Seh Hong Lim and Tsyr-Jiuan Wang contributed equally to this work.
The Distribution of Muscles Fibers and Their Types in the Female Rat Urethra: Cytoarchitecture and Three-Dimensional Reconstruction
Article first published online: 5 JUL 2013
Copyright © 2013 Wiley Periodicals, Inc.
The Anatomical Record
Volume 296, Issue 10, pages 1640–1649, October 2013
How to Cite
Lim, S. H., Wang, T.-J., Tseng, G.-F., Lee, Y. F., Huang, Y.-S., Chen, J.-R. and Cheng, C.-L. (2013), The Distribution of Muscles Fibers and Their Types in the Female Rat Urethra: Cytoarchitecture and Three-Dimensional Reconstruction. Anat Rec, 296: 1640–1649. doi: 10.1002/ar.22740
- Issue published online: 13 SEP 2013
- Article first published online: 5 JUL 2013
- Manuscript Received: 16 OCT 2013
- Manuscript Accepted: 16 MAY 2013
- Taichung Veterans General Hospital and National Chung-Hsing University . Grant Number: TCVGH-NCHU957614
- National Science Council of Taiwan . Grant Number: NSC99-2320-B-005-005-MY3
- 3D reconstruction;
- female rat urethra;
- internal urethral sphincter;
- striated urethral sphincter
An attempt to explore urethral cytoarchitecture including the distribution of smooth muscles and fast and slow striated muscles of adult female Sprague Dawley rat—a popular model in studying lower urinary tract function. Histological and immunohistochemical stainings were carried out to investigate the distribution of urethral muscle fibers and motor end plates. The urethral sphincter was furthermore three-dimensionally reconstructed from serial histological sections. The mucosa at the distal urethra was significantly thicker than that of other segments. A prominent inner longitudinal and outer circular layer of smooth muscles covered the proximal end of urethra. Thick circular smooth muscles of the bladder neck region (urethral portion) decreased significantly distalward and longitudinal smooth muscles became 2- to 3-fold thicker in the rest of the urethra. An additional layer of striated muscles appeared externally after neck region (urethra) and in association with motor end plates ran throughout the remaining urethra as the striated sphincter layer. Most striated muscles were fast fibers while relatively fewer slow fibers often concentrated at the periphery. A pair of extraneous striated muscles, resembling the human urethrovaginal sphincter muscles, connected both sides of mainly the distal vagina to the dorsal striated muscles in the wall of the middle urethra. The tension provided by this pair of muscles, and in conjunction with the striated sphincter of the urethral wall, was likely to function to suspend the middle urethra and facilitates its closure. Comprehensive morphological data of urethral sphincter offers solid basis for researchers conducting studies on dysfunction of bladder outlet. Anat Rec, 296:1640–1649, 2013. © 2013 Wiley Periodicals, Inc.
external urethral sphincter
lower urinary tract
myosin heavy chain
The function of the lower urinary tract (LUT) to store and periodically eliminate urine is regulated by a complex neural control system located in the brainstem and spinal cord. During urine storage, the urinary bladder serves as a reservoir and the bladder muscle relax, and the outlet (bladder neck, smooth and striated muscles of the urethra) is closed. During voiding, the muscles of outlet relax and detrusor muscles contract, raising intravesical pressure and including urine flow. This reciprocal response is achieved by the coordination between the bladder per se and the outlet (de Groat et al., 2001). A variety of neurologic diseases can induce dysfunction of bladder outlet thereby altering the coordination between bladder and external urethral sphincter (EUS), which impedes urine outflow and compromise voiding efficiency (Cheng and de Groat, 2004). They can also damage the motor innervation of EUS resulting in loss of guarding function and urinary incontinence during storage phase (Hijaz et al., 2008). All of these disorders may lead to urological complications such as urinary retention, bladder hypertrophy, urinary infection, and deterioration of renal function.
The mammalian urethra is a complex organ with a wall consisting of mucosa, submucosa, smooth, and striated muscle laminae. Dysfunction or damage to these structures can result in the development of stress urinary incontinence. A better understanding of the various muscles and systems controlling the urethra would be desirable for the development of drugs targeting at the treatment of stress incontinence. In view of the shortage of available normal human urethral tissue, identification of a suitable animal model is of paramount importance in the quest to increase our current understanding on the cytoarchitecture of urethra.
Rat, a readily available laboratory animal has been used extensively in studies of lower urinary tract function, in particular for investigation of the physiology and pathophysiology of the bladder (Oki et al., 2004; Palea et al., 2004; de Seze et al., 2007; FitzGerald and Graziano, 2007; Yoshimura, 2007). Existing anatomical studies have focused on the urethral sphincter of female rats microscopically (Russell et al., 1996; Praud et al., 2003) leaving the composition and distribution of muscle fibers along the entire length of the urethra at large. A more global understanding of the distribution of muscle fibers and their types along the length of the urethra will not only serve the basis for further studies using the same animal model but also help to resolve whether and how different fibers and their localities along the urethra contribute to the control of urine voiding (Elbadawi, 1996). This in addition, provides the basis for further dysfunctional studies. The aim of our study is to investigate the cytoarchitecture including three-dimensional reconstruction and the immunochemical properties of the urethral sphincter fibers in adult female rats.
MATERIALS AND METHODS
A total of 10 adult female Sprague Dawley rats, aged 10–12 weeks (250–300 g) were used. Rats were housed and cared for according to guidelines of the animal research committee of the National Chung-Hsing University. All efforts were taken to minimize animal suffering and reduce the number of animal used.
Histological and Immunohistochemical Procedures
Rats were anesthetized with ketamine and xylazine (8 mg ketamine and 1 mg xylazine/100 g body weight) and bladders were manually emptied. Rats were then perfused transcardially first with normal saline, followed by a fixative containing 4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.3, at room temperature for 30 min. After perfusion, the urethra and vagina were removed together and postfixed in the same fixative for 4 hr. Following this, tissue was cryoprotected in 30% sucrose in 0.1M PB. The urethra diaphragm was not visible on rat urethra; the urethra was divided into four parts that contained the bladder neck region, proximal, middle and distal segments of the urethra. The bladder neck is the region that connected the urethra and bladder. The V-shape structure of the bladder neck could be easily visualized from the ventral aspect. The urethra was defined as a right circular cylinder structure. The border between the bladder neck and urethra lies on the location where the V-shape of the bladder neck ends and the right circular cylinder structure of the urethra starts (Fig. 1A, left panel). Cryosections (n = 5), 8-μm-thick, were divided into five sets (16 sections per series) and mounted directly in series on silane-coated slides (Dako, Carpinteria, CA). The sets of sections were processed for HE-staining for the overall cytoarchitecture, acetyl cholinesterase (AChE) histochemistry for identifying motor end plates and immunofluorescence stainings of actin, slow myosin heavy chain (MHC) and fast MHC muscle fibers for distinguishing smooth and striated muscle fibers. Antisera to actin (α-smooth muscle fibers, clone 1A4; Sigma), slow (type I skeletal muscle fibers, clone N0Q7.5.4D; Sigma) and fast (type II skeletal muscle fibers, clone MY-32; Sigma) MHC were used for the immunohistochemistry; the copper thiocholine method of Koelle and Friedenwald was used in the AChE histochemistry. To process for actin, fast MHC and slow MHC, slides were treated in 10% normal horse serum in 0.1M PBS for 1 hr at room temperature. Following three rinses in 0.1M PBS, sections were incubated in solution containing the mouse anti-actin (1:400, Sigma, St. Louis, MO), mouse anti-fast MHC (1:400, Sigma) and mouse anti-slow MHC (1:400, Sigma) in 0.1 M PBS for 18 hr at 4°C, respectively. They were then washed with three changes of 0.1M PBS and incubated in secondary antibody solution containing Texas red-conjugated horse anti-mouse IgG (1:100, Vector, Burlingame, CA) and 0.5% Triton X-100 in 0.1 M PBS at room temperature for 1 hr. All reacted sections were washed, counterstained with DAPI and cover-slipped subsequently with a fluorescent mounting medium (Dako, Carpinteria, CA).
Three-Dimensional Reconstruction and Data Analysis
To reconstruct the entire urethra, the layers of the urethral wall in HE-stained serial cross sections of entire urethra (n = 5), 80-µm apart, were traced with a PC-based 3D reconstruction software NeuroLucida 7 (MicroBrightField, Williston, VT) to establish the three-dimensional reconstruction of the urethra. Reconstruction was performed with a 40× objective lens under 1,200 pixels per inch resolution. To investigate the distribution of slow and fast muscle fibers, quantitative analysis of the immunopositive area of the imunoreacted cross sections were performed with Image Pro Plus v.4.5 image analysis system (Media Cybernetics, Silver Spring MD) and the size of the immunopositive area was normalized to the total area of each corresponding section and expressed as percentage to reduce variation between sections. Motor end plates in randomly selected 50 sections (10 sections from each rat) of neck region (urethra), proximal, middle and distal segments were calculated and analyzed. All data are presented as mean ± standard error (SE), and two-tailed Student′s t tests were used to test for statistical differences.
In adult female rats, the urethra extending from the internal urethral opening to the external urethral opening was 15.8 ± 0.15 mm (n = 5) in length. The beginning part, approximate 720 ± 50 µm in length, is connected to the urinary bladder and is named the neck region; the remaining is the principal part of the urethra (Fig. 1A, left panel). Internal and external urethral sphincters were not to be distinguished under naked eyes. Here we further divided the principal part of the urethra into proximal, middle and distal segments of ∼5mm in length each. In HE-stained sections, the wall of the neck region urethral wall consisted of three layers, from the lumen outward: mucosa, submucosa and smooth muscle layer (Fig. 1D′,E′). The smooth muscle layer of the neck region contained a prominent outer layer of circular fibers and a considerably thinner layer of inner longitudinal fibers (Table 1). The striated muscles initially encased external to the urethra in the proximal segment so that it became a four-layered structure (Fig. 2E,I). The circular smooth muscle layer of neck region became thin and discontinuous; on the other hand, the longitudinal fibers became prominent in the urethra (Fig. 1 and Table 1). To quantify our morphological observation in details, dorsal, ventral and lateral quadrants of each layer were distinguished and their thicknesses measured as listed in Table 1. Curve graphs that represent the thickness of different layer of urethral wall were drawn (Fig. 2F) to provide a better sense on how the thickness of these layers varied along the urethra. The mucosa of the distal urethra was thicker than that of other segments while the submucosa layers of all three segments were of comparable thickness (Table 1).
|Neck region||Proximal segment||Middle segment||Distal segment|
|Mucosa layer||27 ± 1||24 ± 1||26 ± 1||25 ± 1||27 ± 1||24 ± 1||23 ± 1||22 ± 1 #||25 ± 1||35 ± 2#||31 ± 1 #||38 ± 2 #|
|Submucosa layer||58 ± 12||65 ± 15||55 ± 9||66 ± 12||55 ± 10||66 ± 12||52 ± 10||58 ± 9||61 ± 10||50 ± 18||59 ± 10||55 ± 12|
|Smooth sphincter layer||Longitudinal||80 ± 5#*||53 ± 3 #||55 ± 2 #||234 ± 14*||118 ± 3||140 ± 3||174 ± 6#*||164 ± 5#*||207 ± 5#||398 ± 13#*||223 ± 10#||227 ± 7#|
|Circular||162 ± 5#*||150 ± 3#*||142 ± 3#||81 ± 9*||39 ± 3||34 ± 2||53 ± 10#*||20 ± 6#||32 ± 3||17 ± 2#||40 ± 3*||19 ± 3#|
|Total||242 ± 10#*||203 ± 6#||197 ± 5#||315 ± 23*||157 ± 6||174 ± 5||227 ± 16#||184 ± 11#*||239 ± 8#||415 ± 15#*||263 ± 13#||246 ± 10#|
|Striated sphincter layer||–||–||–||115 ± 3*||92 ± 3*||106 ±3||191 ± 10#*||126 ± 10#||136 ± 4#||104 ± 3||119 ± 3#*||103 ± 3|
The composition of the smooth muscle layer differed markedly from that of the neck region. First, the thick circular smooth muscles typical of the neck region became extremely thin or discontinuous throughout all three segments of the urethra (Fig. 1 and Table 1). Secondly, the thin longitudinal smooth muscles typical of the neck region were thickened in all three segments of the urethra and their thicknesses varied considerably between segments (Fig. 1 and Table 1). On closer examination, the longitudinal smooth muscle layer was thickest at the distal urethra and was more prominent dorsally than on the lateral and ventral walls of the proximal and distal urethra (Table 1). The outermost layer of the main part of the urethra composed of striated muscle fibers was relatively thick and extended from the proximal urethra all the way distally (Fig. 2D,E). The striated muscle on the dorsal wall of the middle urethra was mostly well developed, almost twice as thick as that of the other parts (Table 1). Different layers and surrounding structures of the segments of urethra were traced and color-filled in Fig. 2A′–C′. The three-dimensional reconstruction of the urethra thus constructed is shown in Fig. 2D,E. Utilizing the three-dimensional reconstruction software, we were able to analyze the estimated volume of different layers along the urethra (Fig. 2E).
In addition to the intrinsic urethral wall muscles, a pair of urethrovaginal sphincter-like (UVS-like) striated muscle bundles was found extending on the ventral surface of vagina and ended on the dorsolateral surface of vagina (Figs. 2 and 3F). They ran along the vaginal wall toward where the middle urethra meets the vagina (Fig. 3B,C), i.e., dorsal wall of the middle urethra and appeared to crisscross with fibers of the opposite side and in addition merged into the striated muscle wall of the middle urethra (Fig. 3F), hence forming the thickening of the striated muscles on the dorsal aspect of the middle urethra (Fig. 3B and Table 1).
To find out how motor end plates are distributed in the urethral wall and the UVS-like striated muscle, AChE histochemistry was performed and each section of the proximal (Fig. 4A), middle and distal urethra contained 64.9 ± 2.7, 84.2 ± 2.8, and 56.2 ± 1.5 motor end plates, respectively (Fig. 4B). The higher density of motor end plates at the middle urethra is consistent with the finding that this segment of the urethra contained thicker striated muscle. Motor end plates were also found at the UVS-like striated muscle. The proximal, middle and distal UVS-like striated muscle contained 10.5 ± 0.7, 26.4 ± 3.1, and 27.0 ± 2.1 motor end plates (Fig. 4B). The middle and distal UVS-like striated muscle significantly contained more motor end plates than the proximal, as UVS-like striated muscle are more abundant in the middle and distal urethra (Fig. 2F,G,I).
Immunohistochemical Identification of Muscle Fiber Types
The smooth muscle fibers of the urethral wall expressed actin (Fig. 3A″–C″). Actin-immunopositive area occupied 13.0% ± 1.3% (52 sections), 14.3% ± 0.8% (50 sections), and 10.6% ± 0.1% (50 sections) of the total cross-sectional area of the proximal, middle, and distal urethra, respectively (Fig. 3G, left panel). On the other hand, most striated muscle fibers were immunoreactive to fast MHC (Fig. 3A–C) and only a few fibers stained positive to the slow MHC (Fig. 3A′–C′). Fast MHC and slow MHC-immunopositive area constituted 10.4% ± 1.0% (42 sections) and 3.5% ± 0.4% (30 sections), 15.3% ± 0.7% (38 sections), and 3.3% ± 0.3% (34 sections), 13.1% ± 0.9% (44 sections), and 4.6% ± 0.5% (46 sections) of the cross-sectional area of the proximal, middle, and distal urethra, respectively (Fig. 3G right panel). Detail quantification showed that fast MHC muscle fibers made up the bulk of the inner circular striated muscle layer and was approximately three folds of that of slow MHC-positive fibers. Slow MHC fibers tended to be more dispersed and many of them concentrated at the periphery of the striated muscle layer especially at the distal urethra. In most cases, fast and slow MHC stained different muscle fibers, although occasional colocalization was observed at the light microscopic level (Fig. 3D,E).
In this study, we provide detail morphological quantification of different tissue layer along the urethra. As majority of the previous studies only focused on urethral muscle tissue, our study demonstrated a more comprehensive observation including the mucosa and submucosa layers. In addition, our study not only focused on the urethra itself, but also the histological connection between urethra and its surrounding organs (vagina). Morphological observations of female rat urethra were arranged and compared with previous studies in Table 2. We found that a circular smooth muscle layer (internal urethral sphincter) was only present at the wall of the neck region. This corresponds to the internal urethral sphincter of the urethra. Although previous study has indicated that external urethral sphincter can be observed (Cruz and Downie, 2005), neither internal urethral sphincter nor external urethral sphincter was visible to naked eyes in our study. With the exception of the neck region, striated muscles were found throughout the main part of the urethra being most prominent around the middle urethra. Thus, this layer of external striated muscles that ran the entire length except for the neck region of the urethra might play a sphincteric function for the bulk of the entire urethra. In addition, our results also suggest that the middle urethra might be more important in modulating micturition. Previous microscopic study described an inner longitudinal smooth muscle layer and an outer circular striated muscle layers surrounding the urethral lumen (Russell et al., 1996). In consistent with our study, Praud et al. (2003), however, described the middle urethra as a thin outer circular and a thick inner longitudinal smooth muscle fiber that separated from the lumen epithelium by a dense connective tissue. This smooth muscle was present along the whole length of the urethra and, near the bladder neck, the outer layer became thicker and replaced the striated sphincter (Praud et al., 2003).
|Morphological observations||Consistency with our findings||Reference|
|Smooth muscle is an inner longitudinal muscle and striated muscle is an outer circular muscle||Partially consistent||Russell et al., 1996, Jankowski et al., 2006|
|Urethral muscle layer is circular oriented where middle urethra is mainly composed of circular striated muscle||Inconsistent||Andersson et al., 1990|
|The amount of muscle in distal urethra is relatively small||Consistent||Andersson et al., 1990|
|Smooth muscle is identified along the entire urethral wall||Consistent||Gosling and Dixon, 1975, Praud et al., 2003|
|Smooth muscle is thickest in the proximal and thinnest in the middle urethra||Consistent||Andersson et al., 1990|
|The ratio of smooth muscle area / total urethral cross section area is found highest in the distal segment||Inconsistent||Jankowski et al., 2004|
|Striated muscle is attached along the entire urethral wall (from proximal to distal)||Consistent||Chen et al., 2012|
|Urethral striated muscle does not have anchorage points like skeletal striated muscle||Consistent||Praud et al., 2003|
|Striated muscle is absent at bladder neck||Consistent||Phillips and Davies, 1980, Andersson et al., 1990, Praud et al., 2003|
|Striated muscle is undetectable in the proximal segment of the urethra||Consistent||Praud et al., 2003|
|Striated muscle can only be found at the middle segment||Inconsistent||Cruz and Downie, 2005|
|Striated muscle is thickest in the middle segment||Consistent||Andersson et al., 1990|
|Striated muscle is found most abundant in the proximal segment that is close to middle segment||Partially consistent||Russell et al., 1996, Kim et al., 2007|
|The ratio of striated muscle area / total urethral cross section area is found highest in the middle segment||Consistent||Jankowski et al., 2004|
|Striated muscle (suggested the existence of UVS-like) is also found to be abundant in the distal segment||Partially consistent||Russell et al., 1996, Kim et al., 2007|
|Dorsal and ventral striated muscle is thicker than the lateral||Inconsistent||Praud et al., 2003|
|The dorsal striated muscle is becoming thinner than the ventral in the distal segment||Consistent||Praud et al., 2003|
|Striated muscle is thicker in the dorsal part of the urethra||Consistent||Praud et al., 2003|
|Most of the striated muscle are MHC fast type fiber and only a few of them are slow MHC||Consistent||Praud et al., 2003, Jankowski et al., 2004, Buffini et al., 2010, Chen et al., 2012|
|Striated muscle is composed of slow type fiber only||Inconsistent||Russell et al., 1996|
|Motor end plates|
|The highest density of motor end plates is found in the proximal segment that is close to middle segment.||Consistent||Kim et al., 2007|
|The highest density of motor end plates is found in the proximal segment||Inconsistent||Praud et al., 2003|
To the best of our knowledge, we are the first to provide solid evidence to demonstrate the existence of UVS-like striated muscle in female rat urethra. This pair of muscles resembles the urethrovaginal sphincter muscle of human that originates from the vagina bilaterally and covers on the urethra (Perucchini et al., 2002). In female rats, an implication of the abundant distal urethral striated muscle may correspond to the existence of the UVS-like striated muscle in female rat urethra had been drawn (Kim et al., 2007). The UVS-like striated muscle extended from the dorsal urethral towards the lateral of vaginal wall. They initially extended from the proximal urethra, fully encased the ventral surface of vagina in the middle urethra and eventually develop toward the dorsolateral of the vagina in the distal urethra. Together with the circular urethral striated muscle wall, they are in a position to suspend and constrict the urethra. The distribution of the UVS-like striated muscle suggested that their contraction may squash the urethra. Urethral striated muscle shares high similarity with skeletal striated muscle (Bierinx and Sebille, 2006) but urethral striated muscle does not have anchorage points as myotendinous junctions of skeletal striated muscle. The discovery of UVS-like striated muscle may implicate that urethral striated muscle could produce contractile force even under the absence of myotendinous junctions. The circular striated muscles along the urethra and the UVS-like striated muscles seems to work together to tightly close the female urethra of rats and may play an important role in the urinary continence.
Pudendal nerve is long known to innervate the urethral striated muscle (McKenna and Nadelhaft, 1986; Ueyama et al., 1987; Marson, 1997). In female rats, retrograde tracing proved that pudendal motor neurons originate from dorsalateral column of L6 spinal cord and supply the urethral striated muscle through its axon (Kane et al., 2002). In morphological studies, the transection and crushing of pudendal nerve were found to cause the atrophy of urethral striated muscle (Heidkamp et al., 1998; Peng et al., 2006) and the decrease of innervated motor end plates (Kong et al., 2009). Our study revealed that the middle urethra, which is composed of thicker striated muscle, contained significantly more motor end plates. We also revealed that UVS-like striated muscle contained motor end plates. Significantly more motor end plates were found in the middle and distal urethra as these segments contain more abundant UVS-like striated muscle. The visualization of motor end plates within the UVS-like striated muscle even further strengthen our speculation that this muscle may play a orchestrating role in the complex neural control of micturition. More studies may need to confirm whether the innervations of striated muscles of urethra and UVS-like striated muscle were all come from the same nerve.
We also found that the circular striated muscle layer contained three times more fast-MHC labeled striated muscle fibers than slow-MHC labeled striated muscle fibers. This is consistent with the finding that the female rat external urethral sphincter contained more fast MHC fibers (Praud et al., 2003; Buffini et al., 2010). This is also in consistent with the findings in male rat urethra (Bierinx and Sebille, 2006). However the proportion of fast and slow MHC fibers in urethral striated muscle may varies between species. Studies showed that the striated urethral sphincter of rabbit (Tokunaka et al., 1993) and dog (Augsburger and Cruz-Orive, 1998) present a similar feature we observed in rat urethra, contains a high proportional of fast fibers. On contrary, human sphincter contains a mixture of slow and fast fibers with slow fibers being the dominant type (Tokunaka et al., 1990). An earlier histochemical study also shows that human striated sphincter contains slow fibers (Gosling et al., 1981). In the rat, both slow and fast fibers were detected using slow and fast myosin immunohistochemical staining method (Russell et al., 1996; Praud et al., 2003; Jankowski et al., 2004). Fast fibers are believed to be involved in the state of continence as intravenous injection of α-bungarotoxin reduced the voiding of paraplegic rats presumably by suppressing high frequency phasic sphincter activity (Yoshiyama et al., 2000). An important role of the presence of fast MHC fibers in the maintenance of urinary continence is also indicated by the finding that there is an association between aging and loss of MHC fast fibers within the urethra (Pandit et al., 2000) or the transformation of most MHC fast into MHC slow fibers during aging (Fujimoto et al., 1994). It is of interest to learn that switching of muscle types also occurs with other striated muscles (Serrano et al., 2001). Apart from age, loss of fast MHC muscle type could also result from parity, disease, prior surgery and genetic and environmental factors (Pette, 1992; Gurgel-Giannetti et al., 2003). Thus, the presence of a relatively thick circular layer of mostly fast MHC type striated muscles along the entire length of the principal part of the female rat urethra is likely to be important in the animal′s urinary continence control. Changes of its muscle fiber types and thickness should be closely examined in future experiments that study the compromise and treatment of urinary incontinence using this animal model. We have illustrated the detail morphological structure of normal female urethra, in either quantitative data or three-dimensional structure. This can be served as a basis for further study in morphological, physiological, pharmacological and biomedical engineering aspects of the injury-induced urine incontinence model.
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