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Histochemical Study of Urethral Striated Musculature in the Dog
0022-5347 /82/1282-0406$02.00/0
THE
Vol. 128, August Printed in U.S.A.
JOURNAL OF UROLOGY
Copyright© 1982 by The Williams & Wilkins Co.
HISTOCHEMICAL STUDY OF URETHRAL STRIATED MUSCULATURE IN THE DOG MAHMOUD A. BAZEED, *t JOACHIM W. THUROFF,:j: RICHARD A. SCHMIDT EMIL A. TANAGHO
AND
From the Department of Urology, University of California School of Medicine, San Francisco, California
ABSTRACT
The striated musculature of the dog urethra was studied histochemically. Two main groups of muscle fibers could be identified: 1 with slow twitch oxidative fibers, which are fatigue resistant (type 1), and 1 with fast twitch fibers (type 2). The fast twitch fibers were subdivided into glycolytic (fatiguable) and oxidative glycolytic (fatigue resistant) fibers: the latter constitute 20 per cent of all fast twitch fibers. Type 1 constitutes 35 per cent of the whole musculature and its proportion tends to decrease toward the distal end of the external urethral sphincter. From these observations we infer that type 1 fibers are likely responsible for continence at rest and that type 2 fibers are recruited in stress conditions, for example, during coughing or sneezing. Additional studies are needed to confirm this conjecture. The clinical implications of these studies for the control of incontinence, urinary retention and dyssynergic urethral sphincter are presented. It has been shown that the major contribution to urethral resistance is derived from the smooth and striated muscle components, the striated external sphincter providing more than 50 per cent of the static urethral resistance. 1• 2 The rise in pressure in midurethra is due to combined function of the smooth musculature and the striated muscle fibers around it. 3 During the course of our studies of electric stimulation of the sacral nerve roots innervating the bladder and urethral sphincter, 4-6 we found it essential to study the histochemical characterization of the urethral striated musculature in our experimental canine model: knowledge of this histochemical composition is important for better understanding of the mechanism of continence, to predict the latter's responses to sacral root stimulation, and to interpret this response. It is also important as a baseline for the investigation of effect of prolonged stimulation on the striated urethral muscles. The twitch characteristics of striated muscle fibers correlate with the specific activity of myosin ATP'ase (adenosine triphosphatase) and histochemical assessment of their oxidative and glycolytic capacity. 1- 11 Bernard and associates 9 found that the speed of contraction of striated muscle fibers correlates with the histochemical reaction for ATP'ase at alkaline pH. Fibers that were strongly reactive for ATP'ase were fast twitch whereas weakly reactive fibers were slow twitch. Gauthier 12 and Schiaffino and associates 13 concluded that the resistance to fatigue in a single muscle unit is directly related to the intensity of the oxidative enzyme staining in the same fiber. Oxidative staining is in turn correlated with the interfiber density of mitochondria. On the basis of these observations, type l fibers, which are pale on alkaline ATP'ase and strongly reactive on NADH-TR, are slow twitch oxidative fatigue resistant fibers. Type 2 fibers, which are strongly reactive to alkaline ATP'ase and strongly reactive to phosphorylase, are fast twitch glycolytic. Accepted for publication December 18, 1981. Read at annual meeting of the American Urological Association, Boston, Massachusetts, May 10-14, 1981. Supported by grant NOl-NS-2307 from the National Institutes of Health. * Requests for reprints: Urology, M-553, University of California, San Francisco, California 94143. Current address: Department of Urology, Mansoura University Hospital, Mansoura, Egypt. t Supported by the Agency for International Development through the Egyptian Government and AMIDEAST. :j: Supported by the Deutsche Forschungs Gemeinschaft grant TH274/l, from the Federal Republic of Germany. 406
MATERIALS AND METHODS
Ten mongrel dogs (5 male, 5 female, weight 10-25 kg.) were anesthetized with pentobarbital sodium (25 mg./kg. of body weight). The abdomen was opened through a suprapubic incision including a pubic symphysiotomy. The bladder and urethra were mobilized. In the female animal the whole urethra was removed; in the male animal the prostatic and membranous portions of the urethra were removed; the urethras were divided into 3 equal segments labeled proximal, middle and distal, and immediately frozen into cryon maintained fluid by liquid nitrogen. Six serial sections 10 µ thick were cut by microtome at a temperature of -25C and stained with l of the following stains: trichrome stain, 14 ATP' ase activity after alkaline preincubation at pH 10.4, 15 ATP'ase activity after acid preincubation at pH 4.3, 15 reduced nicotinamide-adenine-dinucleotide-tetrazolium reductase (NADH-TR) as an indication of oxidative enzymes, 16 periodic acid-Schiff (PAS) reaction for glycogen, and phosphorylase stain17 as an indication for glycolytic activity. Slides stained for phosphorylase were interpreted and photographed immediately because phosphorylase fades with time. The staining characteristics of single fibers were examined in the serial sections and classified as slow twitch oxidative or fast twitch glycolytic according to the classification of Peter and associates. 18 We estimated the percentage of each type by sampling at least 500 fibers per section. RESULTS
The dog urethral musculature is composed of 2 layers: the inner layer is made up of smooth musculature and the outer layer of striated musculature (fig. 1). The striated muscle layer increases and the smooth layer decreases toward the distal urethral sphincter. It is easy to identify the smooth fibers from the striated fibers by their smaller diameter and their staining characteristics. Smooth muscles appear pink in trichrome stain whereas striated muscles appear red and connective tissue bluish green. In alkaline ATP'ase smooth muscle fibers appear as circles of dark periphery and clear center (fig. 2, A) because ATP' ase in the center of the fibers is alkali labile. In acid ATP'ase smooth muscle fibers appear homogenously dark (fig. 2, B). Gosling's work showed a detectable size difference between slow and fast twitch fibers. 19 On the basis of their histochemical properties, striated muscle
407
FIG. L Cross section of dog urethra at end of prostatic urethra stained for ATP'ase activity after alkaline preincubation at pH 10.4. Relation between striated and smooth muscle layers is shown. Striated muscles are outermost layer, smooth muscles are darker middle layer, and innermost layer is submucosa. Reduced from x5.
On. acid .A1TF·j~se 4.3) c,ccurna,,s; chaTactcristics are re~ so th2;t type l dark and type 2 is VVe studied the metabolism of these 2 types examining them for activity of mitochondrial oxidative enzymes (N.ADH-diaphorase). Type l fibers were strongly readive to NADH-TR; thus, they are depending on aerobic energy and, consequently, are fatigue resistant, 12 Phosphorylase activity was higher in 2 fibers; thus, they are glycolytic, depending on anaeromc for energy. PAS stain for glycogen showed that type 2 is rich in glycogen content-another indication of its glycolytic nature. As we became more familiar with the histochemical techniques, we noticed that nearly 20 per cent of the fast twitch fibers stain as darkly as (or slightly less than) the slow twitch fibers in the oxidative (NADH-TR) and also are positive for phosphorylase stain (fig. 4, A and B). Thus, this variety of fast twitch fibers is rich in oxidative enzymes in addition to its glycolytic enzymes. This population is fast oxidative glycolytic and fatigue resistant because it can use both aerobic and anaerobic energy pathways. 18 The same population could also be identified in ATP'ase preparations (fig. 4, A) inasmuch as the fibers appear intermediate in staining density between slow and fast twitch fatiguable fibers. Gosling's findings show the external sphincter to be almost pure slow twitch fibers. 19 We found that slow twitch oxidative fibers (type 1) represent approximately 35 per cent of the whole urethral striated musculature and their nr,m,r.rtrn,n decreases toward the distal external sphincter (fig. 5), thus assume that slow twitch oxidative fibers contribute to the mechanism of continence at rest, Fast twitch fibers contribute to the mechanism of continence during stress conditions like or sneezing because can be recruited for short time only. DISCUSSION
Bernard and associates found that the speed of contraction of striated muscle fibers correlate with the histochemical reaction for ATP'ase at alkaline pH, Fibers that were strongly reactive for ATP'ase were fast twitch whereas weakly reactive fibers vvere slow twitch. This correlation has some biochemical basis as shown in additional studies wherein actomyosin and myosin ATP'ase are determined biochemically parallel with the twitch contraction time. 9 • 20 The properties of myosin ATP'ase evaluated biochemically seemed to agree to those of the histochemical reaction for ATP'ase. 21 Such observations are in accord with the hypothesis that the rate of ATP hydrolysis by r:~··:,)i'r,~~~· is an irnportant factor in controlling the intrinsic 21 ' 22 of fiber cocc.,uauuuv and associates 13 concluded that the in a single muscle unit is directly Telated tc oxidative enzyme ,,ca,an.u;; in the same c,cnllrn,H;; is in turn correlated the interfiber 9
type l on alkaline ATP'ase and strongly reactive on NADH-TR, are slow twitch oxidative resistant fibers. Type 2 fibers, which are strongly reactive to alkaline ATP' ase and s:n-oTHnv reactive to phosphorylase, are fast twitch This latter type is fat.iguable because of its low content of oxidative enzymes and its dependence on anaerobic FIG. 2. Staining characteristics of smooth muscle fibers. A, alkalme ATP'ase. B, acid ATP'ase. Reduced from X500.
fibers could be classified into 2 main groups (table, fig. 3): type 1 is pale on alkaline ATP' ase and type 2 is dark, which indicates that type 1 is slow twitch and type 2 fast twitch. 9 • 20 Slow twitch means that when these fibers contract they attain a low amplitude for a long time and fast twitch that they attain a higher amplitude for a shorter time.
Staining characteristics of dog urethral striated musculature (slow twitch fatigue resistant and fast twitch fatiguable) * Muscle Fiber Type
Type I
Type II
Alkaline ATP'ase (pH 10.4) Acid ATP'ase (pH 4.3) NADH-TR PAS Phosphorylase
Pale Dark Dark Pale Red hue
Dark Pale Pale Dark Blue
' Characteristics of fast twitch fatigue resistant fibers are discussed in the text.
408
BAZEED AND ASSOCIATES
FIG. 3. Serial cross sections of striated muscle fibers of dog urethra stained for following reactions. A, ATP' ase activity after alkaline preincubation. B, ATP'ase activity after acid preincubation (pH 4.3). C, NADH-TR activity. D, periodic acid-Schiff staining for glycogen. E, phosphorylase activity. Note that type 1 fibers are pale on alkaline ATP'ase, PAS and phosphorylase and are dark on acid ATP'ase and NADH-TR. Reduced from X500.
energy. However, we found that in the striated urethral sphincter 20 per cent of fast twitch fibers are oxidative glycolytic, which are fatigue resistant because they can utilize both anaerobic and aerobic energy. Some authors believe that both types of fast twitch fibers, the fatiguable (glycolytic) and the fatigue resistant (oxidative glycolytic), are the expression of a dynamic range of properties of these fibers, which are interchangable depending on the demand for their use. 23 Further studies are needed to ascertain whether our findings purely describe different states (glycolytic vs. oxidative-glycolytic) within the same population of fast twitch fibers, or whether there are 3 distinct, different fiber types. We theorize that slow twitch oxidative fibers are an integral part of the continence mechanism during rest because they are
fatigue resistant and can contract for a long time at a low amplitude. Although this assumption parallels the conclusions of other authors, 24 ' 25 who found that in adult muscles, slow twitch oxidative fibers are recruited more often, it is still in need of further investigation. Fast twitch fibers are recruited during stress conditions; this may explain the significance of their increase toward the distal end of the urethral sphincter. The predominance of fast twitch fibers is parallel to and may explain 2 phenomena investigated in our laboratory. The 1st is that, when measuring urethral pressure in dogs under the stress condition of sneezing at 4 different urethral levels, simultaneously with bladder pressure, pressure increase at the level of the striated sphincter was faster and higher than pressure increase in the more proximal regions of the urethra (fig. 6). 26
URETHRAL STRIATED MUSCULATURE
409
the advantage of muscle fatigue: the higher the frequency of the stimulus the faster the fatigue (fig. 7). 28 Stimulating the 2nd sacral root at a frequency of 200 Hz induced sustained bladder contraction and rapid external sphincter fatigue, with resulting micturition. However, this did not decrease the pressure at the external urethral sphincter below that of the prestimulation baseline. This fact may be explained by our assumption that slow twitch fibers are the main striated muscle component contributing to continence at rest. Because they are fatigue resistant, fatigue does not affect the prestimulation baseline pressure. Our continuing histochemical studies should enable us to modulate the metabolism of the urethral striated musculature through prolonged electric stimulation: by decreasing their fatiguability an effective closure could be maintained and a potential treatment for some cases of incontinence be envisioned. Our studies of the histochemistry of the human urethral musculature will, we hope, introduce histochemical techniques as a new tool for the diagnosis of some cases of incontinence, urinary retention and dyssynergic urethral sphincter.
Acknowledgment. We gratefully acknowledge the technical assistance of Mrs. Dung H. Luu, and the help and advice of Douglas M. Wiggin, neurophysiologist, Elwood W. Wright, biomedical engineer (Urology Laboratory, U.C.S.F.), and Daniel A. Riley, Associate Professor of Anatomy, Wisconsin Medical College at Milwaukee.
Fm. 4. Serial cross sections of striated urethral musculature of the dog stained for the following reactions. A, NADH-TR activity. B, phosphorylase stain. C, alkaline ATP'ase. Note that fast twitch fatigue resistant or intermediate fibers (I) are intermediate in staining density in alkaline ATP'ase whereas slow twitch fibers (S) are pale and fast twitch fatiguable (F) dark. Intermediate fibers are dark in both oxidative and phosphorylase stain, indicating that they have both oxidative and glycolytic activities. Reduced from X400.
The 2nd phenomenon is that, after electrostimulation of the sacral roots to increase urethral resistance (a potential method of treatment for urinary incontinence), the pressure increase occurred predominantly in the region of the distal external sphincter. Even with phasic stimulation there was a slight degree of pressure loss after 35 minutes of stimulation because of fatigue. 27 To control micturition in our experimental animals we used
Fm. 5. Alkaline ATP'ase of the dog urethra. A is more proximal to B showing the increase of fast twitch fibers (dark) as we go distally. Reduced from X400.
410
BAZEED AND ASSOCIATES
3.
4.
5. 6. 7. 8.
9. 10.
11.
12.
13.
14. 15. SNEEZE
-FIG. 6. Pressure recording of a sneezing female dog shows that distal urethral pressure increase (U.) precedes and exceeds all other recorded pressures. S2.
STIMULATION
16.
17.
18.
cmH 2 0
]M,DDeR
19.
20. 21.
22. 23.
ON
Oi=F
3s«c
5sec
24.
o.av 30 Hz
FIG. 7. Stimulation of 2nd sacral root with stimuli of increasing frequency shows that a stimulus of 200 Hz induces sustained bladder contraction and rapid fatigue of external urethral sphincter, and initiates micturition.
REFERENCES 1. Tanagho, E. A., Meyers, F. H. and Smith, D.R.: Urethral resistance: its components and implications. I. Smooth muscle component. Invest. Urol., 7: 136, 1969. 2. Tanagho, E. A., Meyers, F. H. and Smith, D.R.: Urethral resistance:
25.
26.
27. 28.
its components and implications. II. Striated muscle components. Invest. Urol., 7: 195, 1969. Tanagho, E. A., Miller, E., Meyers, F. H. and Corbett, R. K.: Observations on the dynamics of the bladder neck. Br. J. Urol., 38: 72, 1966. Schmidt, R. A., Bruschini, H. and Tanagho, E. A.: Urinary bladder and sphincter responses to stimulation of dorsal and ventral sacral roots. Invest. Urol., 16: 300, 1979. Schmidt, R. A., Bruschini, H. and Tanagho, E. A.: Sacral root stimulation in controlled micturition. Peripheral somatic neurotomy and stimulated voiding. Invest. Urol., 17: 130, 1979. Schmidt, R. A. and Tanagho, E. A.: Feasibility of controlled micturition through electric stimulation. Urol. Int., 34: 199, 1979. Edgerton, V. R. and Simpson, D. R.: The intermediate fiber of rats and guinea pigs. J. Histochem. Cytochem., 17: 823, 1969. Gillespie, C. A., Simpson, D. R. and Edgerton, V. R.: High glycogen content of red as opposed to white skeletal muscle fibers of guinea pigs. J. Histochem. Cytochem., 18: 552, 1970. Barnard, R. J. and Peter, J. B.: Effect of exercise on skeletal muscle. 3. Cytochrome changes. J. Appl. Physiol., 31: 904, 1971. Peter, J.B., Kar, N. C., Barnard, R. J., Pearson, C. M. and Edgerton, V. R.: Distribution of acid hydrolases in guinea pig skeletal muscle. Biochem. Med., 6: 257, 1972. Riley, D. A. and Berger, A. J.: A regional histochemical and electromyographic analysis of the cat respiratory diaphragm. Exp. Neurol., 66: 636, 1979. Gauthier, G. F.: On the relationship of ultrastructural and cytochemical features to colour in mammalian skeletal muscle. Z. Zellforsch. Mikrosk. Anat., 95: 462, 1979. Schiaffino, S., Hanzlikova, V. and Pierobon, S.: Relations between structure and function in rat skeletal muscle fibers. J. Cell. Biol., 47: 108, 1970. Engel, W. K. and Cunningham, G. C.: Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections. Neurology (Minneapolis), 13: 919, 1963. Guth, L. and Samaha, F. J.: Procedure for the histochemical demonstration of actomyosin ATP'ase. Exp. Neurol., 28: 365, 1970. Nachlas, M. M., Walker, D. G. and Seligman, A. M.: A histochemical method for the demonstration of diphosphopyridine nucleotide diaphorase. J. Biophysiol. Biochem. Cytol., 4: 29, 1958. Eranko, 0. and Palkama, A.: Improved localization of phosphorylase by the use of polyvinyl pyrrolidone and high substrate concentration. J. Histochem. Cytochem., 9: 586, 1961. Peter, J. B., Barnard, R. J., Edgerton, V. R., Gillespie, C. A. and Stempel, K. E.: Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry, 1:1.: 2627, 1972. Gosling, J. A., Dixon, J. S., Critchley, H. 0. D. and Thompson, S. A.: A comparative study of the human external sphincter and periurethral levator ani muscles. Br. J. Urol., 53: 35, 1981. Barany, M.: ATP'ase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol., 50: 197, 1967. Guth, L. and Samaha, F. J.: Qualitative difference between actomyosin ATP'ase of slow and fast mammalian muscle. Exp. Neurol., 25: 138, 1969. Close, R. I.: Dynamic properties of mammalian skeletal muscles. · Physiol. Rev., 52: 129, 1972. Guth, L. and Y ellin, H.: The dynamic nature of the so-called "Fiber types of Mammalian skeletal muscles." Exp. Neurol., 31: 277, 1971. Gillespie, C. A., Simpson, D. E., and Edgerton, V. R.: Motor unit recruitment as reflected by muscle fiber glycogen loss in a prosimian (bushbaby) after running and jumping. J. Neurol. Neurosurg. Psychiatry, 37: 817, 1974. Edgerton, V. R., Simpson, D. R., Barnard, R. J. and Peter, J. B.: Phosphorylase activity in acutely exercised muscles. Nature, 225: 866, 1970. Thiiroff, J. W., Bazeed, M.A., Schmidt, R. A. and Tanagho, E. A.: Mechanisms of urinary incontinence: an animal model to study urethral response to stress conditions. J. Urol., 127: June, 1982. Thiiroff, J. W., Bazeed, M.A., Schmidt, R. A., Wiggin, D. M. and Tanagho, E. A.: Functional pattern of sacral root stimulation in dogs. II. Urethral closure. J. Urol., 127: May, 1982. Thiiroff, J. W., Bazeed, M.A., Schmidt, R. A., Wiggin, D. M. and Tanagho, E. A.: Functional pattern of sacral root stimulation in dogs. I. Micturition. J. Urol., 127: May, 1982.
THE
Vol. 128, August Printed in U.S.A.
JOURNAL OF UROLOGY
Copyright© 1982 by The Williams & Wilkins Co.
HISTOCHEMICAL STUDY OF URETHRAL STRIATED MUSCULATURE IN THE DOG MAHMOUD A. BAZEED, *t JOACHIM W. THUROFF,:j: RICHARD A. SCHMIDT EMIL A. TANAGHO
AND
From the Department of Urology, University of California School of Medicine, San Francisco, California
ABSTRACT
The striated musculature of the dog urethra was studied histochemically. Two main groups of muscle fibers could be identified: 1 with slow twitch oxidative fibers, which are fatigue resistant (type 1), and 1 with fast twitch fibers (type 2). The fast twitch fibers were subdivided into glycolytic (fatiguable) and oxidative glycolytic (fatigue resistant) fibers: the latter constitute 20 per cent of all fast twitch fibers. Type 1 constitutes 35 per cent of the whole musculature and its proportion tends to decrease toward the distal end of the external urethral sphincter. From these observations we infer that type 1 fibers are likely responsible for continence at rest and that type 2 fibers are recruited in stress conditions, for example, during coughing or sneezing. Additional studies are needed to confirm this conjecture. The clinical implications of these studies for the control of incontinence, urinary retention and dyssynergic urethral sphincter are presented. It has been shown that the major contribution to urethral resistance is derived from the smooth and striated muscle components, the striated external sphincter providing more than 50 per cent of the static urethral resistance. 1• 2 The rise in pressure in midurethra is due to combined function of the smooth musculature and the striated muscle fibers around it. 3 During the course of our studies of electric stimulation of the sacral nerve roots innervating the bladder and urethral sphincter, 4-6 we found it essential to study the histochemical characterization of the urethral striated musculature in our experimental canine model: knowledge of this histochemical composition is important for better understanding of the mechanism of continence, to predict the latter's responses to sacral root stimulation, and to interpret this response. It is also important as a baseline for the investigation of effect of prolonged stimulation on the striated urethral muscles. The twitch characteristics of striated muscle fibers correlate with the specific activity of myosin ATP'ase (adenosine triphosphatase) and histochemical assessment of their oxidative and glycolytic capacity. 1- 11 Bernard and associates 9 found that the speed of contraction of striated muscle fibers correlates with the histochemical reaction for ATP'ase at alkaline pH. Fibers that were strongly reactive for ATP'ase were fast twitch whereas weakly reactive fibers were slow twitch. Gauthier 12 and Schiaffino and associates 13 concluded that the resistance to fatigue in a single muscle unit is directly related to the intensity of the oxidative enzyme staining in the same fiber. Oxidative staining is in turn correlated with the interfiber density of mitochondria. On the basis of these observations, type l fibers, which are pale on alkaline ATP'ase and strongly reactive on NADH-TR, are slow twitch oxidative fatigue resistant fibers. Type 2 fibers, which are strongly reactive to alkaline ATP'ase and strongly reactive to phosphorylase, are fast twitch glycolytic. Accepted for publication December 18, 1981. Read at annual meeting of the American Urological Association, Boston, Massachusetts, May 10-14, 1981. Supported by grant NOl-NS-2307 from the National Institutes of Health. * Requests for reprints: Urology, M-553, University of California, San Francisco, California 94143. Current address: Department of Urology, Mansoura University Hospital, Mansoura, Egypt. t Supported by the Agency for International Development through the Egyptian Government and AMIDEAST. :j: Supported by the Deutsche Forschungs Gemeinschaft grant TH274/l, from the Federal Republic of Germany. 406
MATERIALS AND METHODS
Ten mongrel dogs (5 male, 5 female, weight 10-25 kg.) were anesthetized with pentobarbital sodium (25 mg./kg. of body weight). The abdomen was opened through a suprapubic incision including a pubic symphysiotomy. The bladder and urethra were mobilized. In the female animal the whole urethra was removed; in the male animal the prostatic and membranous portions of the urethra were removed; the urethras were divided into 3 equal segments labeled proximal, middle and distal, and immediately frozen into cryon maintained fluid by liquid nitrogen. Six serial sections 10 µ thick were cut by microtome at a temperature of -25C and stained with l of the following stains: trichrome stain, 14 ATP' ase activity after alkaline preincubation at pH 10.4, 15 ATP'ase activity after acid preincubation at pH 4.3, 15 reduced nicotinamide-adenine-dinucleotide-tetrazolium reductase (NADH-TR) as an indication of oxidative enzymes, 16 periodic acid-Schiff (PAS) reaction for glycogen, and phosphorylase stain17 as an indication for glycolytic activity. Slides stained for phosphorylase were interpreted and photographed immediately because phosphorylase fades with time. The staining characteristics of single fibers were examined in the serial sections and classified as slow twitch oxidative or fast twitch glycolytic according to the classification of Peter and associates. 18 We estimated the percentage of each type by sampling at least 500 fibers per section. RESULTS
The dog urethral musculature is composed of 2 layers: the inner layer is made up of smooth musculature and the outer layer of striated musculature (fig. 1). The striated muscle layer increases and the smooth layer decreases toward the distal urethral sphincter. It is easy to identify the smooth fibers from the striated fibers by their smaller diameter and their staining characteristics. Smooth muscles appear pink in trichrome stain whereas striated muscles appear red and connective tissue bluish green. In alkaline ATP'ase smooth muscle fibers appear as circles of dark periphery and clear center (fig. 2, A) because ATP' ase in the center of the fibers is alkali labile. In acid ATP'ase smooth muscle fibers appear homogenously dark (fig. 2, B). Gosling's work showed a detectable size difference between slow and fast twitch fibers. 19 On the basis of their histochemical properties, striated muscle
407
FIG. L Cross section of dog urethra at end of prostatic urethra stained for ATP'ase activity after alkaline preincubation at pH 10.4. Relation between striated and smooth muscle layers is shown. Striated muscles are outermost layer, smooth muscles are darker middle layer, and innermost layer is submucosa. Reduced from x5.
On. acid .A1TF·j~se 4.3) c,ccurna,,s; chaTactcristics are re~ so th2;t type l dark and type 2 is VVe studied the metabolism of these 2 types examining them for activity of mitochondrial oxidative enzymes (N.ADH-diaphorase). Type l fibers were strongly readive to NADH-TR; thus, they are depending on aerobic energy and, consequently, are fatigue resistant, 12 Phosphorylase activity was higher in 2 fibers; thus, they are glycolytic, depending on anaeromc for energy. PAS stain for glycogen showed that type 2 is rich in glycogen content-another indication of its glycolytic nature. As we became more familiar with the histochemical techniques, we noticed that nearly 20 per cent of the fast twitch fibers stain as darkly as (or slightly less than) the slow twitch fibers in the oxidative (NADH-TR) and also are positive for phosphorylase stain (fig. 4, A and B). Thus, this variety of fast twitch fibers is rich in oxidative enzymes in addition to its glycolytic enzymes. This population is fast oxidative glycolytic and fatigue resistant because it can use both aerobic and anaerobic energy pathways. 18 The same population could also be identified in ATP'ase preparations (fig. 4, A) inasmuch as the fibers appear intermediate in staining density between slow and fast twitch fatiguable fibers. Gosling's findings show the external sphincter to be almost pure slow twitch fibers. 19 We found that slow twitch oxidative fibers (type 1) represent approximately 35 per cent of the whole urethral striated musculature and their nr,m,r.rtrn,n decreases toward the distal external sphincter (fig. 5), thus assume that slow twitch oxidative fibers contribute to the mechanism of continence at rest, Fast twitch fibers contribute to the mechanism of continence during stress conditions like or sneezing because can be recruited for short time only. DISCUSSION
Bernard and associates found that the speed of contraction of striated muscle fibers correlate with the histochemical reaction for ATP'ase at alkaline pH, Fibers that were strongly reactive for ATP'ase were fast twitch whereas weakly reactive fibers vvere slow twitch. This correlation has some biochemical basis as shown in additional studies wherein actomyosin and myosin ATP'ase are determined biochemically parallel with the twitch contraction time. 9 • 20 The properties of myosin ATP'ase evaluated biochemically seemed to agree to those of the histochemical reaction for ATP'ase. 21 Such observations are in accord with the hypothesis that the rate of ATP hydrolysis by r:~··:,)i'r,~~~· is an irnportant factor in controlling the intrinsic 21 ' 22 of fiber cocc.,uauuuv and associates 13 concluded that the in a single muscle unit is directly Telated tc oxidative enzyme ,,ca,an.u;; in the same c,cnllrn,H;; is in turn correlated the interfiber 9
type l on alkaline ATP'ase and strongly reactive on NADH-TR, are slow twitch oxidative resistant fibers. Type 2 fibers, which are strongly reactive to alkaline ATP' ase and s:n-oTHnv reactive to phosphorylase, are fast twitch This latter type is fat.iguable because of its low content of oxidative enzymes and its dependence on anaerobic FIG. 2. Staining characteristics of smooth muscle fibers. A, alkalme ATP'ase. B, acid ATP'ase. Reduced from X500.
fibers could be classified into 2 main groups (table, fig. 3): type 1 is pale on alkaline ATP' ase and type 2 is dark, which indicates that type 1 is slow twitch and type 2 fast twitch. 9 • 20 Slow twitch means that when these fibers contract they attain a low amplitude for a long time and fast twitch that they attain a higher amplitude for a shorter time.
Staining characteristics of dog urethral striated musculature (slow twitch fatigue resistant and fast twitch fatiguable) * Muscle Fiber Type
Type I
Type II
Alkaline ATP'ase (pH 10.4) Acid ATP'ase (pH 4.3) NADH-TR PAS Phosphorylase
Pale Dark Dark Pale Red hue
Dark Pale Pale Dark Blue
' Characteristics of fast twitch fatigue resistant fibers are discussed in the text.
408
BAZEED AND ASSOCIATES
FIG. 3. Serial cross sections of striated muscle fibers of dog urethra stained for following reactions. A, ATP' ase activity after alkaline preincubation. B, ATP'ase activity after acid preincubation (pH 4.3). C, NADH-TR activity. D, periodic acid-Schiff staining for glycogen. E, phosphorylase activity. Note that type 1 fibers are pale on alkaline ATP'ase, PAS and phosphorylase and are dark on acid ATP'ase and NADH-TR. Reduced from X500.
energy. However, we found that in the striated urethral sphincter 20 per cent of fast twitch fibers are oxidative glycolytic, which are fatigue resistant because they can utilize both anaerobic and aerobic energy. Some authors believe that both types of fast twitch fibers, the fatiguable (glycolytic) and the fatigue resistant (oxidative glycolytic), are the expression of a dynamic range of properties of these fibers, which are interchangable depending on the demand for their use. 23 Further studies are needed to ascertain whether our findings purely describe different states (glycolytic vs. oxidative-glycolytic) within the same population of fast twitch fibers, or whether there are 3 distinct, different fiber types. We theorize that slow twitch oxidative fibers are an integral part of the continence mechanism during rest because they are
fatigue resistant and can contract for a long time at a low amplitude. Although this assumption parallels the conclusions of other authors, 24 ' 25 who found that in adult muscles, slow twitch oxidative fibers are recruited more often, it is still in need of further investigation. Fast twitch fibers are recruited during stress conditions; this may explain the significance of their increase toward the distal end of the urethral sphincter. The predominance of fast twitch fibers is parallel to and may explain 2 phenomena investigated in our laboratory. The 1st is that, when measuring urethral pressure in dogs under the stress condition of sneezing at 4 different urethral levels, simultaneously with bladder pressure, pressure increase at the level of the striated sphincter was faster and higher than pressure increase in the more proximal regions of the urethra (fig. 6). 26
URETHRAL STRIATED MUSCULATURE
409
the advantage of muscle fatigue: the higher the frequency of the stimulus the faster the fatigue (fig. 7). 28 Stimulating the 2nd sacral root at a frequency of 200 Hz induced sustained bladder contraction and rapid external sphincter fatigue, with resulting micturition. However, this did not decrease the pressure at the external urethral sphincter below that of the prestimulation baseline. This fact may be explained by our assumption that slow twitch fibers are the main striated muscle component contributing to continence at rest. Because they are fatigue resistant, fatigue does not affect the prestimulation baseline pressure. Our continuing histochemical studies should enable us to modulate the metabolism of the urethral striated musculature through prolonged electric stimulation: by decreasing their fatiguability an effective closure could be maintained and a potential treatment for some cases of incontinence be envisioned. Our studies of the histochemistry of the human urethral musculature will, we hope, introduce histochemical techniques as a new tool for the diagnosis of some cases of incontinence, urinary retention and dyssynergic urethral sphincter.
Acknowledgment. We gratefully acknowledge the technical assistance of Mrs. Dung H. Luu, and the help and advice of Douglas M. Wiggin, neurophysiologist, Elwood W. Wright, biomedical engineer (Urology Laboratory, U.C.S.F.), and Daniel A. Riley, Associate Professor of Anatomy, Wisconsin Medical College at Milwaukee.
Fm. 4. Serial cross sections of striated urethral musculature of the dog stained for the following reactions. A, NADH-TR activity. B, phosphorylase stain. C, alkaline ATP'ase. Note that fast twitch fatigue resistant or intermediate fibers (I) are intermediate in staining density in alkaline ATP'ase whereas slow twitch fibers (S) are pale and fast twitch fatiguable (F) dark. Intermediate fibers are dark in both oxidative and phosphorylase stain, indicating that they have both oxidative and glycolytic activities. Reduced from X400.
The 2nd phenomenon is that, after electrostimulation of the sacral roots to increase urethral resistance (a potential method of treatment for urinary incontinence), the pressure increase occurred predominantly in the region of the distal external sphincter. Even with phasic stimulation there was a slight degree of pressure loss after 35 minutes of stimulation because of fatigue. 27 To control micturition in our experimental animals we used
Fm. 5. Alkaline ATP'ase of the dog urethra. A is more proximal to B showing the increase of fast twitch fibers (dark) as we go distally. Reduced from X400.
410
BAZEED AND ASSOCIATES
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4.
5. 6. 7. 8.
9. 10.
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14. 15. SNEEZE
-FIG. 6. Pressure recording of a sneezing female dog shows that distal urethral pressure increase (U.) precedes and exceeds all other recorded pressures. S2.
STIMULATION
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FIG. 7. Stimulation of 2nd sacral root with stimuli of increasing frequency shows that a stimulus of 200 Hz induces sustained bladder contraction and rapid fatigue of external urethral sphincter, and initiates micturition.
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25.
26.
27. 28.
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