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Morphological analysis of the acute effects of overdistension on the extracellular matrix of the rat urinary bladder wall
ANNALS OF ANATOMY
Ann Anat 186 (2004): 55-59 http://www.elsevier-deutschland,de
ELSEVIER
Morphological analysis of the acute effects of overdistension on the extracellular matrix of the rat urinary bladder wall* Graciela M. P. de Souza, Waldemar S. Costa, Homero Bruschini, and Francisco J. B. Sampaio From the Urogenital Research Unit, State University of Rio de Janeiro, Av. 28 de Setembro, No. 87, Fundos-FCM-t6rreo, 20551-030 Rio de Janeiro, RJ, Brazil, and Section of Urological Neurology, Federal University of S~o Paulo, Silo Paulo, Brazil
Summary. Purpose: To investigate the morphological effects of acute overdistension in the structure of the extracellular matrix of the bladder wall in rats. - Materials and Methods: The bladders of a group of 6 male Wistar rats were transurethrally overdistended for 3 hours. Another identical group (the control group) was only submitted to a sham operation. Specimens from the bladder dome were analyzed with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). - Results: LM - The control group bladders had a 4 to 5 layer urothelium, a lamina propria, and a smooth muscle layer with longitudinal and transversal fibers. The overdistended bladders presented an intense interstitial infiltrate in the lamina propria, and a less intense infiltrate among the smooth muscle fibers. TEM The cells of the overdistended bladders had a significant amount of vacuoles, unlike the control bladders, where such vacuoles were scarce or absent. SEM - A delicate three-dimensional mesh of collagen fibrils was observed in the lamina propria of the bladder walls from the control group. Whilst for the control group this mesh consisted of distinct geometric structures, with mostly circular cellular spaces surrounded by the fibrils, the overdistended group showed evidence of distortion of the
* Supported by Grants from the National Council of Scientific and Technological Development (CNPq-Brazil), and from the Foundation for Research Support of Rio de Janeiro (FAPERJ) Correspondence to: Fransisco J. B. Sampaio E-mail: [email protected]
mesh, with flattened and elongated cellular spaces. - Conclusions: Acute bladder overdistension induces structural modifications, altering the arrangement and interaction of collagen fibrils, as well as incipient tissue damage as edema in the lamina propria and smooth muscle layers.
Key words: Bladder - Overdistension - Urological neurology - Urodynomics - Extracellular matrix - Collagen Rats
Introduction Several pathologies may cause disarrangement in the bladder's extracellular matrix fibrillar structure and may lead to vesical compliance alterations. As a consequence, these bladders will store urine under increased intravesicular pressure (Leppilahti etal. 1997; Deveaud etal. 1998). In men, benign prostatic hyperplasia is the more prevalent cause of voiding symptoms, whereas in women it is detrusor hyperactivity and/or in continence disturbances (Holm et al. 1991). Acut bladder overdistension has been used to treat vesicular instability, urge-incontinence and interstitial cystitis (Brading et al. 1999; Taub et al. 1994). Nevertheless, it has been demonstrated that partial obstruction and bladder overdistension may cause damage to the mucosal epithelium (urothelium) and detrusor smooth muscle (Hass et al. 1999; Damaser et al. 1998). The extracellular matrix components, such as glycoproteins and many types of collagen and elastic system fibers,
0940-9602/04/186101-055 $30.00/0
5 days at room temperature (about 25 °C). Afterwards, the samples were postfixed in 1% aqueous solution of osmium tetroxide for 1 hour at room temperature. The tissues were rinsed in three consecutive 1 hour baths of distilled water and examined in a low vacuum scanning electron microscope (LEO 950).
are essential for m a i n t e n a n c e of b l a d d e r proprieties, playing a f u n d a m e n t a l role in tissue biomechanics ( H o l m et al. 1991). In addition, the extracellular matrix is involved in m a n y pathological conditions that i m p a i r normal tissue function (Ewalt et al. 1992; H a y et al. 1991). In the b l a d d e r , quantitative alterations in the extracellular matrix c o m p o n e n t s are usually r e l a t e d to obstruction, incontinence or contractility p r o b l e m s (Chang et al. 1998). To u n d e r s t a n d the consequences of b l a d d e r overdistension, e x p e r i m e n t a l studies have b e e n p e r f o r m e d t h r o u g h m o l e c u l a r analysis and enzyme histochemistry, d e m o n strating negative effects on the b l a d d e r wall extracellular matrix ( L e p p i l a h t i et al. 1997; Chen et al. 1994; L e p p i l a h t i et al. 1994). Also, e x p e r i m e n t s on b l a d d e r overdistension have d e m o n s t r a t e d d a m a g e to the a u t o n o m i c innenervation of the b l a d d e r matrix (Leppilahti et al. 1997; L a s a n e n et al. 1992). In the p r e s e n t study, we have investigated the m o r p h o logical consequences of acute overdistension on the structure and integrity of the extracellular matrix of the rat b l a d d e r wall.
Results Light microscopy: The b l a d d e r s of the control group presented an u r o t h e l i u m of 4 to 5 layers, a lamina propria, and a s m o o t h muscle layer with longitudinal and transversal fibers (Fig. 1 A ) . The o v e r d i s t e n d e d b l a d d e r s presented a conspicuous interstitial infiltrate in the lamina propria, and a discrete infiltrate a m o n g the s m o o t h muscle fibers (Fig. 1B). Transmission electron microscopy: The observations in T E M d e m o n s t r a t e d that in b o t h groups there was great a quantity of collagen fibrils distributed in different spatial orientations exhibiting c o m p a c t or spaced bundles of col-
Materials and methods A total of 12 male adult Wistar rats, weighing 260 to 320 g (mean = 290 g) were anesthetized with 25 mg/kg sodium pentobarbital given intraperitoneally. The posterior bladder region was accessed through a midline suprapubic incision and the distal ureters were identified and ligated. In 6 rats, the bladder was transurethrally catheterized (0.6 mm x 1.05 mm catheter) and filled by gravity with Ringer solution until a pressure of 100 cm of water was achieved and maintained for 3 hours. The control group consisted of 6 rats, which did not undergo bladder overdistension and were submitted to a sham operation. After the overdistension period, the Ringer solution was gradually changed by the fixative solution of glutaraldehyde 2.5 % in 0.1 M in sodium phosphate buffer (pH7.4) with tannic acid, maintaining the bladder under the initial levels of overdistension (100 cm of water) while fixing. Some specimens were fixed in phosphate buffer formalin solution and processed according to routine histological methods. From the paraffin-embedded samples, 5 gm thick sections were obtained and stained with Gomori's trichrome. For transmission electron microscopy (TEM) bladder dome specimens were postfixed with a buffered 2% osmium tetroxide solution for 1.5 hour at room temperature and washed in PBS. Dehydration was performed using a graded series of ethanol solution and then embedded in Epon resin. Semithin sections (1 gm) from each block of the bladder dome were stained with toluidine blue and examined by light microscopy to select the most appropriate blocks. Ultrathin sections were obtained in a Leica ultramicrotome and collected on copper grids that were counterstained with uranyl acetate and lead citrate, for examination in a Zeiss EM 906 transmission electron microscope (80100 kV). For scanning electron microscopy (SEM) the bladder dome specimens were initially fixed in glutaraldehyde 2.5% in 0.1 M in phosphate buffer (pH 7.4) with 1% tannic acid solution, and then immersed in a 10% NaOH- phosphate buffer solution for 3 to
Fig. 1. Light microscopy of the rat bladder wall stained with Gomori's trichrome. A: Control group (× 400). B: Overdistended bladder, demonstrating interstitial infiltrate (arrow) (× 400). 56
lagen fibrils with scattered elastic fibers but no characteristic difference was found between them (Figs. 2 A, 2 B). Low vacuum scanning electron microscopy: The observation of the normal rat bladder wall at low magnification demonstrated 3 distinct layers: 1) the epithelium, visualized as a thin line, whose cells were removed after treatment with N a O H - phosphate buffer solution for elimination of the cellular components, 2) the lamina propria, with interlacing fibrils, and with some spaces that were previously occupied by blood vessels, and 3) a layer of smooth muscle fibers which presented a lighter image because they are more compact (Fig. 3). The bladder wall of the control specimens treated with N a O H solution presented a three-dimensional meshwork of collagen fibrils and various cavities, which were occupied by cells and blood vessels (Fig. 4 A). In the lamina
Fig. 3. Low vacuum scanning electron microscopy of a bladder from the control group demonstrating the different layers of the bladder wall (x 330). propria we observed a delicate fibrillar mesh formed by components of varying diameters and formed defined geometric forms (Fig. 4 B). In the overdistended bladder, the threedimensional meshwork of collagen fibrils changed: the cellular spaces, encircled by the fibrils, which were for the most part circular, became flattened and elongated in the overdistended bladders, characterizing a response to the bladder stretching (Fig. 4C). Thus, the collagen fibrillar meshwork lost the geometric shape that was observed in the bladders of the control group (Fig. 4D).
Discussion Collagen and elastic system fibers are abundant in the bladder and play a key role in its compliance property (Ewalt et al. 1992). Diverse experimental models have been used for investigating the morphofunctional changes in the bladder (Capolicchio et al. 2001; Chang et al. 1998; Leppilahti et al. 1997). Leppilahti et al. (1999), promoted bladder overdistension for 3 hours in rats, by catheter urethral obstruction and forced diuresis. In that study the bladders were processed 12 hours after the overdistension period. In our present study, we used the same period of overdistension but immediately after overdistension the bladders were fixed by gradual substitution of the Ringer solution by glutaraldehyde. This procedure maintained the initial pressure of overdistension without any interruption. Leppilahti et al. (1997, 1999) demonstrated the occurrence of vacuoles and edemas in the vesicular submucosa of overdistended bladders. These findings agree with our present work, in which we observed a significant amount of edemas and vacuoles, in contrast with the control bladders, in which such vacuoles were rare or absent.
Fig. 2. Transmission electron microscopy demonstrating the general aspect of collagen in the rat bladder wall. A: Control group. Compact bundles of collagen fibrils are arranged horizontally and transversally (x20500). B: Overdistended bladder. The collagen fibrils rtm in various directions. Elastic fibers can be identified among the collagen fibrils (arrow), (x 22 500). 57
Fig. 4. Low vacuum scanning electron microscopy demonstrating the fibrillar structure in the rat bladder wall. A: Control group. The arrangement of collagen fibrils forms a skeleton of circular spaces (asterisk) that were occupied by smooth muscle cells, dissolved by NaOH (x 1500). B: Control group. Note collagen fibrils of different diameters limiting geometric spaces (x 3000). C: Overdistended bladder. Note that the cellular spaces described in the control group are compressed and elongated (arrows), (x 1500). D: Overdistended bladder. Collagen fibrils are elongated and have lost their distinctive structure (× 3000). Grossklaus et al. (2000) and Taub et al. (1994) have already demonstrated that bladder overdistension, also used for treatment of interstitial cystitis, may cause tissue necrosis. Observations under light microscopy using an ex vivo rat model of bladder stretch injury demonstrated that after 8 hours of overdistension, there was an increased abundance of interfascicular spaces in the smooth muscle layer, consistent with edema and recent damage following the stretch injury (Capolicchio et al. 2001). Our current study also demonstrated an interstitial infiltrate in the lamina propria and a slight infiltrate among the smooth muscle fibers after 3 hours of overdistension. Ultrastructural studies have contributed to the understanding of the cellular changes resulting from infravesicular obstruction in humans (Hailemariam et al. 1997; Tse et al. 2000) and obstructed bladders in an experimental model (Gosling et al. 2000). However, little is known about the ultrastructural effects of acute bladder overdis-
tension on the extracellular matrix, which may be a consequence of partial infravesicular obstruction, such as acute urinary retention suffered by patients with benign prostatic hyperplasia. H o l m et al. (2002), did not find any specific ultrastructural features in the smooth bladder muscle (detrusor) of human patients with acute urinary retention when compared to controls. In the present experimental study using the rat as a model, our observations in T E M also did not find any morphological differences in the arrangement of collagen fibrils. Strauss et al. (2000), recently demonstrated the role of various types of collagen in the human bladder wall, as well as the collagen changes resulting from infravesicular obstruction. Anomalies in the distribution of the various types of collagen fibrils may have considerable influence on the mechanical properties of the bladder extracellular matrix (Kim et al. 2000; Baskin et al. 1994; Ewalt et al. 1992). In our SEM experimental data we also saw evidence that an overdistended bladder showed at least tern58
porary changes in E C M fibrillar components following acute bladder overdistension. M u r a k u m o et al. (1995) studied the empty, distended and contracted urinary bladders of the guinea pig using high vacuum SEM and demonstrated that the arrangement of collagen fibrils is drastically altered dependent upon the muscle cell extension and contraction. In the empty bladder, the contours of the smooth muscle collagen fibrillar sheaths were usually oval or polygonal, whereas in the distended bladder the sheaths' contours were polygonal. In our study, changes to the shape of the collagen fibrils were evident in the overdistended rat bladders, which lost the polygonal nature of their collagen fibrils. Thus, the results of our three-dimensional analysis morphologically support the findings of Kim et al. (2000) and Chang et al. (1998), which demonstrated that alterations in the vesicular collagen would interfere with bladder function. M u r a k u m o et al. (1995), analyzed the h u m a n bladder wall in S E M and described how the mucosa and the muscular layers had different collagen organization. O u r S E M observations in the rat bladder wall of the control group confirmed the same collagen organization as demonstrated by these authors, and a clear stretching of the collagen fibrils in the overdistended bladder. The present study showed structural modifications of the fibrillar components of the ECM. Despite the fact that no experiment was done to determine whether or not these alterations are reversible, we postulate that these changes, when they occur several times, could cause irreversible changes in the fibrillar components of the E C M of these bladders. In conclusion, our current study has demonstrated that acute bladder overdistension induces striking trends in the collagen fibrils arrangement and their interaction, as well as incipient tissue damage such as edema in the lamina propria and smooth muscle layers. These changes should be considering when overdistension is used as treatment method. However, additional investigations are necessary for better understanding of the extracellular matrix response to acute and chronic bladder overdistension, as well as for the development of novel therapeutic strategies or for preventing the occurrence of irreversible damage to the vesicular tissue.
References Baskin LS, Constantinescu S, Duckett JW et al. (1994) Type III collagen decreases in normal fetal bovine bladder development. J Urol 152:688 Brading AF, Greenland JE, Mills IW et al. (1999) Blood supply to the bladder during filling. Scand J Urol Nephrol 201:25 Capolicchio G, Aitken KJ, Gu JX, et al. (2001) Extracellular gene responses in a novel ex vivo model of bladder stretch injury. J Urol 165:2235 Chang SL, Howard PSH, Koo HP et al. (1998) Role of type III collagen in bladder filling. Neurourol Urodynam 17:135
Chen MW, Krasnapolsky L, Levin R et al. (1994) An early molecular response induced by acute overdistension of the rabbit urinary bladder. Mol Cell Biochem 132:39 Damaser MS, Brzezinski K, Longhurst PA (1999) Filling mechanics of obstructed and de-obstructed rat urinary bladders. Neurourol Urodynam 18:659 Deveaud CM, Macarak EJ, Kucich U et al. (1998) Molecular analysis of collagens in bladder fibrosis. J Urol 160:1518 Ewalt DH, Howard PS, Blyth B (1992) Is lamina propria matrix responsible for normal compliance? J Urol 145:544 Gosling JA, Kung LS, Dixon JS et al. (2000) Correlation between the structure and function of the rabbit urinary bladder following partial outlet obstruction. J Urol 163:1349 Grossklaus D J, Franke JJ (2000) Vesical necrosis after hydrodistension of the urinary bladder in a patient with interstitial cystitis. BJU Int 86:140 Hailemariam S, Elbadawi A, Yalla SV et al. (1997) Structural basis of geriatric voiding dysfunction. V. Standardized protocols for routine nltrastructural study and diagnosis of endoscopic detrusor biopsies. J Urol 157:1783 Hass MA, Elena L, Levin RM (1999) Fatty acid profiles in normal and obstructed bladder smooth muscle and mucosa. Neurourol Urodynam 18:697 Hay ED (1991) Cell Biology of Extracellnlar Matrix. Massachusetts, Harvard Medical School, 2nd Ed, pp 1--72 Holm NR, Horn T, Smedts F et al. (2002) Does structural morphology of human detrusor smooth muscle cells characterize acute urinary retention? J Urol 167:1705 Holm NR, Horn T, Hald T (1995) Detrusor in aging and obstruction. Scand J Urol Nephrol 29:45 Kim JC, Yoon JY, Hwang TK et al. (2000) Effects of partial outlet obstruction and its relief on types I and II collagen and detrusor contractility in the rat. Neurourol Urodynam 19:29 Lasanen LT, Tammela TLJ, Kallioinen M e t al. (1992) Effect of acute distension on cholinergic innervation of the rat urinary bladder. Urol Res 20:59 Lasanen LT, Tammela TLJ, Liesi P et al. (1992) The effect of acute distension on vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP) immunoreactive nerves in the female rat urinary bladder. Urol Res 20:259 Leppilahti M, Hirvonen J, Tammela T (1997) Influence of transient overdistension on bladder wall morphology and enzyme histochemistry. Scand J Urol Nephrol 31:517 Leppilahti M, Kallioinem M, Tammela, TLJ (1999) Duration of increased mucosal permeability of the urinary bladder after acute overdistension: an experimental study in rats. Urol Res 27:272 Murakumo M, Ushiki T, Koyanagy T et al. (1993) Scanning electron microscopic studies of smooth muscle cells and their collagen fibrillar sheaths in empty, distended and contracted urinary bladders of the guinea pig. Arch Histol Cytol 56:441 Murakumo M, Ushiki T, Abe K et al. (1995) Three-dimensional arrangement of collagen and elastin fibers in the urinary bladder: a scanning electron microscopic study. J Urol 154:251 Strauss L, Paranko J, Salmi S et al. (2000) Distribution of collagen XII and XIV in the bladder wall of the male rat with outlet obstruction. J Urol 163:1304 Taub HC, Stein M (1994) Bladder distension therapy for symptomatic relief of frequency and urgency: a ten year review. Urology 43:36 Tse V, Wills E, Szonyi G e t al. (2000) The application of ultrastructural studies in the diagnosis of bladder dysfunction in a clinical setting. J Urol 163:535 Accepted August 12, 2003 59
Ann Anat 186 (2004): 55-59 http://www.elsevier-deutschland,de
ELSEVIER
Morphological analysis of the acute effects of overdistension on the extracellular matrix of the rat urinary bladder wall* Graciela M. P. de Souza, Waldemar S. Costa, Homero Bruschini, and Francisco J. B. Sampaio From the Urogenital Research Unit, State University of Rio de Janeiro, Av. 28 de Setembro, No. 87, Fundos-FCM-t6rreo, 20551-030 Rio de Janeiro, RJ, Brazil, and Section of Urological Neurology, Federal University of S~o Paulo, Silo Paulo, Brazil
Summary. Purpose: To investigate the morphological effects of acute overdistension in the structure of the extracellular matrix of the bladder wall in rats. - Materials and Methods: The bladders of a group of 6 male Wistar rats were transurethrally overdistended for 3 hours. Another identical group (the control group) was only submitted to a sham operation. Specimens from the bladder dome were analyzed with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). - Results: LM - The control group bladders had a 4 to 5 layer urothelium, a lamina propria, and a smooth muscle layer with longitudinal and transversal fibers. The overdistended bladders presented an intense interstitial infiltrate in the lamina propria, and a less intense infiltrate among the smooth muscle fibers. TEM The cells of the overdistended bladders had a significant amount of vacuoles, unlike the control bladders, where such vacuoles were scarce or absent. SEM - A delicate three-dimensional mesh of collagen fibrils was observed in the lamina propria of the bladder walls from the control group. Whilst for the control group this mesh consisted of distinct geometric structures, with mostly circular cellular spaces surrounded by the fibrils, the overdistended group showed evidence of distortion of the
* Supported by Grants from the National Council of Scientific and Technological Development (CNPq-Brazil), and from the Foundation for Research Support of Rio de Janeiro (FAPERJ) Correspondence to: Fransisco J. B. Sampaio E-mail: [email protected]
mesh, with flattened and elongated cellular spaces. - Conclusions: Acute bladder overdistension induces structural modifications, altering the arrangement and interaction of collagen fibrils, as well as incipient tissue damage as edema in the lamina propria and smooth muscle layers.
Key words: Bladder - Overdistension - Urological neurology - Urodynomics - Extracellular matrix - Collagen Rats
Introduction Several pathologies may cause disarrangement in the bladder's extracellular matrix fibrillar structure and may lead to vesical compliance alterations. As a consequence, these bladders will store urine under increased intravesicular pressure (Leppilahti etal. 1997; Deveaud etal. 1998). In men, benign prostatic hyperplasia is the more prevalent cause of voiding symptoms, whereas in women it is detrusor hyperactivity and/or in continence disturbances (Holm et al. 1991). Acut bladder overdistension has been used to treat vesicular instability, urge-incontinence and interstitial cystitis (Brading et al. 1999; Taub et al. 1994). Nevertheless, it has been demonstrated that partial obstruction and bladder overdistension may cause damage to the mucosal epithelium (urothelium) and detrusor smooth muscle (Hass et al. 1999; Damaser et al. 1998). The extracellular matrix components, such as glycoproteins and many types of collagen and elastic system fibers,
0940-9602/04/186101-055 $30.00/0
5 days at room temperature (about 25 °C). Afterwards, the samples were postfixed in 1% aqueous solution of osmium tetroxide for 1 hour at room temperature. The tissues were rinsed in three consecutive 1 hour baths of distilled water and examined in a low vacuum scanning electron microscope (LEO 950).
are essential for m a i n t e n a n c e of b l a d d e r proprieties, playing a f u n d a m e n t a l role in tissue biomechanics ( H o l m et al. 1991). In addition, the extracellular matrix is involved in m a n y pathological conditions that i m p a i r normal tissue function (Ewalt et al. 1992; H a y et al. 1991). In the b l a d d e r , quantitative alterations in the extracellular matrix c o m p o n e n t s are usually r e l a t e d to obstruction, incontinence or contractility p r o b l e m s (Chang et al. 1998). To u n d e r s t a n d the consequences of b l a d d e r overdistension, e x p e r i m e n t a l studies have b e e n p e r f o r m e d t h r o u g h m o l e c u l a r analysis and enzyme histochemistry, d e m o n strating negative effects on the b l a d d e r wall extracellular matrix ( L e p p i l a h t i et al. 1997; Chen et al. 1994; L e p p i l a h t i et al. 1994). Also, e x p e r i m e n t s on b l a d d e r overdistension have d e m o n s t r a t e d d a m a g e to the a u t o n o m i c innenervation of the b l a d d e r matrix (Leppilahti et al. 1997; L a s a n e n et al. 1992). In the p r e s e n t study, we have investigated the m o r p h o logical consequences of acute overdistension on the structure and integrity of the extracellular matrix of the rat b l a d d e r wall.
Results Light microscopy: The b l a d d e r s of the control group presented an u r o t h e l i u m of 4 to 5 layers, a lamina propria, and a s m o o t h muscle layer with longitudinal and transversal fibers (Fig. 1 A ) . The o v e r d i s t e n d e d b l a d d e r s presented a conspicuous interstitial infiltrate in the lamina propria, and a discrete infiltrate a m o n g the s m o o t h muscle fibers (Fig. 1B). Transmission electron microscopy: The observations in T E M d e m o n s t r a t e d that in b o t h groups there was great a quantity of collagen fibrils distributed in different spatial orientations exhibiting c o m p a c t or spaced bundles of col-
Materials and methods A total of 12 male adult Wistar rats, weighing 260 to 320 g (mean = 290 g) were anesthetized with 25 mg/kg sodium pentobarbital given intraperitoneally. The posterior bladder region was accessed through a midline suprapubic incision and the distal ureters were identified and ligated. In 6 rats, the bladder was transurethrally catheterized (0.6 mm x 1.05 mm catheter) and filled by gravity with Ringer solution until a pressure of 100 cm of water was achieved and maintained for 3 hours. The control group consisted of 6 rats, which did not undergo bladder overdistension and were submitted to a sham operation. After the overdistension period, the Ringer solution was gradually changed by the fixative solution of glutaraldehyde 2.5 % in 0.1 M in sodium phosphate buffer (pH7.4) with tannic acid, maintaining the bladder under the initial levels of overdistension (100 cm of water) while fixing. Some specimens were fixed in phosphate buffer formalin solution and processed according to routine histological methods. From the paraffin-embedded samples, 5 gm thick sections were obtained and stained with Gomori's trichrome. For transmission electron microscopy (TEM) bladder dome specimens were postfixed with a buffered 2% osmium tetroxide solution for 1.5 hour at room temperature and washed in PBS. Dehydration was performed using a graded series of ethanol solution and then embedded in Epon resin. Semithin sections (1 gm) from each block of the bladder dome were stained with toluidine blue and examined by light microscopy to select the most appropriate blocks. Ultrathin sections were obtained in a Leica ultramicrotome and collected on copper grids that were counterstained with uranyl acetate and lead citrate, for examination in a Zeiss EM 906 transmission electron microscope (80100 kV). For scanning electron microscopy (SEM) the bladder dome specimens were initially fixed in glutaraldehyde 2.5% in 0.1 M in phosphate buffer (pH 7.4) with 1% tannic acid solution, and then immersed in a 10% NaOH- phosphate buffer solution for 3 to
Fig. 1. Light microscopy of the rat bladder wall stained with Gomori's trichrome. A: Control group (× 400). B: Overdistended bladder, demonstrating interstitial infiltrate (arrow) (× 400). 56
lagen fibrils with scattered elastic fibers but no characteristic difference was found between them (Figs. 2 A, 2 B). Low vacuum scanning electron microscopy: The observation of the normal rat bladder wall at low magnification demonstrated 3 distinct layers: 1) the epithelium, visualized as a thin line, whose cells were removed after treatment with N a O H - phosphate buffer solution for elimination of the cellular components, 2) the lamina propria, with interlacing fibrils, and with some spaces that were previously occupied by blood vessels, and 3) a layer of smooth muscle fibers which presented a lighter image because they are more compact (Fig. 3). The bladder wall of the control specimens treated with N a O H solution presented a three-dimensional meshwork of collagen fibrils and various cavities, which were occupied by cells and blood vessels (Fig. 4 A). In the lamina
Fig. 3. Low vacuum scanning electron microscopy of a bladder from the control group demonstrating the different layers of the bladder wall (x 330). propria we observed a delicate fibrillar mesh formed by components of varying diameters and formed defined geometric forms (Fig. 4 B). In the overdistended bladder, the threedimensional meshwork of collagen fibrils changed: the cellular spaces, encircled by the fibrils, which were for the most part circular, became flattened and elongated in the overdistended bladders, characterizing a response to the bladder stretching (Fig. 4C). Thus, the collagen fibrillar meshwork lost the geometric shape that was observed in the bladders of the control group (Fig. 4D).
Discussion Collagen and elastic system fibers are abundant in the bladder and play a key role in its compliance property (Ewalt et al. 1992). Diverse experimental models have been used for investigating the morphofunctional changes in the bladder (Capolicchio et al. 2001; Chang et al. 1998; Leppilahti et al. 1997). Leppilahti et al. (1999), promoted bladder overdistension for 3 hours in rats, by catheter urethral obstruction and forced diuresis. In that study the bladders were processed 12 hours after the overdistension period. In our present study, we used the same period of overdistension but immediately after overdistension the bladders were fixed by gradual substitution of the Ringer solution by glutaraldehyde. This procedure maintained the initial pressure of overdistension without any interruption. Leppilahti et al. (1997, 1999) demonstrated the occurrence of vacuoles and edemas in the vesicular submucosa of overdistended bladders. These findings agree with our present work, in which we observed a significant amount of edemas and vacuoles, in contrast with the control bladders, in which such vacuoles were rare or absent.
Fig. 2. Transmission electron microscopy demonstrating the general aspect of collagen in the rat bladder wall. A: Control group. Compact bundles of collagen fibrils are arranged horizontally and transversally (x20500). B: Overdistended bladder. The collagen fibrils rtm in various directions. Elastic fibers can be identified among the collagen fibrils (arrow), (x 22 500). 57
Fig. 4. Low vacuum scanning electron microscopy demonstrating the fibrillar structure in the rat bladder wall. A: Control group. The arrangement of collagen fibrils forms a skeleton of circular spaces (asterisk) that were occupied by smooth muscle cells, dissolved by NaOH (x 1500). B: Control group. Note collagen fibrils of different diameters limiting geometric spaces (x 3000). C: Overdistended bladder. Note that the cellular spaces described in the control group are compressed and elongated (arrows), (x 1500). D: Overdistended bladder. Collagen fibrils are elongated and have lost their distinctive structure (× 3000). Grossklaus et al. (2000) and Taub et al. (1994) have already demonstrated that bladder overdistension, also used for treatment of interstitial cystitis, may cause tissue necrosis. Observations under light microscopy using an ex vivo rat model of bladder stretch injury demonstrated that after 8 hours of overdistension, there was an increased abundance of interfascicular spaces in the smooth muscle layer, consistent with edema and recent damage following the stretch injury (Capolicchio et al. 2001). Our current study also demonstrated an interstitial infiltrate in the lamina propria and a slight infiltrate among the smooth muscle fibers after 3 hours of overdistension. Ultrastructural studies have contributed to the understanding of the cellular changes resulting from infravesicular obstruction in humans (Hailemariam et al. 1997; Tse et al. 2000) and obstructed bladders in an experimental model (Gosling et al. 2000). However, little is known about the ultrastructural effects of acute bladder overdis-
tension on the extracellular matrix, which may be a consequence of partial infravesicular obstruction, such as acute urinary retention suffered by patients with benign prostatic hyperplasia. H o l m et al. (2002), did not find any specific ultrastructural features in the smooth bladder muscle (detrusor) of human patients with acute urinary retention when compared to controls. In the present experimental study using the rat as a model, our observations in T E M also did not find any morphological differences in the arrangement of collagen fibrils. Strauss et al. (2000), recently demonstrated the role of various types of collagen in the human bladder wall, as well as the collagen changes resulting from infravesicular obstruction. Anomalies in the distribution of the various types of collagen fibrils may have considerable influence on the mechanical properties of the bladder extracellular matrix (Kim et al. 2000; Baskin et al. 1994; Ewalt et al. 1992). In our SEM experimental data we also saw evidence that an overdistended bladder showed at least tern58
porary changes in E C M fibrillar components following acute bladder overdistension. M u r a k u m o et al. (1995) studied the empty, distended and contracted urinary bladders of the guinea pig using high vacuum SEM and demonstrated that the arrangement of collagen fibrils is drastically altered dependent upon the muscle cell extension and contraction. In the empty bladder, the contours of the smooth muscle collagen fibrillar sheaths were usually oval or polygonal, whereas in the distended bladder the sheaths' contours were polygonal. In our study, changes to the shape of the collagen fibrils were evident in the overdistended rat bladders, which lost the polygonal nature of their collagen fibrils. Thus, the results of our three-dimensional analysis morphologically support the findings of Kim et al. (2000) and Chang et al. (1998), which demonstrated that alterations in the vesicular collagen would interfere with bladder function. M u r a k u m o et al. (1995), analyzed the h u m a n bladder wall in S E M and described how the mucosa and the muscular layers had different collagen organization. O u r S E M observations in the rat bladder wall of the control group confirmed the same collagen organization as demonstrated by these authors, and a clear stretching of the collagen fibrils in the overdistended bladder. The present study showed structural modifications of the fibrillar components of the ECM. Despite the fact that no experiment was done to determine whether or not these alterations are reversible, we postulate that these changes, when they occur several times, could cause irreversible changes in the fibrillar components of the E C M of these bladders. In conclusion, our current study has demonstrated that acute bladder overdistension induces striking trends in the collagen fibrils arrangement and their interaction, as well as incipient tissue damage such as edema in the lamina propria and smooth muscle layers. These changes should be considering when overdistension is used as treatment method. However, additional investigations are necessary for better understanding of the extracellular matrix response to acute and chronic bladder overdistension, as well as for the development of novel therapeutic strategies or for preventing the occurrence of irreversible damage to the vesicular tissue.
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