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Keywords:

  • rhabdosphincter;
  • structure;
  • histology;
  • cystometry;
  • electromyography;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

In order to understand the structure-function relationship in the male rat rhabdosphincter, the 3D structure of the striated muscle and associated dense connective tissue was reconstructed from representative serial sections cut from the proximal urethra harboring the muscle. The 3D structure was correlated with electromyography (EMG) of the rhabdosphincter, urodynamic parameters (bladder pressure and flow rate), and longitudinal contraction force of the proximal urethra. The muscular component of the rhabdosphincter consisted of a homogeneous population of the fast-twitch-type fibers. In the cranial part, striated muscle formed a complete ring encircling the urethra, deferent ducts, and ducts from seminal vesicles and prostatic lobes. Toward the middle part, the amount of densely packed connective tissue lacking type III collagen increased anteriorly and posteriorly and penetrated the muscular ring that became divided first posteriorly and then anteriorly into two symmetrical halves. In the caudal part, a thin midsagittal dense connective tissue septum remained posteriorly. EMG recordings suggested that the rhabdosphincter muscle was functionally divided into two parts. Unlike the cranial and middle parts, the caudal part did not show the first depolarization peak. It appears that rapid oscillatory oblique-to-circular muscular contractions proceeding in craniocaudal direction in the cranial and middle part draw the anterior wall supported by arch-like dense connective tissue closer to the posterior wall supported by a more rigid rhomboidal raphe. Longitudinal contractions of the urethra are possibly evoked from the proximal and caudal parts of rhabdosphincter. These could lead to simultaneous increase in urethral pressure ensuring rapid urine flow rate. The caudal part could augment the opening of urethral lumen during oscillatory voiding. Anat Rec Part A, 2006. © 2006 Wiley-Liss, Inc.

The rhabdosphincter (RB) composed of specialized striated muscle is found in the proximal urethra. The muscle extends without interruption from the bladder neck to the pelvic floor in all species studied so far. However, its relation to the prostate varies considerably, causing marked species-specific differences in the gross structure of the RB. The prostate may locate inside the RB or be partially overlaid by RB fibers as in cat, dog, and man (Oelrich,1980; Cullen et al.,1983; Wang et al.,1999). The other possibility is the location of the prostatic glands completely outside a more or less cylindrical RB as in the rat, mouse, and guinea pig (Watanabe and Yamamoto,1979; Neuhaus et al.,2001). In rhesus monkeys, the upper third of the RB is circular and bordered by the prostate gland, i.e., prostate lays partly outside the RB (Ganzer et al.,2002)

In studies on the role of RB in voiding, it is vital to understand the structure-function relationships and to know the structures that are homologous between the animal species and humans. This is particularly important when applying the results of animal experiments to micturition problems in man. In order to achieve the goals, serial sections were cut from the proximal urethra and the 3D structure of the RB with the associated dense connective tissue was reconstructed from the digitized light microscopic images. Longitudinal contraction force of the urethra during volume-evoked micturition, electromyography (EMG) at three different sites representing the cranial, middle, and caudal parts of the proximal urethra, and measurements of bladder pressure and urine flow rate were recorded. Our findings show that the proximal urethra of the adult rats has a complex fascial inner structure, which accompanies the division of the RB into structurally and functionally differentiated parts.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Animals

Adult male Noble strain rats (300–400 g) were used for the study, approved by the Animal Care and Use Committee of Turku University. The rats were maintained under standard laboratory conditions at 12:12 light/dark cycle. They had free access to soy-free food pellets (SDS; Witham, Essex, U.K.) and tap water.

Histology and 3D Reconstructions

Bladder, RB, and prostatic lobes dissected as a block from six adult male rats were immediately fixed in 10% buffered formalin for 24 hr at room temperature. After fixation, the samples were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. At 250 μm intervals, 5 μm sections were cut perpendicular to the longitudinal axis of the urethra. The sections were stained with Weigert-Van Gieson method to indicate collagenous connective tissue. The images were taken under the Olympus SZX9 microscope (Olympus Optical, Tokyo, Japan) and digitized with the Olympus DP-Soft imaging program (Soft Imaging system, Münster, Germany). The 3D structure of RB was reconstructed from eight representative serial sections with 3D Studio Max R3 program (Kinetix; Autodesk).

Immunohistochemstry

For immunohistochemical visualization of type III collagen and fast- and slow-type skeletal myosin, 7 μm thick cryosections were prepared from the tissues frozen in liquid nitrogen. The sections were fixed for 4 min in methanol at −20°C and for 2 min in acetone at −20°C. Endogenous peroxidase activity was blocked with 20-min incubation in 1% H2O2. Masking of unspecific immunoglobulin binding sites was carried out at 4°C with 1-hr incubation in PBS containing 10% normal rabbit serum (Sigma Chemical, St. Louis, MO) for anticollagen III staining, and in 10% normal goat serum for antimyosin staining. Washed sections were then incubated over night at 4°C with the primary antibody diluted in PBS-3% bovine serum albumin (BSA; Sigma Chemical). Goat antitype III collagen antibody (Southern Biotechnology Associates, Birmingham, AL) was diluted 1:1,000; monoclonal antiskeletal slow-type myosin and antiskeletal fast-type myosin antibodies (Sigma Chemical) were diluted 1:500 in PBS-BSA. Secondary biotinylated rabbit antigoat IgGs (Zymed Laboratories, South San Francisco, CA) and goat antimouse IgGs (DakoCytomation, Glostrup, Denmark) antibodies were diluted 1:200 in PBS-2% normal rat serum. Immunoreactions were visualized with the avidin-biotin method (Vectastain ABC-kit; Vector Laboratories, Burlingame, CA) using diaminobenzidine (Zymed Laboratories) as a chromogen. For smooth muscle specific α-actin staining, 5 μm thick paraffin sections were used. Endogenous peroxidase activity was blocked 1% H2O2. Antismooth muscle α-actin (A-2547; Sigma Chemical) antibody diluted 1:2,000 in dilution buffer was applied for an overnight incubation at 4°C. Biotinylated goat antimouse IgGs (DakoCytomation) were used at 1:200 dilution. Immunoreaction was visualized with avidin-biotin method (DakoCytomation). Negative controls were incubated in the absence of the primary antibodies.

Urodynamical Recordings, RB EMG, and Longitudinal Contraction Force of Urethra

Rats (n = 10) were anesthetized with chloral hydrate (0.9 g/kg i.p.; Sigma Chemical). The anesthesia was maintained with i.v. urethane (0.32 g/kg; Sigma Chemical). Body temperature was kept constantly at 36–38°C by means of a thermostatically controlled animal blanket (Harvard Animal Blanket Control Unit, type 50-7061; Harvard Apparatus, Edgenbridge, U.K.) and, if needed, with a heating lamp as described earlier (Streng et al.,2001). During the recordings, the rat was lying on the back. The bladder, anterior surface of the RB, and distal urethra were exposed by a midline incision of the lower abdomen. The bone of symphysis pubis was cut in vertical direction in order to expose the RB. For bladder pressure measurements, a 20 G i.v. cannula was inserted through the bladder apex into the lumen. The cannula was connected to an infusion pump (SP 100i Syringe Pump; World Precision Instruments, Sarasota, CA) and to a pressure transducer (Statham P23XL, Hato Ray, Puerto Rico) connected to an amplifier (Model 7P122B; Grass Instruments, Quincy, MA). The whole system was filled with saline (0.9% NaCl). After an equilibration time (10 min), continuous infusion of warm (37°C) saline into the bladder (0.185 ml/min) was started. Micturition was evoked by the infusion of saline. Tissues were kept moist with saline (37°C). The urine flow rate was measured by using an ultrasonic flow probe (no. 2, 5SB178; Transonic Systems, Ithaca, NY). The probe inserted around the distal urethra was connected to a flow meter (Animal Research Flowmeter Type, T206; Transonic Systems). The EMG activity was recorded simultaneously from the proximal, middle, and distal parts of the RB by monopolar suction electrodes (i.d. 1.10 mm and o.d. 2.00 mm). The signals were filtered by 50 Hz notch filter of the Grass amplifier. Movement artifacts were minimized by using a flexible tube following the tissue movements and by carrying the electrical signals from the tissue to the Ag/AgCl wire located inside the tubing in saline. To prevent the occurrence of electrocardiographic (ECG) signal, reference and the ground electrodes were placed on the edge of the wound close to the recording electrode. To record the longitudinal force of the RB, two hooks were connected to the anterior surface of the RB (one to the proximal and another to the distal part of the RB), and to a force displacement transducer (FT03; Grass Instruments). Continuous recordings were obtained by using Acq Knowledge 3.5.3 software (Biopac Systems, Santa Barbara, CA). A sampling rate of 400 Hz by the computer software was used. Flow meter signals were filtered to 100 Hz. The recordings lasted for 30–60 min and at least 10 micturitions were collected and analyzed from each rat.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Distribution of Dense Connective Tissue and Striated Muscle

The stack of serial histological cross-sections from the cranial, middle, and caudal parts of the male rat proximal urethra is shown in Figure 1. Striated muscle, the RB, represented the largest tissue component throughout. However, the amount and distribution of dense connective tissue and its relation to striated muscle varied considerably along the craniocaudal axis. In the cranial-most part next to the bladder neck, the striated muscle embraced the urethra only anterolaterally (not shown). In the following cranial sections, the urethra was encircled by a uniform striated muscle ring, a structure covering the urethral area having ductal outlets. In this part, dense connective tissue formed an arch-like submuscular layer delineating also the opposing anterior surface of the urethra. Posteriorly, at the caudal margin of the ductal area, a rhomboid-shaped dense connective tissue with an edge projecting into urethral crest became recognizable. In the middle part of the RB, the anterior dense connective tissue was relatively thick and medially penetrated the muscular ring. When also on the opposing posterior side an intramuscular dense connective tissue septum was formed, the rhabdosphincter became divided into two symmetrical C-shaped halves (Fig. 1). The halves were composed of circularly and oblique-orientated muscle fibers.

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Figure 1. Left: Anterior view of the male rat lower urinary tract showing bladder, rhabdosphincter, distal urethra, penis, and the bulb of penis. Right: Serial sections from the rhabdosphincter stained with Weigert-Van Gieson. Cranially (a and b), dense connective tissue (deep purple, arrows in b) forms anteriorly an arch-like submuscular sheet. Posteriorly (b), at the margin of the ductal area (a), a bulk of a rhomboid-shaped posterior connective tissue is apparent. In the middle part (c and d), penetration of the muscular ring anteriorly and posteriorly causes the division of the rhabdosphincter into two symmetrical halves. Caudally (e and f), the decrease in anterior connective tissue enables the partial reunion of the muscle. U, urethra; E, ejaculatory ducts; P, prostatic ducts.

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In the beginning of the caudal part, the posterior connective tissue septum was at the widest and thereafter tapered again (Fig. 1). The anterior connective tissue septum became considerably reduced and only slender intermuscular septae could be identified in the caudal end of the proximal urethra. Thus, the RB became anteriorly reunited, relatively thick, and had distinct intramuscular fasciculi. Starting already from the middle part of the RB, oblique orientation of the majority of muscle fibers was evident on the posterior side. The outer layer appeared with fibers running laterocaudally from the anterior aspect toward the posterior midsagittal septum (not observable in Fig. 1). Separate superficial striated muscle fiber bundles were seen on the posterior corners of the RB.

Three-Dimensional Structure of Dense Connective Tissue

The 3D structure of the RB constructed from representative serial sections is presented in two rotation angles in Figure 2. As can be seen in the model, the dense connective tissue forms an uninterrupted fascial complex on the anterior and posterior sides of the RB. While the anterior connective tissue sheet tapers, penetrates, and caudally forms intramuscular fasciculi, the outermost connective tissue layer serving for vasculature widens. Craniocaudal variation in the structure of the posterior rhomboid-shaped intramuscular raphe and its relation to the striated muscle tissue was revealed.

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Figure 2. The 3D visualization of the male rat RB (blue) and its connective tissue (purple) reconstructed from eight serial sections. a: Anterior view. b: Rotated lateral view. The model includes an external anterior connective tissue layer (O) shown in green. Tapering of the anterior dense connective tissue sheet (A) occurs in craniocaudal direction. The posterior raphe (P) is a relatively thick rhomboid-shaped structure, which divides the rhabdosphincter posteriorly into two halves. Broken arrow indicates intramuscular growth of the anterior connective tissue.

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Immunocytochemistry

The striated RB was entirely composed of fast-twitch muscle fibers (Fig. 3). The anterior and posterior dense connective tissues were negative for type III collagen and smooth muscle-type α-actin (Fig. 4). Positive labeling for α-actin was seen in the smooth muscle cells of urethral glands and in a thin layer of smooth muscle tissue adjacent to the inner surface of the RB (Fig. 4c and d).

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Figure 3. Immunohistochemical staining of fast- (ac) and slow- (df) type striated muscle fibers in the cranial, middle, and caudal parts of rhabdosphincter. Positive labeling is seen for fast-type fibers only. Ant, anterior; post, posterior side.

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Figure 4. Collagen type III (a and b) and smooth muscle α-actin (c and d) immunocytochemistry in the middle part of rhabdosphincter. The anterior (a and c) and the rhomboid posterior (b and d) dense connective tissue structures (arrows) are devoid of collagen type III and smooth muscle α-actin. Muscular wall of the ducts (D) and urethral glands (G) and a slender smooth muscle tissue (asterisk) under the rhabdosphincter serve as positive controls for α-actin labeling. S, striated muscle of rhabdosphincter.

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Urodynamical Recordings, RB EMG, and Longitudinal Contraction Force of Urethra

A total of 88 micturitions from 10 rats with RB EMG from the cranial, middle, and caudal parts of the muscle were analyzed. The four phases described (Chien et al.,2000) were seen in all the bladder pressure waves (A in Fig. 5A). The high-amplitude RB EMG was seen during the second phase of micturition and therefore only this phase is dealt with in the present results (B–D in Fig. 5). The phases of EMG activity peaks included the simultaneous high-frequency oscillations (HFOs) of bladder intraluminal pressure and urine flow peaks as is shown in details in Figure 5B. In the cranial and middle parts of the RB, an “M”-shaped electrical activation of the muscle was observed. The activity started with the first depolarizing potential (FDP) and a concomitant increase in the HFO. A transient negative repolarization (TRP) spike interrupted the depolarization. After the TRP, the second depolarizing potential (SDP) was seen. The flow rate peaks (E in Fig. 5) occurred during the TRPs, in line with our earlier observations (Streng et al.,2001). The amplitude of FDPs, SDPs, and TRPs showed interindividual variation. In some cases, the amplitude of the FDPs and SDPs was lower in the middle than in the cranial RB. The EMG activity considerably differed in the caudal part of the RB, where the FDP was absent or markedly reduced. However, the TRPs and SDPs occurred simultaneously in all parts of the muscle. Even though the shape of the RB EMG was different in various parts, the total EMG amplitude showed no differences, being 0.8 ± 0.19 mV in the cranial, 0.9 ± 0.17 mV in the middle, and 0.8 ± 0.33 mV in the caudal area. In all animals studied, the RB contracted toward the middle part of the muscle in synchrony with the HFOs and urine flow peaks (Fig. 6).

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Figure 5. A: One micturition cycle of an adult male rat. The voiding phases are shown by numbers 1–4. High-frequency oscillations (A) of the bladder pressure, urine flow peaks (E), and EMG activity of cranial (B), middle (C), and caudal (D) parts of the rhabdosphincter all occur during the second oscillatory phase. Oscillations boxed in panel A are shown enlarged in panel B. In the proximal and middle parts of rhabdosphincter, the FDP starts the EMG activity. This activity is cut off by a TRP. The period of EMG activity ends up with the SDP. In the middle part, the depolarization amplitude shows some variation. In the distal part, the FDP is absent or markedly reduced. The dashed box in panel B frames the sequential events during one contraction-relaxation period.

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Figure 6. Longitudinal contractions of proximal urethra, i.e., RB during voiding. High-frequency oscillations (A) of bladder (BL) pressure occur simultaneously with longitudinal contractions of cranial (B) and caudal (C) parts of RB. Contractions proceed in opposite directions (arrows). Urine flow rate (D) is recorded from the distal part of the urethra (DU). Synchrony of high-frequency oscillations, longitudinal urethral contractions, and urine flow peaks is indicated by a dashed line. BS, bulbospongiosus muscle.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The morphological descriptions published so far have given an oversimplified picture of the male rat RB. According to Watanabe and Yamamoto (1979), the striated muscle layer is thick at the anterior and lateral aspects of urethra, but may be less complete posteriorly and the fibers run mostly in a circular direction. This agrees with our findings but has left open the question of the structure-function relationships. The present findings extend earlier observations and show that the structure of the male rat RB contains an intriguing dense connective tissue fascial inner structure, which accompanies the division of the RB into structurally and functionally differentiated parts.

The EMGs recorded from the three consecutive urethral parts suggest that the RB is divided into at least two functionally differentiated parts. EMG measurements of the cranial and middle parts of the rat RB support an active role for these parts (Streng et al.,2001). The transient repolarization interrupts the depolarization wave and causes the relaxation of the urethra. As indicated, the urethra at the level of RB contracts longitudinally in synchrony with the high-frequency oscillations of the intraluminal bladder pressure (HFOs) and urine flow peaks. The opening and closure of the RB must be very fast and the opening period short enough to ensure rapid oscillatory urine flow peaks, as has also been described earlier (Streng et al.,2001). The contractions occurring toward the middle part of the muscle may facilitate the urine flow. In the absence of major longitudinal striated muscle fiber bundles, the longitudinal contractions could be attributable to the oblique-oriented fibers originating from the anterior and posterior aspects of the cranial and middle parts of the RB. The muscle fibers may be attached posteriorly to the raphe formed by the densely packed connective tissue. The raphe, which does not contain type III collagen indicating rigidity, may offer a structural backbone for the muscular contraction. Cranially and in the middle part, the contractions of the muscle fibers could bring the anterior wall supported by the underlying dense connective tissue layer closer to the posterior raphe. The cranial part (except for structures in the cranial-most part) of the RB is composed of circular fibers forming a complete striated muscular ring. In addition to voiding, the cranial part may have a role in blocking the retrograde ejaculation. The orientation of the muscle fibers in the caudal part appeared complex and needs to be studied more thoroughly.

EMG recordings showed the lack of the first depolarization peak in the caudal part of the RB. In the cranial and middle parts, the role of the first depolarization is to prevent leakage of urine into the urethra when the bladder pressure (HFO) is increasing. The lack of the FDP suggests that the urethral wall is not contracting prior to the urine flow peak and therefore the caudal part of RB may have a less active role in voiding. Relaxation, i.e., the transient repolarization of the caudal and middle parts in synchrony with the HFOs and urine flow peaks would, accordingly, augment the opening of the urethral lumen during voiding (Streng et al.,2001). TRP was followed by SDP in all parts of the RB, apparently causing the closure of the urethral lumen, as could also be judged from periodic cessation of urine flow.

The three structurally different parts described now in the male rat RB can be identified in the guinea pig even though there are no clear landmarks for the borders of the hypothetical parts in either species (Neuhaus et al.,2001). In marked contrast to the rat and guinea pig, the feline, canine, and human striated sphincter embraces the prostate, leaving the dorsal circumference open (Oelrich,1980; Cullen et al.,1983; Wang et al.,1999). Oelrich (1980) divided the human male RB into the prostatic (internal) and membranous (external) parts. The prostatic part overlies or is embedded within the anterolateral aspects of human adult prostate as shown for the first time by Henle (1866) and confirmed later by numerous other researchers. According to Oelrich (1980), the most proximal fibers persist as a bundle lying between the bladder and the basis of the prostate. They possibly correspond to the cranial part of the male rat RB. The amount of the prostatic striated musculature is relatively low at the basis of the prostate just below the bladder neck but increases in craniocaudal direction. The fibers cover the anterolateral surface of the prostate and extend to its posterolateral border. They could be the remnants of the middle part of human RB and be homologous with the muscular structures of the middle part of the rat. The lowest part of the human RB, which could be equivalent to the caudal part of the rat RB, consists of omega-shaped muscle located at the apex of the prostate and membranous urethra. Whether the prostatic part of the human RB has any role in the voiding mechanism, as the homologous part of the rat has, remains an intriguing question.

The ratio of the fast-to-slow-twitch myofibers varies among the species and there may also be interindividual differences. Male rat RB is entirely composed of a homogeneous population of the fast-twitch-type fibers. In canine RB, the majority of the fibers are fast-contracting as well (Creed and Van der Werf,2001). Gosling et al. (1981) found only slowly contracting fibers in the urethral sphincter of human males, but according to more recent studies (Schrøder and Reske-Nielsen,1983; Tokunaka et al.,1990; Elbadawi et al.,1997; Light et al.,1997; Ho et al.,1998), about one-third of the fibers are fast-contracting. The myofiber composition may be significant for voiding mechanism. For instance, the dominance of fast-contracting fibers could be one prerequisite for the auxillary role of RB in voiding.

In conclusion, the structure-function analysis of the male rat rhabdosphincter suggests that the muscular component consists of a homogeneous population of fast-twitch striated fibers and that the rhabdosphincter can be divided structurally into three different parts according to the orientation of muscular fibers and the location and amount of dense connective tissue fascial structures. Correlations of the EMG recordings with oscillations of bladder pressure and urine flow suggest that functionally the muscle may be divided into two parts. The present findings increase our understanding on the voiding mechanisms in rat. The may also increase the value of the rat as an experimental animal for the studies of voiding and continence in man.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED