Acta physiol. scand. 1978. 103. 19-30 From the Laboratory of Experimental Biology, Department of Anatomy, University of Goteborg, Sweden
Capillary supply of the muscle fibre population in hindlimb muscles of the cat BY
RHAGNAR MYRHAGE Received 28 October 1977
Abstract MYRHAGE, R. Capillary supply of the muscle fibre population in hindlimb muscles of the cat. Acta physiol. scand. 1978. 103. 19-30. Comparative analyses of the fibre content (FG, FOG, and SO fibres) and the capillary density (the number of capillaries surrounding individual fibres and the capillary/fibre ratio) were performed in hind limb muscles of the cat. Cross-sections from the tenuissimus, the biceps femoris, the lateral head (LG) and the medial head (MG) of the gastrocnemius and the soleus were cut in a cryostat. The sections were stained histochemically for the NADH,-diaphorase and alkaline (pH 9.4) actomyosin ATPase activity, which enables differentiation of different types of fibres. The endothelium of the capillaries was identified uia staining for unspecific alkaline ATPase activity. The number of capillaries surrounding each individual muscle fibre had a positive correlation, first to the oxidative capacity and secondly to the average diameter of the fibres. The thin tenuissimus muscle did not differ in this respect from the thicker muscles. The highest proportion of SO fibres was found in the soleus and the MG muscles. FG fibres of two different types were dominating the fibre mass in the biceps femoris and the LG muscles, while the tenuissimus contained more FOG fibres than these muscles. In general the F G fibres had a larger diameter than the FOG and the SO fibres. The soleus and the M G muscles contained larger fibres than the other examined muscles. FG fibres were surrounded by fewer capillaries than FOG and SO fibres. The soleus and the MG muscles, with a higher percentage of SO fibres and also larger fibres, had the largest number of capillaries around the fibres and the highest capillary/fibre ratio. Key words: Hind limb muscles, muscle fibre population, muscle fibre diameter, capillary density.
The microvascular bed in the cat tenuissimus muscle has previously been analysed with intravital microscopy (Eriksson and Myrhage 1972). Since this muscle is rather thin and small it is not unequivocally representing skeletal muscle tissue in wider aspects. The present study deals with comparative analyses of the muscle fibre population and the capillary supply in the tenuissimus and thicker hind limb muscles in cats. Detailed knowledge of the fibre composition in various skeletal muscles has been provided by histochemical techniques (see review by Close 1972, Khan 1976). Quantitative biochemical analyses have also been performed on muscles with a known fibre content (Dawson and Romanul 1964, Peter et al. 1972). 19
20
RHAGNAR MYRHAGE
Ronianul ( 1965) found that the number of capillaries, surrounding each individual muscle fibre, was directly proportionate to the activity of oxidative enzymes in the fibres. Similar observations have been reported by Andersen (1975). Contradictory to this, Plyley and Groom (1975) stated that the diameter of the muscle fibre is the only factor determinating the number of capillaries in muscle tissue. However, this statement was not based on actual measurements of the muscle fibre diameter. Ariano et al. (1973) have described the percentage distribution of different muscle fibre types in various cat muscles. There is, however, no comparative study available concerning the capillary density in these muscles, even if quite a few authors give data on capillary density in muscles of different fibre composition (see e.g. Hudlicka 1973). The present study will discuss the relationship between fibre type, fibre dimension and capillarization of different fibres in some cat hind limb muscles. The different inuscle fibre types will be described as: FG (fast twitch - glycolytic), FOG (fast twitch - oxidative-glycolytic) and SO (slow twitch -oxidative). These terms were proposed by Petex et al. (1972) and are corresponding to the FF, FR, and S fibres defined by Burke e t a / . (1971). FG, FOG and SO are also corresponding to the terms A, B and C fibres, respectively, in previous publications (Eriksson and Myrhage 1972, Myrhage and Hudlicka 1976).
Materials and Methods 8 adult cats of both sexes, weighing about 2.5 kg, were used. They were anesthetized with chloralose (i.v.
injcction of 50 mgjkg) after induction with ether. The examined muscles (the tenuissimus, the dorsal part of the biceps femoris, both heads of the gastrocnemius, and the soleus) were carefully exposed and transverse segments, 10 mm long, were excised from 3 different parts of each muscle (15 mm distal to the origin and 15 mm proximal to the insertion and one segment from the centre of the muscle belly). In the tenuissimus muscle, however, one segment was taken from the usual area of intravital microscopy, which is located about 10 mm distal to the nutritive artery, emerging from the popliteal f a t pad (cf. Myrhage and Eriksson 1977). The other two segments were taken 20 mm proximal respectively distal to this area. The muscle segments were kept in icecold Hisrocon (Histo-Lab, Goteborg, Sweden) for up to I h, whereafter freezing was performed in liquid propane-propene (Gasol). After mounting o n cryostat chucks the specimens were stored at - 70.C. I n a cryostat ( - 20 C), serial sections (7-10 p n thick) were cut perpendicularly 10 the muscle fibre direction. T o enable qualitative fibre differentiation, the sections were stained hisrochemically for the activity of NADH,-diaphorase (showing the oxidative capacity (Chayen c i ul. 1973) and alkaline (pH 9.4) actomyosin ATPase (Padykula and Herman 1955). The method of unspecific alkaline ATPase described by Guth and Saniaha (1969, 1970) was used for histochemical staining of capillary endothelium. I n i of the 8 animals the hind legs were fixed b) 3 ” ” glutaraldehyde (buffered with 0.075 M sodium cacod!latc to pH 7.2) via perfusion, at a pressure of 13.3 kPa (100 mmHg), through a cannula placed in abdominal aorta. To optimize the filling of the microvascular bed, vasodilation was induced with papaverin (0.2 m g k g ) and preperfusion was undertaken with PerfadexR (cf Myrhage and Eriksson 1977). Muscle segments, as described above, were excised and embedded in Epon and serial sections ( 1 p n thick) were cut, perpendicular to the muscle fibres, in an ultrotome. In sections, stained with toludine blue. the capillaries could cn.;ily be idtntified (Eriksson and Myrhage 1972). For description of the fibre population in the examined muscles, 10 entire muscle fibre bundles (i.e. 300~400fibres) were evaluated in each of the serial sectioned muscle segments. From each section, a train of hundles, extending from the central area towards the periphery of the muscle belly, was photographed in a Leitz Orthomat automatic microscope camera (Leitz-Wetzlar, W. Germany). By comparing photographs from serial sections, alternately stained for diaphorase-, actomyosin ATPase- and unspecific alkaline ATPale activity, different fibre types could be distinguished and quantified. Fibre diameters were calculated
CAPILLARY SUPPLY OF MUSCLE FIBRES
21
Fig. I . Ultrotome section from the medial head of the gastrocnemius muscle (glutaraldehyde fixation). Filled arrows indicate the boundary of a muscle fibre “bundle”; open arrows show the capillaries.
by taking the mean of 4 polygonal diameter measurements, on each of the studied fibres, in calibrated photographs (Cotter et al. 1973). The number of capillaries surrounding each muscle fibre was simultaneously registered. Both glutaraldehyde fixed sections and sections stained for unspecific alkaline ATPase activity were used for evaluation of capillary density. The numbers of capillaries per muscle fibre (the capillary/fibre ratio) and per cross sectional area were calculated in 20 adjacent fibre bundles from each muscle segment.
Comments on methods The muscle fibre bundle
Because of its fairly uniform fibre arrangement, the bundle (Fig. 1 and 2) was judged to be the most representative unit for descriptions of the fibre population. The bundle is enclosed by a delicate sheath of perimysium and contains about 20-80 muscle fibres, which are surrounded by a thin layer of endomysium (Myrhage and Eriksson 1977). The entire fibre mass was grouped into fibre bundles in all the examined muscles. In the tenuissimus, biceps femoris and gastrocnemius muscles most of the large fibres, with low oxidative capacity, were located close to the periphery of the bundles, while the smaller fibres with higher oxidative capacity were situated deeper in the bundles. C a p i h r y cisualization In muscle sections stained ad niodum Padykula and Herman (1955), the alkaline ATPase activity in the different muscle fibres is marked quite intensively. The capillaries are, however, only weakly and unevenly visualized. In the sections stained for unspecific alkaline ATPase activity (Guth and Samaha 1970) the capillaries are distinctly delineated (Fig. 2d). Capillary endothelium has been shown to contain a high activity of alkaline phosphatase (Gomori 1939). ATP (which is used as substrate in methods for ATPase) can be hydrolyzed by alkaline phosphatase as well as by different specific ATPases (Padykula and Herman 1955, Freiman and Kaplan 1960). Since the method by Guth and Samaha (1970) contains no specific inhibitor for alkaline phosphatase, it seems to be quite comparable with the method of Gomori (1939) and all capillaries, present in the section, should be stained by either of these two methods. The capillaries can also be identified in muscle sections fixed by glutaraldehyde (Fig. 1 ; see also Eriksson and Myrhage 1972).
Results Muscle fibre orientation
The muscle fibres in the tenuissimus muscle ran parallel to the longitudinal axis of the muscle. A similar relationship existed in the dorsal part of the biceps femoris and in the
RHAGNAR MYRHAGE
Fig. 2. Cryostat sections from the lateral head (a. c and d) and the medial head (b) of the gastrocnemius muscle. Histochemical staining for NADH,-diaphorase activity (a, b), alkaline ATPase activity ntl nzodurn Padykula and Herman (c) and crtl m o h m Guth and Saniaha (d). Fibre types: FG ( I ) , FG/FOG ( 2 ) . FOG (3) a n d SO (4).
lateral head of the gastrocnemius (LG). In the medial head of the gastrocnemius (MG) a n d i n the soleus muscle the fibres were, however, oriented obliquely to the longitudinal axis of the muscle (Myrhage and Eriksson 1977). F i l m popirlcrtions
Different fibre types could be distinguished by their staining intensity for alkaline ATPaseand NADH,-diaphorase-activity (denoted here as ATPase activity/diaphorase activity): FG high/low, FOG - moderate/high close to the sarcolemma but low to moderate in the centre of the fibre, FG/FOG - high/as in FOG fibres, SO -low/moderate t o high, but evenly distributed (Fig. 2 ) . FG and FOG fibres, taken together, were most common in the tenuissimus, the biceps femoris and the LG muscles, where such fibres constituted about 8 5 % of the total fibre population. More than half of this amount consisted of FOG fibres in all examined tenuisTAIW I . Muscle fibre populations (mean 5 S.E.; n-4). Fihre lqpes
(
Capillary supply of the muscle fibre population in hindlimb muscles of the cat BY
RHAGNAR MYRHAGE Received 28 October 1977
Abstract MYRHAGE, R. Capillary supply of the muscle fibre population in hindlimb muscles of the cat. Acta physiol. scand. 1978. 103. 19-30. Comparative analyses of the fibre content (FG, FOG, and SO fibres) and the capillary density (the number of capillaries surrounding individual fibres and the capillary/fibre ratio) were performed in hind limb muscles of the cat. Cross-sections from the tenuissimus, the biceps femoris, the lateral head (LG) and the medial head (MG) of the gastrocnemius and the soleus were cut in a cryostat. The sections were stained histochemically for the NADH,-diaphorase and alkaline (pH 9.4) actomyosin ATPase activity, which enables differentiation of different types of fibres. The endothelium of the capillaries was identified uia staining for unspecific alkaline ATPase activity. The number of capillaries surrounding each individual muscle fibre had a positive correlation, first to the oxidative capacity and secondly to the average diameter of the fibres. The thin tenuissimus muscle did not differ in this respect from the thicker muscles. The highest proportion of SO fibres was found in the soleus and the MG muscles. FG fibres of two different types were dominating the fibre mass in the biceps femoris and the LG muscles, while the tenuissimus contained more FOG fibres than these muscles. In general the F G fibres had a larger diameter than the FOG and the SO fibres. The soleus and the M G muscles contained larger fibres than the other examined muscles. FG fibres were surrounded by fewer capillaries than FOG and SO fibres. The soleus and the MG muscles, with a higher percentage of SO fibres and also larger fibres, had the largest number of capillaries around the fibres and the highest capillary/fibre ratio. Key words: Hind limb muscles, muscle fibre population, muscle fibre diameter, capillary density.
The microvascular bed in the cat tenuissimus muscle has previously been analysed with intravital microscopy (Eriksson and Myrhage 1972). Since this muscle is rather thin and small it is not unequivocally representing skeletal muscle tissue in wider aspects. The present study deals with comparative analyses of the muscle fibre population and the capillary supply in the tenuissimus and thicker hind limb muscles in cats. Detailed knowledge of the fibre composition in various skeletal muscles has been provided by histochemical techniques (see review by Close 1972, Khan 1976). Quantitative biochemical analyses have also been performed on muscles with a known fibre content (Dawson and Romanul 1964, Peter et al. 1972). 19
20
RHAGNAR MYRHAGE
Ronianul ( 1965) found that the number of capillaries, surrounding each individual muscle fibre, was directly proportionate to the activity of oxidative enzymes in the fibres. Similar observations have been reported by Andersen (1975). Contradictory to this, Plyley and Groom (1975) stated that the diameter of the muscle fibre is the only factor determinating the number of capillaries in muscle tissue. However, this statement was not based on actual measurements of the muscle fibre diameter. Ariano et al. (1973) have described the percentage distribution of different muscle fibre types in various cat muscles. There is, however, no comparative study available concerning the capillary density in these muscles, even if quite a few authors give data on capillary density in muscles of different fibre composition (see e.g. Hudlicka 1973). The present study will discuss the relationship between fibre type, fibre dimension and capillarization of different fibres in some cat hind limb muscles. The different inuscle fibre types will be described as: FG (fast twitch - glycolytic), FOG (fast twitch - oxidative-glycolytic) and SO (slow twitch -oxidative). These terms were proposed by Petex et al. (1972) and are corresponding to the FF, FR, and S fibres defined by Burke e t a / . (1971). FG, FOG and SO are also corresponding to the terms A, B and C fibres, respectively, in previous publications (Eriksson and Myrhage 1972, Myrhage and Hudlicka 1976).
Materials and Methods 8 adult cats of both sexes, weighing about 2.5 kg, were used. They were anesthetized with chloralose (i.v.
injcction of 50 mgjkg) after induction with ether. The examined muscles (the tenuissimus, the dorsal part of the biceps femoris, both heads of the gastrocnemius, and the soleus) were carefully exposed and transverse segments, 10 mm long, were excised from 3 different parts of each muscle (15 mm distal to the origin and 15 mm proximal to the insertion and one segment from the centre of the muscle belly). In the tenuissimus muscle, however, one segment was taken from the usual area of intravital microscopy, which is located about 10 mm distal to the nutritive artery, emerging from the popliteal f a t pad (cf. Myrhage and Eriksson 1977). The other two segments were taken 20 mm proximal respectively distal to this area. The muscle segments were kept in icecold Hisrocon (Histo-Lab, Goteborg, Sweden) for up to I h, whereafter freezing was performed in liquid propane-propene (Gasol). After mounting o n cryostat chucks the specimens were stored at - 70.C. I n a cryostat ( - 20 C), serial sections (7-10 p n thick) were cut perpendicularly 10 the muscle fibre direction. T o enable qualitative fibre differentiation, the sections were stained hisrochemically for the activity of NADH,-diaphorase (showing the oxidative capacity (Chayen c i ul. 1973) and alkaline (pH 9.4) actomyosin ATPase (Padykula and Herman 1955). The method of unspecific alkaline ATPase described by Guth and Saniaha (1969, 1970) was used for histochemical staining of capillary endothelium. I n i of the 8 animals the hind legs were fixed b) 3 ” ” glutaraldehyde (buffered with 0.075 M sodium cacod!latc to pH 7.2) via perfusion, at a pressure of 13.3 kPa (100 mmHg), through a cannula placed in abdominal aorta. To optimize the filling of the microvascular bed, vasodilation was induced with papaverin (0.2 m g k g ) and preperfusion was undertaken with PerfadexR (cf Myrhage and Eriksson 1977). Muscle segments, as described above, were excised and embedded in Epon and serial sections ( 1 p n thick) were cut, perpendicular to the muscle fibres, in an ultrotome. In sections, stained with toludine blue. the capillaries could cn.;ily be idtntified (Eriksson and Myrhage 1972). For description of the fibre population in the examined muscles, 10 entire muscle fibre bundles (i.e. 300~400fibres) were evaluated in each of the serial sectioned muscle segments. From each section, a train of hundles, extending from the central area towards the periphery of the muscle belly, was photographed in a Leitz Orthomat automatic microscope camera (Leitz-Wetzlar, W. Germany). By comparing photographs from serial sections, alternately stained for diaphorase-, actomyosin ATPase- and unspecific alkaline ATPale activity, different fibre types could be distinguished and quantified. Fibre diameters were calculated
CAPILLARY SUPPLY OF MUSCLE FIBRES
21
Fig. I . Ultrotome section from the medial head of the gastrocnemius muscle (glutaraldehyde fixation). Filled arrows indicate the boundary of a muscle fibre “bundle”; open arrows show the capillaries.
by taking the mean of 4 polygonal diameter measurements, on each of the studied fibres, in calibrated photographs (Cotter et al. 1973). The number of capillaries surrounding each muscle fibre was simultaneously registered. Both glutaraldehyde fixed sections and sections stained for unspecific alkaline ATPase activity were used for evaluation of capillary density. The numbers of capillaries per muscle fibre (the capillary/fibre ratio) and per cross sectional area were calculated in 20 adjacent fibre bundles from each muscle segment.
Comments on methods The muscle fibre bundle
Because of its fairly uniform fibre arrangement, the bundle (Fig. 1 and 2) was judged to be the most representative unit for descriptions of the fibre population. The bundle is enclosed by a delicate sheath of perimysium and contains about 20-80 muscle fibres, which are surrounded by a thin layer of endomysium (Myrhage and Eriksson 1977). The entire fibre mass was grouped into fibre bundles in all the examined muscles. In the tenuissimus, biceps femoris and gastrocnemius muscles most of the large fibres, with low oxidative capacity, were located close to the periphery of the bundles, while the smaller fibres with higher oxidative capacity were situated deeper in the bundles. C a p i h r y cisualization In muscle sections stained ad niodum Padykula and Herman (1955), the alkaline ATPase activity in the different muscle fibres is marked quite intensively. The capillaries are, however, only weakly and unevenly visualized. In the sections stained for unspecific alkaline ATPase activity (Guth and Samaha 1970) the capillaries are distinctly delineated (Fig. 2d). Capillary endothelium has been shown to contain a high activity of alkaline phosphatase (Gomori 1939). ATP (which is used as substrate in methods for ATPase) can be hydrolyzed by alkaline phosphatase as well as by different specific ATPases (Padykula and Herman 1955, Freiman and Kaplan 1960). Since the method by Guth and Samaha (1970) contains no specific inhibitor for alkaline phosphatase, it seems to be quite comparable with the method of Gomori (1939) and all capillaries, present in the section, should be stained by either of these two methods. The capillaries can also be identified in muscle sections fixed by glutaraldehyde (Fig. 1 ; see also Eriksson and Myrhage 1972).
Results Muscle fibre orientation
The muscle fibres in the tenuissimus muscle ran parallel to the longitudinal axis of the muscle. A similar relationship existed in the dorsal part of the biceps femoris and in the
RHAGNAR MYRHAGE
Fig. 2. Cryostat sections from the lateral head (a. c and d) and the medial head (b) of the gastrocnemius muscle. Histochemical staining for NADH,-diaphorase activity (a, b), alkaline ATPase activity ntl nzodurn Padykula and Herman (c) and crtl m o h m Guth and Saniaha (d). Fibre types: FG ( I ) , FG/FOG ( 2 ) . FOG (3) a n d SO (4).
lateral head of the gastrocnemius (LG). In the medial head of the gastrocnemius (MG) a n d i n the soleus muscle the fibres were, however, oriented obliquely to the longitudinal axis of the muscle (Myrhage and Eriksson 1977). F i l m popirlcrtions
Different fibre types could be distinguished by their staining intensity for alkaline ATPaseand NADH,-diaphorase-activity (denoted here as ATPase activity/diaphorase activity): FG high/low, FOG - moderate/high close to the sarcolemma but low to moderate in the centre of the fibre, FG/FOG - high/as in FOG fibres, SO -low/moderate t o high, but evenly distributed (Fig. 2 ) . FG and FOG fibres, taken together, were most common in the tenuissimus, the biceps femoris and the LG muscles, where such fibres constituted about 8 5 % of the total fibre population. More than half of this amount consisted of FOG fibres in all examined tenuisTAIW I . Muscle fibre populations (mean 5 S.E.; n-4). Fihre lqpes
(
