Cell Tissue Res (1990) 262:67-79

Researr 9 Springer-Verlag 1990

Urinary bladder of rat: fine structure of normal and hypertrophic musculature Giorgio Gabella and Bengt Uvelius Department of Anatomy, University College London, Gower Street, London, UK; Department of Urology, University Hospital, Lund, Sweden Accepted May 28, 1990

Summary. The fine structure of the muscle of the urinary bladder in female rats is similar to that of other visceral muscles, although it is arranged in bundles of variable length, cross-section and orientation, forming a meshwork. When distended, the musculature is 100-120 ktm thick, with some variation and occasional discontinuity. Extended areas of cell-to-cell apposition with uniform intercellular space occur between muscle cells, whereas attachment plaques for mechanical coupling are less common than in other visceral muscles. There are no gap junctions between muscle cells. Many bundles of microfilaments and small elastic fibres run between the muscle cells. After chronic partial obstruction of the urethra, the bladder enlarges and is about 15 times heavier, but has the same shape as in controls; the growth is mainly accounted for by muscle hypertrophy. The outer surface of the hypertrophic bladder is increased 6-fold over the controls; the muscle is increased 3-fold in thickness, and is more compact. Mitoses are not found, but there is a massive increase in muscle cell size. There is a modest decrease in percentage volume of mitochondria, an increase in sarcoplasmic reticulum, and no appreciable change in the pattern o f myofilaments. Gap junctions between hypertrophic muscle cells are virtually absent. Intramuscular nerve fibres and vesicle-containing varicosities appear as c o m m o n in the hypertrophic muscle as in controls. There is no infiltration of the muscle by connective tissue and no significant occurrence of muscle cell death. Key words: Urinary b l a d d e r - Smooth muscle - Hypertrophy - Ultrastructure - Rat

We have studied the ultrastructure of the urinary bladder after experimentally inducing outflow obstruction. Send offprint requests to: Dr. Giorgio Gabella, Department of Anatomy, University College London, Gower Street, London WCIE 6BT, UK

Under these circumstances, the bladder muscle cells hypertrophy; this is well established from previous experiments on animals and also occurs in man as a result of benign prostatic hypertrophy in aged males (Gilpin et al. 1985). The response of the organ is undoubtedly complex, probably varying with age, clinical history, and animal species; in the case of animal experiments, it is also probably affected by the type of surgery used. We are thus aware that great caution is needed when making comparisons with other studies. However, the hypertrophic response that we have obtained in rats seems consistent, thus providing the possibility of a more systematic and partly quantitative approach to the problem. To this end, we have also analyzed the structure of the control musculature in detail. There are very few and limited studies of the fine structure of smooth muscle in the urinary bladder of mammals (Nagasawa and Suzuki 1967; Gosling and Dixon 1975; Larsen 1977) in comparison with the numerous investigations on muscles of blood vessels and the intestine. Whereas the basic cytological aspects of bladder muscle are similar to those of other visceral muscles, and are therefore unremarkable, it is not clear whether characteristic structural features are also present. It would be strange if there were none, since distinctive features have been found in every smooth muscle studied (Gabella 1981). With respect to both the control and the hypertrophic muscle of the bladder, we have made comparisons with the normal structure and hypertrophic changes of other visceral muscles, especially in the intestine. In the latter, on the oral side of a chronic partial obstruction, the musculature undergoes a massive hypertrophy, accompanied by neuronal hypertrophy (Gabella 1987 a). The microscopic appearance of a smooth muscle is greatly affected by the degree of distension or by the occurrence of contraction at the time of fixation. A technical difficulty of previous investigations on the bladder has been the absence of standard conditions of muscle distension, a drawback even more serious in experiments with muscle strips excised from the wall. One of the

68 objectives o f the p r e s e n t i n v e s t i g a t i o n has been to overc o m e this p r o b l e m a n d to establish s t a n d a r d a n d c o n t r o l l e d c o n d i t i o n s o f d i s t e n s i o n , i n d e p e n d e n t o f the degree o f b l a d d e r e n l a r g e m e n t .

processes for thin-section electron microscopy and for freeze-fracture, as described above.

Results Materials and methods Normal bladder. Adult female albino rats were used (body weight : 225-250 g). After several preliminary experiments, 9 animals were selected for electron microscopy. Five animals were killed by cervical dislocation. The abdomen was opened and the bladder was dissected out with a segment of the urethra, freed of adjacent structures, emptied of fluid and weighed. A ligature of cotton thread was tied around an indwelling needle in the urethra near the neck of the bladder, and fluid was injected: I ml per 100 mg bladder wet weight. The bladder was then immersed in fixative and after a few minutes cut in half; it was kept in fixative for several days (while being mailed from Lund to London) and then embedded in resin. Another 4 rats were killed with an overdose of anaesthetic. The abdomen was opened along the midline with an incision from the sternum to the pelvis. A bulbed needle was inserted into the urethra, through its opening at the perineum, and into the bladder. The exposed outer surface of the bladder was kept moist by dripping oxygenated Krebs solution at room temperature. The bladder was emptied of urine through the needle, refilled with Krebs solution into it and re-emptied several times; it was then distended with Krebs solution, by injecting an amount proportional to its estimated weight (see above), and immersed in fixative for 2-18 h prior to embedding in resin. During fixation, the bladder was cut into strips 1-3 mm wide with known orientation and position in the bladder. The fixative was 5% glutaraldehyde in 100 mM Na cacodylate buffer, pH 7.4, at room temperature. After primary fixation, the specimens were thoroughly washed in buffer and post-fixed in 1% osmium tetroxide in the same buffer for 1-2 h, block-stained in an aqueous saturated solution of uranyl acetate, dehydrated in ethanol and epoxy-propane, and embedded in Araldite. The same blocks were used for semithin sections (stained with thionine or toluidine blue) or for ultrathin sections (stained with uranyl acetate and lead citrate). Some of the specimens of bladder wall fixed in glutaraldehyde were trimmed down to squares of 1 x I mm and were prepared for freeze-fracture. After a wash in cacodylate buffer, they were glycerinated for 2 h, mounted on gold-nickel alloy studs and frozen in Freon 22 cooled to -- 150~ C with liquid nitrogen. The specimens were fractured in a freeze-fracturer, operating at 3-4 x 10 -7 torr, and rotary shadowed with the source at an angle of 25 ~. The replicated tissue was treated with methanolic KOH and digested in sodium hypochlorite. The replicas were cleaned in distilled water and mounted on uncoated copper grids.

Hypertrophic bladder. After several preliminary experiments, 9 female Sprague-Dawley rats were used (age about 4 months, body weight at time of surgery 200-225 g) to study bladder hypertrophy. They were anaesthetized with methohexital sodium (Brietal), 70 mg/kg body weight i.p. The abdominal cavity was opened by a lower midline incision; urethral stenosis was induced according to our standard technique (Mattiasson and Uvelius 1982) by placing a metal rod of 1 mm diameter along the initial portion of the urethra and then tying a ligature with silk thread n. 4-0. The rod was then pulled out and the ligature left in situ. The abdominal wall was sutured with Dexon stitches. After 10 weeks the rats, weighing by then 270-280 g, were killed by cervical dislocation and their bladder with a small segment of the urethra attached was excised, emptied and weighed. The bladder was then cannulated, and oxygenated Krebs solution was injected into the lumen; the amount injected was based on the bladder weight, viz. I ml for every 100 rag. The bladder was then immersed in fixative and

Normal bladder T h e w e i g h t o f the r a t u r i n a r y b l a d d e r w h e n e m p t y was a b o u t 70 m g ; we d i s t e n d e d it w i t h 0.7 m l K r e b s s o l u t i o n b e f o r e fixation. A b o u t 1.4 ml c o u l d be injected i n t o the b l a d d e r b e f o r e it burst. T h e d i s t e n d e d b l a d d e r was a p p r o x i m a t e l y o v o i d , w i t h the d o r s a l wall slightly less c o n vex t h a n the v e n t r a l wall. T h e g r e a t e r d i a m e t e r was in the c r a n i o - c a u d a l axis ( ~ 14 m m ) ; the lesser d i a m e t e r s were o n the t r a n s v e r s e ( ~ 9.5 m m ) a n d the v e n t r o - d o r s a l axes ( ~ 9.0 ram), a n d the r a t i o b e t w e e n g r e a t e r a n d lesser d i a m e t e r was ~ 1 . 5 . B o t h the i n n e r a n d the o u t e r surfaces o f the b l a d d e r were s m o o t h .

Smooth muscle in normal bladder T h e m u s c u l a t u r e c o n s i s t e d o f b u n d l e s t h a t were h e t e r o g e n e o u s in size, l e n g t h a n d o r i e n t a t i o n . A d j a c e n t b u n d l e s r a n f r o m n e a r l y p a r a l l e l to o r t h o g o n a l to o n e a n o t h e r . O n the v e n t r a l a s p e c t o f the b l a d d e r , there was a large c o m p o n e n t o f l o n g i t u d i n a l m u s c u l a t u r e (i.e., r u n n i n g f r o m the c r a n i a l to the c a u d a l p o l e o f the b l a d d e r ) , w h e r e a s d o r s a l l y , a large sheet-like c o m p o n e n t o f circular m u s c u l a t u r e c o u l d be f o u n d . O n the sides o f the b l a d d e r a n d at the c r a n i a l pole, the muscle b u n d l e s crisscrossed and ran without a preferred orientation. Under the s t a n d a r d c o n d i t i o n o f d i s t e n s i o n used, the m u s c u l a ture m e a s u r e d 100-120 gm, b u t t h i c k e r a r e a s were present t o g e t h e r with small a r e a s o f the wall d e v o i d o f m u s cle. M u s c l e cells r a n p a r a l l e l a n d closely p a c k e d w i t h i n each b u n d l e (Fig. 1). O n l y u n i n u c l e a t e d m u s c l e cells were f o u n d . A few c o n v o l u t e d o r s t a r - s h a p e d profiles r e p r e s e n t e d t e r m i n a l p o r t i o n s o f m u s c l e cells with a n e x t e n d e d i n s e r t i o n o n t o the s t r o m a (Fig. 2B). M u s c l e cells were s i m i l a r in fine s t r u c t u r e to t h o s e o f o t h e r viscera, e.g. the g u t (Fig. 1). Small l a m i n a r o r c y l i n d r i c a l i n v a g i n a t i o n s o f the cell surface, a l t h o u g h n o t a p r o m i n e n t feature, were c h a r a c t e r i s t i c o f this m u s c l e (Fig. 1). C a v e o l a e were d i s t r i b u t e d in i r r e g u l a r rows o v e r the cell surface a n d were i d e n t i c a l in size a n d s h a p e to t h o s e o f o t h e r s m o o t h muscles. M i t o c h o n d r i a o c c u p i e d 4 % - 6 % o f the c y t o p l a s m v o l u m e . T h e r e were n u m e r o u s sacs o f s m o o t h s a r c o p l a s m i c r e t i c u l u m , m a n y lying p a r allel a n d close to the cell m e m b r a n e (Fig. 2 A ) . Intermediate cell-to-cell j u n c t i o n s (attachment p l a q u e s ) were less c o m m o n t h a n in i n t e s t i n a l m u s c l e cells, as the m a j o r i t y o f dense b a n d s d i d n o t m a t c h each o t h e r in a d j a c e n t cells (Fig. 1). T h e r e were extensive areas o f m e m b r a n e - t o - m e m b r a n e a p p o s i t i o n w i t h a g a p o c c u p i e d b y a m o r p h o u s m a t e r i a l o f faint e l e c t r o n density (Fig. 2 A ) . L a m i n a r a n d finger-like processes a b u t ting o n a d j a c e n t cells were c o m m o n . G a p j u n c t i o n s between m u s c l e cells were n o t f o u n d , either in thin sections o r in freeze-fracture replicas.

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Fig. 1. Electron micrograph showing the main cytological features of the musculature in the rat urinary bladder; a an area of extensive apposition between two muscle cells, with apparent fusion of the basal laminae; c caveolae; e elastic fibres;f collagen fibrils; i small

invaginations of the cell surface; m mitochondrion ; n unmyelinated nerve fibre, associated with small process of Schwann cell and mainly containing small clear vesicles; r sarcoplasmic reticulum. Bar: 1 Jim

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71

Fig. 3A-C. Transverse sections of Araldite-embedded bladders photographed with phase-contrast microscopy. A Control bladder. The mucosa is at the top, the serosa at the bottom. Arrows point to smooth muscle bundles. Bar: 100 gin. B Bundle of hypertrophic musculature. Note the enlarged cells, the occurrence of numerous

C o l l a g e n fibrils, m e a s u r i n g 30-50 n m in diameter, lay between muscle cells (Figs. 1, 2); m i c r o f i l a m e n t s , a b o u t 10 n m in diameter, a n d m a n y small elastic fibres r a n b e t w e e n muscle cells (Fig. 2 B). B u n d l e s o f collagen fibrils r a n b e t w e e n the muscle b u n d l e s a n d were a b u n -

Fig. 2A-F. The muscle cells are packed with myofilaments and also display dense bands (b), caveolae (c), mitochondria (m), and sarcoplasmic reticulum (r). Various forms of contact between adjacent cells are seen. Collagen fibrils (J) lie in the intercellular space. B The highly corrugated profile in the centre is the terminal region of a muscle cell; e elastic fibres; n unmyelinated nerve fibre. C Longitudinal section of a varicosity lying close to a muscle cell. The junctional gap measures about 20 nm. D Three single axons (varicosities packed with small clear vesicles and devoid of Schwann cell wrapping) occur close to muscle cells. E A varicosity packed with small clear vesicles lies at a considerable distance from the nearest muscle cell; f collagen fibrils; m mitochondrion; s Schwann cell process. F Another varicosity with vesicles and mitochondfia is found in intimate relation with a muscle cell. The gap between the two measures about 30 nm and is occupied by a single basal lamina. Arrow another axon tunnelling through a muscle cell; s Schwann cell process. Bars: I gm

deep invaginations of the cell membrane, and the presence of blood vessels. Bar: 50 gm. C Hypertrophic bladder wall with the same orientation as in A (same magnification); m mucosa; s serosa. Note the thick compact musculature (arrows). Bar: I00 gm

d a n t b e n e a t h the e p i t h e l i u m a n d in the serosa, where the collagen fibrils m e a s u r e d 4 0 - 1 0 0 n m . W i t h i n the muscle, there was a small n u m b e r o f n o n - m u s c l e cells, lacking p r o m i n e n t specializations a n d r e s e m b l i n g quiescent f i b r o b l a s t s ; n o cells r e m i n i s c e n t o f interstitial cells of the gut were f o u n d . B l o o d vessels, m o s t l y capillaries with n o n - f e n e s t r a t e d e n d o t h e l i u m , occurred only between bundles. T h e m a j o r vessels o f the b l a d d e r r a n in the c o n n e c t i v e tissue between the e p i t h e l i u m a n d m u s cle.

Muscle innervation in normal bladder

The i n n e r v a t i o n o f the b l a d d e r m u s c u l a t u r e was extensive. Nerves were f o u n d in all the layers o f the wall (serosa, m u s c u l a r i s , t u n i c a p r o p r i a o f the m u c o s a ) ; they were well sheathed b y c o n n e c t i v e tissue a n d some contained m y e l i n a t e d fibres. T h o s e w i t h i n the m u s c u l a t u r e r a n m a i n l y b e t w e e n muscle bundles. G a n g l i o n n e u r o n s were n o t observed in the b l a d d e r wall.

Fig. 4A, B. Electron micrographs of the hypertrophic musculature in transverse section. A These cells were among the largest found. There are prominent invaginations of the cell surface, especially where the cells face a wide intercellular space. N nucleus. B A

large muscle cell with eccentric nucleus and a very deep invagination of the cell surface. The dark bodies in the cytoplasm are mitochondria. Bars: 2 g m

73 Table 1. Quantitative data on control and hypertrophic bladders

Weight Outer surface Muscle coat thickness Muscle cells : Area of largest cell profiles Area of largest nucleus profiles Nucleus length Percentage of nucleated profiles Nncleus area in nucleated profiles

Control bladder

Hypertrophic bladder

70 mg 82 mm2 100-120 Ixm

1021 nag 490 mmz 250-350 gm

20 ttrnz 6 l.tm 2 21 gm 7,5% 33%

Numerous nerve fibres occurred within the muscle bundles. They were either individual fibres, often devoid of the Schwann cell sheath (Fig. 2D), or arranged in bundles of 2~4 axons with small Schwann cell processes. Many intramuscular nerve fibres were varicose axons, 1.2 gm in diameter at their widest, 0.2 ~tm at their narrowest. Varicosities were up to 3.5 ~tm long; they were packed with clear vesicles and also contained microtubules, neurofilaments and mitochondria (Fig. 2C). Dense-cored vesicles were only rarely seen, and varicosities rich in granular vesicles were not found. The junctional cleft of some varicosities measured about 2 nm and was occupied by a single basal lamina (Fig. 2F); other varicosities lay 1000 nm or more from the nearest muscle cell (Fig. 2E). There were no junctional specializations in either the muscle cell or the nerve ending.

Hypertrophic response Of the 9 rats operated with urethral stenosis, 7 developed a massive enlargement of the lumen and hypertrophy of the wall. The weight of the bladder (when empty) ranged between 870 and 1180mg (mean: 1021, S.D. 118). By injecting each bladder with an amount of fluid proportional to its weight, a relatively consistent distension of the wall was achieved in all the samples. Two bladders hypertrophied to 350 mg only and were not used in this study. The observations of the 7 hypertrophic bladders were pooled, as there were only minor differences between them. The shape o f the enlarged bladders was similar to the controls, viz. elongated with the cranio-caudal axis about 1.5 times the transverse and the dorso-ventral axes, the latter being marginally smaller than the transverse diameter. Although the outer surface of the hypertrophic bladder was smooth, as in the controls, the inner surface displayed prominent long ridges (trabeculations) mainly running parallel to the equator o f the organ; they were formed by some large muscle bundles projecting into the lumen. These folds persisted even if the bladder was distended further.

Hypertrophic musculature The basic arrangement of the musculature was the same as in controls, although larger and longer muscle bundles

240 gm2 12 g m 2 22 gm 4.6% 7-15%

were found (Fig. 3). The muscle coat was 250-350 gm thick and generally more compact and without discontinuities. There was also an increase in the thickness of the serosa and epithelium (which was not analysed in this study), but the spaces between muscle bundles were narrower. The muscle cells greatly increased in size (as judged from transverse sectional profiles) (Figs. 3 C, 4) (Table 1). The degree of hypertrophy appeared similar in all the cells of a bundle, but there was some variation between bundles, apparently not correlated with the position or orientation of the bundle. Many of the enlarged cell profiles were polygonal, crescent-like, very flattened, indented by other cells, or wedge-shaped, in contrast to the ovoid or simple polygonal shape of control muscle profiles. The hypertrophic muscle cells had prominent invaginations of the cell membrane, often radially arranged and shaped as wedge-like or finger-like inward folds. The invaginations could be found at any point along the cell and seemed to be present in s o m e ceils to a greater extent than in others. They reached as far as half way into the deepest point of the cell profile and their membrane bore dense bands and caveolae, and was coated by a basal lamina (Fig. 5C). Whereas in some cells these invaginations were evenly distributed around the circumference, in others they occurred only over the side facing the edge of a bundle (Fig. 4 A). Mitoses among muscle cells were not found, but binucleated muscle cells (the two nuclei being aligned along the cell length) were seen in all preparations. Some muscle cells were split longitudinally by deep furrows that included the nucleus; in extreme cases, the cell was divided into two except for a bridge of cytoplasm that also constricted and divided the nucleus (the distribution of chromatin appeared unchanged) (Fig. 5 A). The cell membrane displayed caveolae and dense bands, the latter occupying a higher proportion of the cell membrane than in control muscle cells. The cells were packed with thin, thick and intermediate filaments, and we noticed no obvious changes in the relative frequency of filament types from the control muscle. Mitochondria occupied about 4.7% of the cell volume. Sarcoplasmic reticulum was more in evidence than in controls. There were many small sacs and tubules of smooth sarcoplasmic reticulum, and some muscle cell profiles showed large cisternae of rough sarcoplasmic reticulum (Fig. 5 B); these cisternae had a uniform content o f medium electron density. Caveolae appeared similar in struc-

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75 ture (both in thin sections and in freeze-fracture preparations) to those of control muscle cells. Gap junctions were virtually absent (in all the preparations only two were seen, one by thin section and the other by freeze-fracture). Intermediate junctions (attachment plaques), which are infrequent in control bladder muscle cells, were c o m m o n between hypertrophic cells. The spaces between muscle cells were occupied by collagen fibrils (more heterogeneous in diameter, range 40-170 nm, than in control muscles, and often not circular in outline), numerous small elastic fibres (more numerous than in controls), and bundles of filaments of about 10 nm diameter. There were only few fibroblasts between muscle cells. The blood vessels, mainly capillaries, were situated between muscle bundles. Rare and isolated intercellular debris identified as remnants of degenerated muscle cells were seen in several preparations.

Innervation of hypertrophic musculature Nerve endings, in the form of varicosities containing small clear vesicles, microtubutes and neurofilaments, were commonly found among muscle cells; the vesicle content of the varicosities and the separation between nerve endings and muscle cells were similar to those of controls (Fig. 5D). No sign of degenerating nerve endings was seen.

Discussion

Fine structure of normal bladder The fine structure of the musculature of the urinary bladder in the rat is similar to that of other visceral muscles (Gabella 1985). There are, however, some structural aspects of the normal musculature that deserve discussion. There are no proper layers of musculature in the bladder. The structural units of the muscle are the bundles, and these are extremely variable in cross section, length, and orientation. There is a tendency in the ventral and dorsal walls o f the bladder for the bundles to run in a preferential direction, whereas at the sides and at the cranial pole, the bundles criss-cross in every direc-

tion and are probably oriented randomly. Many bundles run as geodesic lines (i.e., covering the shortest distance between two points on the wall) and most of them merge with one another, thus forming a mesh of a closed type, i.e., with very few bundles ending in the connective tissue. The manner in which the random arrangement of muscle bundles and the heterogeneity in bundle size and length are achieved during development is unknown; the processes involved are probably even more complex than those of the morphogenesis of regular layers of musculature. The mechanical significance of this spatial arrangement is unclear. The division of the musculature into many bundles with different orientation probably inparts an extraordinary stretchability to the wall, together with a marked ability to increase its thickness and to reduce length and width when the bladder voids. The changes in structure and arrangement o f the bundles taking place during bladder emptying remain, however, to be investigated. Studies of the mechanical behaviour of bladder muscle in vitro often rely on the use of strips cut across the wall. Within any strip of bladder, the orientation of the bundles is very variable and difficult to predict; therefore, the mechanical recordings from muscle strips may not provide an accurate quantitative representation of the mechanical performance of the muscle in situ. The non-spherical shape o f the bladder o f the rat is also mechanically important, as the stress will (according to the law of Laplace) not be equal in orthogonal directions along the bladder wall. This has not been taken into consideration in previous studies of rat bladder motility. One last point related to the mechanics of the bladder is the well-developed mass of collagen that we have found in the mucosa and the serosa. This is obviously important for the uniform redistribution of stress, even when there are small irregularities in muscle thickness. However, a geometric arrangement o f collagen fibres as found in the intestinal submucosa (Gabella 1987b), has not been observed in the bladder. Collagen is also found between muscle bundles (intramuscular septa) and, in small amounts, between muscle cells. Elastic fibres are more c o m m o n than in intestinal muscles, a feature probably contributing to the high distensibility of the bladder.

Absence of gap junctions Fig. 5 A-D. Electron micrographs of hypertrophic musculature in

transverse section. A Muscle cell constricted sideways by two deep invaginations (arrows) that also constrict the nucleus (N). B Welldeveloped rough sarcoplasmic reticulum in a muscle cell. C At the top surface of a muscle cell, displaying caveolae, sarcoplasmic reticulum (r) and dense bands. Arrows point to tubular invaginations of the cell membrane (they too bear caveolae, sarcoplasmic reticulum, dense bands and a basal lamina). Most of the cytoplasm is occupied by myofilaments and dense bodies. D Two vesiclecontaining nerve varicosities, partly covered by a thin Schwann cell process, run close to muscle cells. In addition to small clear vesicles, the axons contain microtubules, neurofilaments, endoplasmic reticulum and mitochondria. Arrow points to a dense projection around which a few vesicles are clustered. Bars: 1 gm

Despite an extensive search for gap junctions in the muscle, both in thin sections and in freeze-fractured specimens, they were virtually absent in all preparations examined. This feature of bladder musculature is at variance with the richness of gap junctions in other muscles (e.g., the circular layer of the small intestine, tracheal muscle). It is well known from the literature that the frequency of gap junctions varies between different muscles (Daniel et al. 1976; Gabella 198D; the urinary bladder, in being devoid o f gap junctions, is therefore at one of the extremes of the range. To what extent electrical coupling exists in the urinary bladder is unknown.

76 However, by studying impedance changes in smooth muscles of the guinea-pig, Brading et al. (1989) have concluded that electrical coupling in the bladder is poor in comparison with intestinal muscles, e.g. the circular muscle of the stomach. Bladder muscle cells may be sensitive to stretching to the extent that contracting cells can activate surrounding cells via their mechanical couplings. This mechanism would account (even in the absence of gap junctions) for the occurrence of coordinated contractions in bladder muscle (Uvelius 1985), to which the richness of innervation would also contribute. Intermediate junctions (also called attachment plaques) are less frequent than in other muscles. Dense extracellular material present in the areas of direct apposition between muscle cells, which are extensive, probably provides an additional form of cell-to-cell adhesion for mechanical coupling.

Innervation of normal bladder Nerve fibres in the bladder muscle are of several types, as demonstrated by histochemistry (see, e.g., Elbadawi and Schenk 1968; Alm and Elmbr 1975) and to some extent also ultrastructurally (Hoyes et al. 1976). Histochemical studies have identified several neuropeptides in bladder intramural nerve fibres (e.g., Alm et al. 1977; Sharkey et al. 1983; Mattiasson et al. 1985). The bladder musculature is well innervated as amply reported in the literature for several species (Elbadawi and Schenk 1968; Gosling and Dixon 1975; Hoyes et al. 1976; Feh6r et al. 1980). We can confirm that this is the case also for the bladder of the female rat. The fibres must all be of extrinsic origin since we have seen no ganglion neurons in the bladder wall. The female rat bladder thus seems comparable to the male rat bladder where removal of the pelvic ganglia results in an almost complete disappearance of cholinergic (Ekstr6m and Elm6r 1977) and non-cholinergic nerves (Mattiasson et al. 1985). In our preparations, a high proportion of nerve fibre profiles are varicosities, packed with vesicles. However, the differences between vesicles in different varicosities are not marked, as all are clear, lucent vesicles; it is therefore impossible to correlate ultrastructuraI features with different types of fibre. The observation of a large variation in width of the synaptic cleft is interesting. The pharmacological characteristics of the nerve-muscle interaction can be expected to be markedly influenced by the distance between nerve terminal and muscle cell, as reported for noradrenergic terminals in the vascular bed (for discussion, see Bevan et al. 1980). In blood vessels, a short distance is consistent with a fast phasic response to nerve stimulation, whereas a long distance favours a slow development of neurogenic tone.

Technical aspects bladder hypertrophy The surgical approach that we used to induce bladder hypertrophy in adult rats, developed in previous studies

(Mattiasson and Uvelius 1982; Uvelius and Mattiasson 1984), produces consistent and extensive hypertrophy while allowing long survival times. The procedure impairs urinary outflow, favouring urine retention and requiring higher intraluminary pressure to void. R e l a t e d methods to induce bladder hypertrophy have been used by other authors in developing (Brent and Stephens 1975; Sibley/985; Mockless et al. 1988) and adult animals (Brent and Stephens 1975; Levin et al. 1984; Steers and De Groat 1988; Elbadawi et al. 1989). Hypertrophy also follows denervation of the bladder (Elliott 1907; Carpenter and Root 1951; Goss etal. 1973; Sharkey et al. 1983); however, this method is complicated by the direct effects of denervation. Others investigators have induced bladder hypertrophy by placing a bolus of paraffin chronically into the bladder (Peterson et al. 1974), or simply by repeatedly injecting extra fluid into the bladder (Carey 1921). In our rat model, the partial obstruction does not develop slowly, unlike human bladder hypertrophy accompanying prostatism, but is established at the time of operation. However, the cystometric characteristics are very similar to those of the human hypertrophic bladder, and, in both cases, there is detrusor instability (Malmgren et al. 1987); hence our interest in this model. Morphological analysis requires that the conditions of distension of the muscle are consistent in different prepartions. Views of a contracted and emptied bladder are difficult to compare with views of a distended bladder. This point has not been taken into account in previous studies and this may explain some of the discrepancies in the literature. We designed a protocol, viz. injection into the lumen of a volume of fluid proportional to the weight of bladder when empty, that allowed a comparable degree of muscle distension independent of the degree of bladder hypertrophy.

Muscle hypertrophy The degree of hypertrophy is very large. In 7 out of 9 experimental animals, the bladder increased about 15fold in weight. Under the standardized conditions of distension, the outer surface of the bladder increased more than 6 times and the thickness of the wall about 3 times. Since histology shows that the musculature represents the same proportion (or a slightly higher proportion) of the bladder wall in the hypertrophic condition as in the control, we conclude that the musculature also increases about 15-fold. The bladder as a whole is enlarged but its characteristic shape remains unchanged. There are no signs of replacement of muscle with connective tissue, unlike results presented by Gosling and Dixon (1980) in man and by Speakman et al. (1987) in the pig. In spite of the enormous increase in size, the high stresses in the wall, and the extensive structural changes of the tissue, histology of the bladder shows no sign of regressive or degenerative changes in the hypertrophic musculature (apart from some debris of degenerated muscle cells), and no signs of inflammation or oedema. In this respect, previous observations on the same experi-

77 mental model are confirmed (Uvelius and Mattiasson 1984; Uvelius et al. 1984). In contrast, Elbadawi et al. (1989) have observed that, in the short-term obstructed bladder of rabbits, over 50% of the cell profiles were disintegrating muscle cells. The 15-fold increase in size of the bladder muscle is probably close to the limit of bladder growth; whether this limit is imposed by metabolic or mechanical constraints on the tissue, or whether it is an intrinsic limit to growth, we cannot tell. Interestingly, in the experiments on intestinal hypertrophy, the maximal hypertrophy that could be achieved with a partial obstruction was also an approximately 15-fold increase in muscle volume (Gabella 1987a). In both the bladder and the intestine, the hypertrophic musculature is well vascularized, i.e. new blood vessels are formed together with the growth of the muscle. Is there an increase in muscle cell number (hyperplasia) during hypertrophy of the bladder, and how extensive is the growth in muscle cell size (cell hypertrophy)? The question of the relative importance of hyperplasia and cell hypertrophy has often been examined in previous studies in this field (Seidel and Weisbrodt 1987). Smooth muscle cells are capable of dividing even when they are fully differentiated, e.g. in the pregnant uterus (Sanborn 1987) and at the edges of surgical cuts in intestinal muscles (McGeachie 1975), and in various conditions of hypertrophy, e.g. in the ureter (Cussen and Tymms 1972). In the hypertrophic ileum, muscle cells in mitosis are observed in both muscle layers (Gabella 1987a). In contrast, mitoses are very rare in adult smooth muscles in control conditions. Mitoses in muscle cells, either in the control or in the hypertrophic bladder at the stage that we studied (10 weeks), are absent and have not been noted in previous investigations (e.g., Elbadawi et al. 1989). However, the total DNA content of the bladder musculature increases 9-fold after a 6-week obstruction and its concentration is decreased only by a factor of 2 (Uvelius et al. 1984). Part of the DNA increase is probably accounted for by the increase in size of the nucleus and polyploidy, as is the case in vascular smooth muscle hypertrophy in hypertensive rats (Owens and Schwartz 1983); some of the increase may be the result of mitoses, although their occurrence is not documented. The significance of binucleate muscle cells that are consistently found is not clear, but it is possible that they eventually indicate cell division. A further possibility is that the longitudinal splitting of a muscle cell and its nucleus leads to a complete cell division (in this case, the daughter cells should be aligned across their length). Complete longitudinal splitting occurs in skeletal muscle fibres that hypertrophy when subject to overload (Goneya et al. 1977); this has also been suggested for intestinal hypertrophic musculature (Gabella 1987a). Nevertheless, in the hypertrophic bladder, the muscle increase is mainly, if not entirely, accounted for by an increase in cell size. There is also a re-arrangement of muscle cells, and their shape is greatly distorted. Cell profiles are not only enlarged but also irregular and heterogeneous, and partly moulded around each other. These changes probably

occur on account of the intense stresses imposed onto each bundle, not only along its length but also across its width (longitudinal and lateral stresses). Moreover, the muscle grows along all its axes, the outer surface of the bladder growing 6-fold; this must involve re-arrangement of the bundles and cells within them.

Cytology of hypertrophic muscle cells An extensive corrugation of the cell membrane with deep infoldings is found in the largest hypertrophic cell profiles. It is interpreted as an adaptation of the cell to reduce the fall of the surface-to-volume ratio, thus providing additional surface membrane for metabolic exchanges and for dense-band insertion. A similar change is found in other hypertrophic smooth muscles (Gabella 1987 a). In our preparations, there is no apparent alteration in the pattern of myofilaments. A recent biochemical study has shown that, in hypertrophic bladder muscle, there is no change in the concentration of actin and a modest fall in the concentration of myosin, whereas the concentration of intermediate filaments (90% desmin, 10% vimentin) increases (Uvelius etal. 1989). These changes are probably too small to be detected by thin-section studies, and are small compared with those found in other hypertrophic muscles, such as the gut (Gabella 1987a) and the portal vein (Berner et al. 1981), where a prominent increase in the relative frequency of intermediate filaments occurs. Another difference in the hypertrophic response of bladder and intestine is that, although in both the total number of mitochondria per cell is much increased, in the bladder the mitochondrial percentage volume remains almost unchanged, whereas in the gut there is a marked fall (Gabella 1987a), reminiscent of that observed in skeletal muscles undergoing hypertrophy of the 'overload' type (Goldspink 1971). Gap junctions are absent in the control bladder, and they are virtually absent in hypertrophy. In contrast, in the hypertrophic intestine, gap junctions increase in number and size within the tissue where they are normally present (the circular muscle), whereas they do not appear within the tissue where they do not already exist in controls (the longitudinal muscle) (Gabella 1987a). In the uterus, a large increase in gap junctions between hypertrophic muscle cells is found in the late stages of pregnancy (Garfield et al. 1977).

Nerves and extracellular materials in hypertrophic muscle There was no apparent increase in the amount of collagen fibrils, although in the 15-fold enlarged tissue many of the fibrils must be newly formed. However, since there is no expansion of the spaces between muscle cells and a vast cell enlargement takes place, the collagen concentration must be reduced. Previous estimates of collagen content in this material showed an absolute 4-fold increase in the total collagen content of the hypertrophic

78 bladder, but a reduction in concentration to about one third o f the control value (Uvelius and Mattiasson 1984). The newly formed intramuscular collagen is probably synthesized by the muscle cells, some of which display prominent cisternae of rough sarcoplasmic reticulum. Other authors have observed an increase in collagen concentration (collagen infiltration) in the bladder of h u m a n subjects with chronic outflow obstruction (Gosling and Dixon 1980; Iacovou et al. 1989), whereas Susset et al. (1978) noted on average a modest decrease; Cortivo et al. (1981) observed an increase in elastin but not in collagen. These differences may reflect not only variation between species, but also the different effects o f an experimental surgical obstruction and a long-term prostatic enlargement. The generalization that the obstructed bladder becomes infiltrated by connective tissue is not warranted (Iacovou et al. 1989). In contrast, a substantial increase in collagen concentration is found in other hypertrophic smooth muscles, e.g. in the uterus (Cullen and Harkness 1968) and the intestine (Gabella 1987a). The innervation o f the hypertrophic muscle appears well maintained; indeed, taking into account the overall volume increase o f the muscle, the nerve fibres must have grown substantially in length and/or number. It has recently been demonstrated that the content of nerve growth factor is higher in hypertrophic bladders than in control bladders (Steers et al. 1989). The increased total a m o u n t of choline acetyltransferase in the hypertrophic bladder muscle can be taken as an indication o f a growth of cholinergic nerves within the bladder wall (Mattiasson et al. 1987). We have not found degenerating nerve fibres. Our results on rat bladder are in this respect inconsistent with the suggestion o f a decreased nerve density secondary to degeneration of nerves in biopsies from obstructed bladder of pig (Sibley 1985; Speakman et al. 1987) and man (Gosling et al. 1986).

Conclusion The hypertrophy of the wall of the bladder following outlet obstruction is not a simple increase in muscle cell size, and possible in cell number. It is a process accompanied by complex structural remodelling of the stroma and the muscle cells with their cell-to-cell contacts, resulting in an increased diameter and surface of the bladder. Many of the structural changes observed in hypertrophic muscles bear witness to specific aspects of the response in different animal species, in different organs, and in different experimental and pathological conditions.

Acknowledgements. The expert technical assistance of Peter Trigg and Christine Davis is acknowledged. The work was supported by grants from the Wellcome Trust, the Medical Research Council (U.K.), and the Medical Faculty of the University of Lund.

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