significant from controls. Each histogram represents mean+/- S.E.M. of 6-10 separate determinations.

30

40

50

60

70

80

90

100

lntravesical Volume (mis.)

FIG. 2. In vitro cystometrogram curves. Each cystometrogram curve represents average curve for 6-8 individual curves.

TABLE 1. Biochemical evaluation of the obstructed urinary bladder Biochemical Component Estimated receptor number (pmoles per bladder) Receptor density (fmoles/mg. protein) DNA concentration (u gm./mg. protein) RNA concentration (u gm./mg. protein) Total lipids (mg./gm. tissue) Hydroxyproline (mg./bladder) Hydroxyproline (µg.jmg. tissue)

Control

1 Day

6.1 (.3)

5.8 (.4)

Days of Obstruction 3 Days 5 Days

7 Days

14 Days

6.3 (.6)*

10.2 (1.2)*

11.8 (2.2)*

12.1 (1.1)*

(2.5)*

10.8 (2.3)*

11.2 (2.1)*

10.1 (1.3)*

21.6 (2.1)*

10.1 (1.4)

15.2 (2.0)*

16.3 (2.1)*

13.3 (1.6)

11.8 (1.5)

9.6 (1.1)

7.4 (1.1)

8.0 (1.2)

21.3 (3.2)*

23.4 (2.8)*

26.4 (2.8)*

8.4 (1.0)

29.5 (3.4) 8.7 (.9) 5.8 (.4)

48.6 (5.1)* 8.2 (1.1) 2.7 (3.1)*

86.4 (9.5)* 19.0 (2.1)* 3.8 (.27)*

71.7 (4.6)* 39.0 (4.5)* 3.9 (.23)*

57.6 (6.2)* 45 (6.1)* 4.1 (.35)*

15.6 (3.4) 21.5 (3.4)* 4.3 (.32)*

38

(3.3)

18

Each value represents the mean+/- (S.E.) for 6-8 individual preparations.*= significantly different from control (p <0.5).

1327

RABBIT BLli,.DDEFt 01J'TLET OBSTRUCTION

EFFE(;T OF EGT,&l~ ON !N-ViTRO CY"STOMETR\f

com/

'

Ca++

, / EGTA

-----

,/

60

40

20

80

100

60 7 DAY

, / ossrnm,o, 40

ca++

20

,./"EGTA

60 14 DAY OBSTRUCTION ;

40

;

;

;

,

/ ca++

20

,,,-"

/,,,,,,,,,,,·"",,~GT A ,,,," 20

80

60

40

100

lntravesical Volume (mis.) FIG. 3. Effect of EGTA on in vitro cystometry. Each curve repre· sents mean of 4 individual bladders. A, curves for control bladder; B, curves for one week obstructed; C, curves for 14 day obstructed bladder.

first seven of obstruction, seven days the peak pressure response to bethanechol stimulation was similar to that of controls. After fourteen days of obstruction the peak pressure response to field and pharmacologic stimulation was similar to that of controls. It should be noted that this chart presents only the peak pressure obtained and does not show the entire contractile response. Figure 5 depicts the ability of the in vitro preparation to expel intravesical saline. An intravesical volume of 15 ml. was used in these studies. The control response to bethanechol and field stimulation was similar and displayed virtually complete emptying. As in the case of pressure generation, the functional properties of the preparation were most impaired after the first day of obstruction. The functional (ability to empty) response to bethanechol improved over the remaining six days yet remained significantly depressed (65% vs 95%). More importantly, the functional response to field stimulation was dramatically impaired during the first seven days of obstruction (25% vs 95% ). At this time the bladder's response to cholinergic stimulation was significantly greater than that seen with field stimulation. At fourteen days, responses to field and pharmacologic stimulation were still significantly impaired, although the field stimulation response had improved from 25% to 40% of volume expulsion. An intravesical volume of fifteen ml. was utilized because of the relative ease with which the bladder can empty this volume. Under normal conditions, the rabbit bladder is capable of effectively emptying volumes greater than 50 ml. Figure 6 displays the ability of bethanechol to empty 15, 30 and 45 ml. The control bladders emptied over 96% of the volume. The seven day obstructed bladder emptied 62, 43 and 25% respectively. Thus utilizing higher volumes in these studies would increase the apparent functional defects present in the obstructed bladders. Pretreatment of the bladder preparation with tetrodotoxin completely inhibited the bladder response to electrical stimulation yet had no effect on cholinergic stimulation (data not shown). This indicates that electrical stimulation acts through the release of excitatory neurotransmitter and not through direct muscle stimulation. Bethanechol acts postsynaptically on the muscarinic cholinergic receptor. DISCUSSION

Experimental models of bladder outlet obstruction vary with respect to the species employed and the degree or duration of Ertect of Obstruction on lntravesical Pressure (EE) Field Stimulation (Bl Bethanechol (0.5mM)

Eflect of Obstruction on Volume El(pelled

25

0 -:i

E'

100-

(B) Bethanechol (0.5mM)

20·

°O

.:3

~

:" :,

a.

"'"'

t

X

15

w

"'

a:

60

E

:,

0

10

>

0

(!)

>

;

80

"'

(!)

oi () ·;;;

(EE) Field Stimulation

40

°#

5

20

C

Control

3

5

7

14

Control

3

5

7

14

Days of Obstruction

Days of Obstruction

FIG. 4. Peak intravesical pressure response to bethanechol and field stimulation. Each histogram represents maximal response to either 200 µM bethanechol (B) or field stimulation (EE) using 80 volts, 1 ms duration. Asterisk represents significant difference from controls. Each histogram represents mean +/- S.E.M. of 6-10 separate determinations.

FIG. 5. Functional response to bethanechol and field stimulation. Each histogram represents maximal response to either 200 µM bethanechol (B) or field stimulation (EE) using 80 volts, 1 ms duration. Asterisk represents significant difference from controls. Each histogram represents mean+/- S.E.M. of 6-10 separate determinations.

1328

MALKOWICZ AND ASSOCIATES C=Control OB= 7 Day Obstructed

100

'O

~

80

ai

a. X

w (I)

60

E

::,

0

>

0

40

~

20

C OB 15 ml

C

OB

30 ml

C OB 45 ml

lntravesical Volume

FIG. 6. Functional response to bethanechol at 15, 30 and 45 ml. intravesical volume. Each histogram represents maximal response to 200 µM bethanechol (B) for either control or 7 day obstructed bladders. Asterisk represents significant difference from controls. Each histogram represents mean+/- S.E.M. of 4-6 separate determinations.

the obstruction. Even so, certain general response patterns exist. 1- 7, 15 An initial dilatation of the bladder is followed by a concentric bladder wall thickening. In previously described models this has been due to variable degrees of collagen deposition along with smooth muscle hyperplasia/hypertrophy. Further morphologic changes associated with longer periods of obstruction vary with the particular model employed. 2• 5 In this model the bladder was grossly dilated at one day but mounted a strong adaptive response to the obstruction, with the greatest relative increase in mass occurring between days 3 to 5. This is consistent with the rat model of Uvelius in which the rate of bladder mass increase was greatest after three days of obstruction. 5 During the period of obstruction the estimated number of muscarinic cholinergic receptors increased over time (fig. 1). During the earliest period of obstruction (3 to 7 days) the receptor density per milligram protein is significantly reduced. Even at day 14 the density is half that of controls. Considering the recent demonstration that spare receptors do not exist in the urinary bladder, 16• 17 the decreased muscarinic receptor density will no doubt contribute to the decreased functional response to both bethanechol and field stimulation. The Kd for QNB binding was .36 nM +/- .11 nM for all samples studied. This value is similar to that reported by Nilvebrant et al. for QNB binding to human and guinea pig urinary bladders. 17• 18 The marked initial bladder dilation with accompanying muscular and neuronal damage will also significantly contribute to the observed functional and contractile pathology. Preliminary histological studies have demonstrated that an acute inflammatory response occurs in the obstructed tissue during the first few days. This inflammatory response may account in part for the rapid changes in DNA, RNA arid lipids observed in the first few days following obstruction. The histological and morphometric responses of the bladder to obstruction will be the subject of subsequent studies. The time course of the increase in intracellular RNA correlates well with the observed increase in bladder mass and the presumed protein synthesis. This increase in intracellular RNA has also been reported in other obstruction models and is corroborated by the demonstration of abundant endoplasmic reticulum observed in electron micrographs of the acutely hypertrophied detrusor. 5 • 19 Without thorough histological evalu-

ation, we cannot give specific details about the morphology of the increased bladder mass. Although edema is probably present, the most likely explanation for the majority of the increased mass is smooth muscle hypertrophy. This would be consistent with the observed increase in the functional response to bethanechol observed between days 3 through 14. The increase in lipid content during the three-to-seven day period is quite striking. This increased lipid may result in part from both an acute inflammatory reaction and the fatty degeneration of muscle and neuronal elements which has been observed in parallel morphological studies. 19 Although the total hydroxyproline content of the obstructed bladders increased, the concentration as a percentage of total bladder protein dropped significantly. These results indicate the major portion of the increased bladder mass is not collagen. Further morphometric studies will be required to determine the composition of the increased bladder mass. This finding, which is similar to the. studies by Uvelius and Mattiasson in rats, 20 may be explained in part by the progressive muscle hypertrophy during the acute phase of obstruction which masks the progression of collagen deposition in the obstructed bladder. These findings are similar to results from detrusor cadaver specimens of obstructed males. 21 Structural changes in the bladder are inferred by the nature of the cystometrograms generated by the control and obstructed tissues. The cystometrograms of the initially obstructed tissue are consistent with a high volume structure compliant to the degree of filling performed (bladder dilation). Over seven days the filling limb develops an increased slope, indicating a decrease in the elastic property of the muscle. The terminal limb is not dissimilar to controls but displays a right shift, implying a larger bladder volume. At fourteen days however, the filling limb is quite steep and a left shift of the terminal arm is noted. This indicates a decrease in volume and a further decrease in compliance which may be due to increased connective tissue content resulting in decreased elastic properties of the hypertrophied muscle. EGTA effectively separates the active vs. passive properties of the bladder smooth muscle in relation to the cystometrograms. The control curves were shifted to the right, although maximum capacity was unchanged. The intravesical pressure monitored during the plateau phase of the cystometrogram was approximately 50% of the pressure in the presence of calcium; thus there appears to be significant active "tone" in the control bladders. The one week obstructed bladders demonstrated a similar amount of tone, whereas the two week obstructed bladders displayed virtually no tone. EGTA in the fourteen day obstructions had no effect on the cystometry curves. This is consistent with the view that the steep cystometric curve is due to a loss of bladder compliance and not related to a increase in bladder tone. The functional response of the bladder consists of two phases: the rapid rise of intravesical pressure followed by a prolonged plateau of increased pressure. The ability of the bladder to empty is directly related to the plateau phase rather than the initial response. 10• 11 The impairment of the plateau phase is best represented in figures 4 and 5, which demonstrate that bladder emptying is significantly impaired at all volumes and in all obstructed tissue regardless of the stimulus employed. Of particular importance is the difference in response seen between electrical and pharmacological stimulation. It is noted from day 3 through day 7 that the response to electrical stimulation is significantly more impaired than the response to bethanechol. A similar relationship is seen at fourteen days, but to a lesser extent. This finding is consistent with electron micrographic studies which display a profound decrease in the density of innervation, degenerative axonal profiles, and very few neuroeffector junctions in the obstructed tissue (days 1, 3, 5 and 7). 19 By day fourteen of obstruction some nerve regener-

ftABB!T BL/d)DER fJl_JTLET OBSTRlJCTIO:N

ation 1s present. In studie£ on. hur.o.a1:. tr2.becu1ated bladder tissue vvas that occur. 22 ·2:1 smooth muscle to acute obstruction is more efficient at one week the neurologic adaption. Although the hypertrophic/hyperplastic tissue is impaired, it is present in sufficient quantity to effect a moderately efficient volume expulsion when the end organ is directly stimulated at the receptor level by a parasympathomimetic agent. Normal neuropathways which would release acetylcholine at the effector junctions are destroyed or impaired by the acute obstruction. Therefore excitation of this effector limb by electrical stimulation results in a poor response during this early obstructive period. The above data suggest that during the early stage of acute partial obstruction the neuropathic contribution to dysfunction may outweigh any myogenic adaption. It is tempting to speculate that voiding function during this stage might be augmented by administration of parasympathomimetic agents. The increase in intravesical pressure produced by these agents is not however, analogous to the potentiation of a coordinated micturition reflex. The amount of bladder hypertrophy observed seems to be inversely proportional to the functional activity of the bladder. The in vivo functional activity of the bladder would be represented best by the in vitro response to field stimulation, since both activities depend on the release of excitatory transmitters. The greatest degree of hypertrophy (increase in bladder mass) was observed during the first seven days of obstruction, at a time when the bladder response to field stimulation was 25% of control. Over the next seven days days) the response to field stimulation significantly improved, simultaneous with both a decrease in RNA concentration and a decrease in bladder mass. It may be that the bladder body is in a continual state of flux which will allow it to rapidly adapt to alterations in the functional activity of the bladder. Acknowledgment. We would like to thank Brenda Barasha, Debra Moore, and Sheila Levin for their expert technical assistance. REFERENCES l. Arbuckle, C. D. Jr.: Experimental bladder neck obstruction. I.

Incidence of vesicoureteral reflux, upper tract dilation, and urinary infection in rabbits. Invest. Urol., 1: 173, 1963. 2. Brent, L. and Stephens, F. D.: The response of smooth muscle cells in the rabbit urinary bladder to outflow obstruction. Invest. Urol., 12: 494, 1975. 3. Mayo, M. E. and Hinman, F.: Structure and function of the rabbit bladder altered by chronic obstruction or cystitis. Invest. Urol., 14: 6, 1975. 4. Mattiasson, A. and Uvelius, B.: Changes in contractile properties in hypertrophic rat urinary bladder. J. UroL, 128: 1340, 1982.

5. Uveliusj B. Persson, Lo and JVIattiasson, A.: Smooth muscle cell and "'""'"""·"'"· in the rat detrusor after short tin,e outflow J. Urol., 131: 173, 1984. 6. Levin, R. M., High, J. and Wein, A. J.: The effect of short-term obstruction on urinary biadder function in the rabbit. J. Urol., 132: 789, 1984. 7. Magasi, P., Cronti, A. and Inozinko, B.: Beitrage Zur Blasen Wamtregeneration. Z. Urol. Nephrol., 62: 209, 1969. 8. Schafer, W.: Detrusor as the energy source of micturition. In: Benign Prostatic Hypertrophy. Edited by Hinman, F. Jr., Springer-Verlag, p. 450, 1983. 9. Dewey, M_ M.: The anatomic basis of propagation in smooth muscle. Gastroenterology, 49: 395, 1965. 10. Levin, R. M. and Wein, A. J.: Response of the in vitro whole bladder (rabbit) preparation to autonomic agonists. J. Urol., 128: 1087, 1982. 11. Levin, R. M., Brendler, K. and Wein, A. J.: Comparative pharmacological response of an in vitro whole bladder preparation (rabbit) with the response of isolated smooth muscle strips. Urology, 30: 377, 1983. 12. Levin, R. M., Shofer, F., Wein, A. J., Atta, M. A. and Elbadawi, A.: Cholinergic, adrenergic, and purinergic response of sequential strips of rabbit urinary bladder. J. Pharmacol. Exp. Ther., 212: 530, 1980. 13. Schneider, W. C.: Determination of nucleic acids in tissues by pentose analysis. In: Methods in Enzymology, vol. 3, Academic Press, New York, 1978. 14. Edwards, C. A. and O'Brien Jr. W. D.: Modified assay for determination of hydroxyproline in a tissue hydrolysate. C!in. Chim. Acta, 104: 161, 1980. 15. Levin, R. M., Malkowicz, S. B. and Wein, A. J.: Recovery from short term obstruction of the rabbit urinary bladder. J. Urol., 134: 388, 1985. 16. Levin, R. M., High, J. and Wein, A. J.: Evidence against the presence of spare muscarinic receptors in the rabbit urinary bladder. Neurourol. Urodynam., 2: 317, 1984. 17. Nilvebrant, L., Andersson, K. E. and Mattiasson, A.: Characterization of the muscarinic cholinoceptors in the human detrusor. J. Urol., 134: 418, 1985. 18. Nilvebrant, L. and Sparf, B.: Muscarinic receptor binding in guinea pig urinary bladder. Acta Pharmacol. Toxicol., 52: 30, 1983. 19. Elbadawi, A., Atta, M. A., Levin, R M., Malkowicz, S. B. and Wein, A. J.: Reversible neuromuscular changes in the rabbit detrusor following one week of outlet obstruction. J. Urol., 133: Abstract 248, 1985. 20. Uvelius, B. and Mattiasson, A,: Collagen content in the rat urinary bladder subjected to infravesical outflow obstruction. J. Urol., 132: 587, 1984. 21. Susset, J. G., Servot-Vigiuer, D., Lamey, F., Madernos, P. and Black, R.: Collagen in 155 human bladders. Invest. Ural., 16: 204, 1978. 22. Gosling, J. A. and Dixon, J. S.: The structure and innervation of trabecu!ated detrusor smooth muscle. Proceedings of the IX Annual of the 1979. 23. Gosling, J. A. J. · Structure of trabeculated detrusor smooth muscle in case of prostatic hypertrophy. UroL Int., 35: 351, 1980. 1