1f.
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Hudllicka
Journal of Phyiology (1991), 444, pp. 1-24 With 18 figures
1
Printed in Great Britain
REVIEW LECTURE* WHAT MAKES BLOOD VESSELS GROW?
BY OLGA HUDLICKA From the Department of Physiology, University of Birmingham, Edgbaston, Birmingham B15 2TT
(Received 31 May 1991) INTRODUCTION
I am very grateful to the Committee of the Physiological Society for the invitation to present the results of my and my collaborator's work in this year's review lecture. To talk about growth of vessels in Oxford is particularly pertinent because of the important contributions made to this field by G. Sanders and G. Schoefl. Sanders greatly improved techniques that enabled direct observation of microcirculation under pathological condition and thus helped to elucidate the problems in development of collateral circulation (North & Sanders, 1958), neovascularization in ocular diseases (Sanders, 1961) and vascularization of tumours (Sanders, 1963). Schoefl (1963; Schoefl & Majno, 1964) studied mostly growth of capillaries during regeneration and inflammation using electron microscopy. Her work represents a classic description of capillary growth by sprouting. Until relatively recently, growth of vessels was studied mainly under pathological conditions and during development. The basic pattern of growth was established during the last century and the beginning of this century (see Hudlicka, 1984): mesenchymal cells develop into angioblasts which differentiate into both blood islands and erythroblasts and into endothelial cells. The latter form capillary networks which grow by sprouting, and larger vessels are formed from these networks by apposition of fibroblasts that later develop into smooth muscle cells (Clark & Clark, 1940). It is thus important to establish factors involved in capillary. growth in the first place.
Capillary growth under physiological circumstances Capillary proliferation is very rare in adult organisms under physiological circumstances. It can be best demonstrated by incorporation of labelled [3H]thymidine into DNA of endothelial cell nuclei using autoradiography. The labelling index, i.e. the number of labelled nuclei expressed as a percentage of all counted nuclei, is extremely low (0 01-1 %) in normal tissues (Denekamp, 1984). The turnover time of endothelial cells calculated on the basis of labelling index is around 10000 days in capillaries in the brain and skin, 1000 in skeletal muscle and bladder * Given at the Meeting of the Physiological Society held in Oxford on 27 July 1990 as the Society's Annual Review Lecture.
MS 9437 1-2
2
0. HUDLICKA
and about 300 days in the heart, kidney or lung. Female reproductive organs are almost the only normal adult tissue with appreciable vessel growth (Reynolds, 1973). The placenta has a turnover time of 10 days - a value similar to that found in malignant tumours (Denekamp, 1984).
I
..
|I
Fig. 1. Cross-section of control (left) and 28 day stimulated (right) rabbit EDL muscle stained for alkaline phosphatase. Capillaries are depicted as black dots. Bar = 100 #sm.
Proliferation of capillaries can also been demonstrated by the presence of capillary sprouts (Cliff, 1963; Schoefl, 1963) or by counting all anatomically present capillaries in tissue samples. Such counting can be based either on histochemical staining for various enzymes present only in capillary endothelium (such as alkaline phosphatase, acid-stable ATPase or PAS (periodic acid-Schiff reagent) in conjunction with amylase, see Hudlicka & Tyler, 1986) or by counting all capillary profiles in lowpower electron micrographs. These methods were used to demonstrate capillary growth induced in skeletal muscles by endurance training (Andersen & Henriksson, 1977; Ingjer, 1979) or long-term exposure to cold (Sillau, Aquin, Lechner & Bui, 1980), and in the heart by long-term exposure to high-altitude hypoxia in combination with cold (Banchero, Kayar & Lechner, 1985), or in endurance training (Tomanek, 1970; Unge, Carlsson, Ljungqvist, Tormling & Adolfsson, 1979; Anversa, Ricci & Olivetti, 1987). So far, the most extensive capillary proliferation was achieved in skeletal muscles by long-term electrical stimulation (Brown, Cotter, Hudlicka & Vrbova, 1976; Myrhage & Hudlicka, 1978; Joplin, Franchi & Salmons, 1987) and in the heart by long-term bradycardial pacing (Wright & Hudlicka, 1981).
WHAT MAKES BLOOD VESSELS GROW?
3
Capillary growth in chronically stimulated skeletal muscles It has been known for a long time that slowly contracting postural muscles have a higher capillary supply than fast muscles used in short-lasting phasic movements (Ranvier, 1874). I came to work in Birmingham shortly after Salmons & Vrbova
-1
E
1.2
~~~~~6001
.2 _; _
0.8E
1 1=1GraciIis stimulated
00
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E 8500 3~~~~400 200
g 60 E 80
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0-61~~~~~~~~~~~~~~~~~~~~Fig. 2. C/F ratio, capillary density (CD), and capillary length (CL) estimated from lowpower electron micrographs in cat gracilis (80 % glycolytic), soleus (100 % oxidative) and chronically stimulated gracilis at 10 Hz for 8 h/day (from Hudlicka et al. 1987).
(1969) demonstrated that long-term stimulation of fast contracting muscles at a frequency occurring naturally in nerves supplying slow muscles transformed the former into the latter. This transformation included changes in myosin isoforms as well as in enzyme patterns (see Pette & Vrbova, 1985) and I was interested to see whether it could also change the vascular supply. Fast muscles in rabbits, rats or cats were subsequently stimulated using electrodes implanted in the vicinity of the appropriate motor nerves and external - mainly portable - stimulators (Tyler & Wright, 1979). The extent of capillary supply was estimated either in frozen sections on the basis of histochemical staining for alkaline phosphatase or in electron micrographs and assessed as increase in capillary density (CD, the number of capillaries/mm2), capillary per fibre ratio, (C/F), or total capillary length (CL) (Hudlicka, Hoppeler & Uhlmann, 1987). Capillary growth occurred remarkably rapidly - after only 4 days of stimulation at 10 Hz for 8 h per day in rabbit extensor digitorum longus, and CD as well as C/F were doubled in muscles stimulated for 28 days (Brown et al. 1976; Hudlicka, Tyler, Wright & Ziada, 1984) (Fig. 1). A similar degree of growth was found in cat fast glycolytic gracilis muscle where the size of the capillary bed eventually equalled that in soleus (Fig. 2). The use of intravital microscopy - direct observation of the vascular bed in transilluminated or epiilluminated muscles - enabled us to identify growth as capillary sprouts. They were found in stimulated rat (extensor hallucis proprius, EHP) and rabbit (tenuissimus) muscles appearing usually at a bend in pre-existing capillaries (Myrhage & Hudlicka, 1978). Within a week, the capillary bed was transformed into one with more numerous and more tortuous capillaries forming loops (Fig. 3). Further experiments (Hudlicka et al. 1984) revealed that capillary growth can be achieved by other types of stimulation, such as trains of tetanic contractions, provided the total amount of
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WHAT MAKES BLOOD VESSELS GROW? 5 stimuli was comparable to stimulation at 10 Hz and was carried out for at least 7 days (Fig. 4). This rapid onset and extent of capillary growth was very different from the increased capillarization described in human muscles where a very small increase in CD was described only after 5 weeks of vigorous endurance training (Andersen & Henriksson, 1977). E E2
40 Hz intermittent, EDL
10 Hz continuous, EDL *
Control
z Stimulated 40 Hz intermittent, soleus
t t
2-
*
Hz ~~~~~~~~~~~~~40 intermittent, FHL
cu
0~~~~~~~~~
3 times/mmn) with the same number of impulses (36000/h). Fast flexor hallucis longus (FHL) or slow soleus were stimulated at 40 Hz (5 s every 100 s) using a much lower total number of impulses (7200/h). *P < 0~01; tP < 0001.
Metabolic factors involved in capillary growth in stimulated muscles Endurance training activates predominantly oxidative fibres (Gollnick, Piehl &r Saltin, 1974) which already have a high capillary supply. In contrast, the type of stimulation used in the above experiments resulted in contraction of all fibres. It is thus possible that activation of glycolytic fibres with a relatively low capillary supply (Gray &; Renkin, 1978) could lead to severe hypoxia which may then act as a stimulus for capillary growth. Hypoxia has been considered as a stimulus for capillary growth since Valdivia (1958) found a considerable increase in capillarization in muscles of guinea-pigs bred at high altitude. Increased activity of oxidative enzymes described in ischaemic muscles was also attributed to hypoxia (Holm, Bjbrntorp & Scherst6n, 1972). Increased activity of cytochrome oxidase and succinate dehydrogenase (SDH) preceded capillary growth in muscles of endurancetrained athletes, and chronic stimulation significantly increased activity of oxidative enzymes at a relatively early stage (Pette, Smith, Staudte & Vrbova, 1973). If hypoxia is an important stimulus for capillary proliferation, growth should start in the vicinity of glycolytic fibres and oxygen tension in stimulated muscles should be low.
0. HUDLICKA The method of Gray & Renkin (1978) was used to localize the initial growth of capillaries. Rabbit fast muscles were stimulated for 8 h/day either continuously at 10 Hz for 2 or 4 days, or intermittently at 40 Hz for 4 or 7 days. Serial frozen sections were stained for myosin ATPase to identify slow or fast fibres, for succinate 6
H Control fl Stimulated at 10 Hz
E Stimulated B
at 40 Hz
7 days
4 days 16 1 4
0
1.2
UY
1.0
08 06 aW
a?
aR
BR
aW
a?
aR
BR
Fig. 5. A, cross-sections of rabbit EDL stained for ATPase after alkaline pre-incubation (a) to depict fast (black) and slow (white) muscle fibres, for succinate dehydrogenase (SDH) (b) to demonstrate darkly stained oxidative and pale glycolytic fibres (with some lightly stained fibres classified as intermediate) and for alkaline phosphatase (c) (black dots). Each capillary was classified as being surrounded by an estimated number of fast glycolytic (aW), fast intermediate (a?), fast oxidative (aR) or slow oxidative (BR) fibres as described by Gray & Renkin (1978). B, C/F ratio in muscles stimulated at 10 Hz for 4 days and those stimulated at 40 Hz for 4 and 7 days (from Hudlicka & Tyler, 1984). * Significantly different from control.
dehydrogenase (SDH) to differentiate oxidative and glycolytic fibres, and for alkaline phosphatase to depict capillaries (Fig. 5A). It was possible to demonstrate increased capillary density in the vicinity of glycolytic fibres in muscles stimulated for only 2 days at 10 Hz (Hudlicka, Dodd, Renkin & Gray, 1982); C/F ratio was also
7 WHAT MAKES BLOOD VESSELS GROW? increased in the vicinity of fast glycolytic or intermediate fibres, and the increase was found earlier in muscles stimulated at 10 than at 40 Hz (Fig. 5B, Hudlicka & Tyler, 1984). These findings would indicate that glycolytic fibres were indeed suffering from hypoxia. However, direct measurements of oxygen tension in the glycolytic part of A
Stimulation 10 Hz
2.016 -
20 fi
* +.
*.
Values at rest and 0-5 min g Postcontraction
~~~~~~~~~~~~~~~~Control
/
XX 0 e ° 1< 2
mfStimulated
0-4
B
4 days
501Stimulation
*2
14 days
4 days
14 days
A
1 0 '40Hzli*neir 30 day
4 days
0-4
-~~~~~~~~~4C
uce
ihra 0()o t4 eesiuae 14dys4das14dy 14 days
z()uigtesm
4 days l4 days
Fig. 6. Left side, oxygen tension (Po2) in control (contralateral) and stimulated rabbit tibialis anterior; muscles were stimulated either at 10 (A) or at 40 Hz (B) using the same number of impulses for 8 h/day for 4 and 14 days. PO was measured at rest and immediately after 5 min of muscle contractions at 4 Hz (lnked with resting values by interrupted lines). Right side, C/F ratio in contralateral and stimulated muscles at 10 Hz (A) or 40 Hz (B). * Significantly different from control.
rabbit tibialis anterior using oxygen electrodes (Hudlicka & Schroeder, 1978; Kanabus, Hudlicka & Tyler, 1980) did not show any changes that could explain capillary growth (Fig. 6): muscles stimulated for 4 days at 10 Hz had similar values of P02 as control muscles, while those stimulated at 40 Hz had higher values. C/F was increased with 10 Hz and not changed with 40 Hz. After 14 days of stimulation, C/F was increased to a similar extent with either frequency, but Po0 was increased in muscles stimulated at 10 Hz and decreased in those stimulated at 40 Hz. The possible role of hypoxia was also refuted by other experiments. In rat extensor digitorum longus, C/F ratio increased after 7 days of stimulation at 10 but not at 40 Hz (Hudlicka, Cotter & Cooper, 1986). Nevertheless, content of lactate (another indicator of muscle hypoxia) was higher in muscles stimulated at 40 Hz and lower in those stimulated at 10 Hz (Fig. 7). Furthermore, stimulation of tibialis anterior using a regime that activated glycolytic, but not oxidative fibres (very low voltage and short trains of tetanic contractions) caused capillary growth in the superficial glycolytic part of the muscle (and not in the oxidative core) that was not accompanied by increased activity of cytochrome oxidase (Egginton & Hudlicka, 1989).
0. HUDLICKA
8 A
30
Control
i
r
20 2
Control, rest
1 035±0 029 mean+S.E.M. n= 56
lo 1 0
-
30
-
C
0
4-
Control,
El
Stimulated at 40 Hz, contracting Stimulated at 10 Hz, contracting
7 days Stimulated at 40 Hz 1.10 - 0.027 ~~n=t 6U
ii;V Frontispiece:
().
Hudllicka
Journal of Phyiology (1991), 444, pp. 1-24 With 18 figures
1
Printed in Great Britain
REVIEW LECTURE* WHAT MAKES BLOOD VESSELS GROW?
BY OLGA HUDLICKA From the Department of Physiology, University of Birmingham, Edgbaston, Birmingham B15 2TT
(Received 31 May 1991) INTRODUCTION
I am very grateful to the Committee of the Physiological Society for the invitation to present the results of my and my collaborator's work in this year's review lecture. To talk about growth of vessels in Oxford is particularly pertinent because of the important contributions made to this field by G. Sanders and G. Schoefl. Sanders greatly improved techniques that enabled direct observation of microcirculation under pathological condition and thus helped to elucidate the problems in development of collateral circulation (North & Sanders, 1958), neovascularization in ocular diseases (Sanders, 1961) and vascularization of tumours (Sanders, 1963). Schoefl (1963; Schoefl & Majno, 1964) studied mostly growth of capillaries during regeneration and inflammation using electron microscopy. Her work represents a classic description of capillary growth by sprouting. Until relatively recently, growth of vessels was studied mainly under pathological conditions and during development. The basic pattern of growth was established during the last century and the beginning of this century (see Hudlicka, 1984): mesenchymal cells develop into angioblasts which differentiate into both blood islands and erythroblasts and into endothelial cells. The latter form capillary networks which grow by sprouting, and larger vessels are formed from these networks by apposition of fibroblasts that later develop into smooth muscle cells (Clark & Clark, 1940). It is thus important to establish factors involved in capillary. growth in the first place.
Capillary growth under physiological circumstances Capillary proliferation is very rare in adult organisms under physiological circumstances. It can be best demonstrated by incorporation of labelled [3H]thymidine into DNA of endothelial cell nuclei using autoradiography. The labelling index, i.e. the number of labelled nuclei expressed as a percentage of all counted nuclei, is extremely low (0 01-1 %) in normal tissues (Denekamp, 1984). The turnover time of endothelial cells calculated on the basis of labelling index is around 10000 days in capillaries in the brain and skin, 1000 in skeletal muscle and bladder * Given at the Meeting of the Physiological Society held in Oxford on 27 July 1990 as the Society's Annual Review Lecture.
MS 9437 1-2
2
0. HUDLICKA
and about 300 days in the heart, kidney or lung. Female reproductive organs are almost the only normal adult tissue with appreciable vessel growth (Reynolds, 1973). The placenta has a turnover time of 10 days - a value similar to that found in malignant tumours (Denekamp, 1984).
I
..
|I
Fig. 1. Cross-section of control (left) and 28 day stimulated (right) rabbit EDL muscle stained for alkaline phosphatase. Capillaries are depicted as black dots. Bar = 100 #sm.
Proliferation of capillaries can also been demonstrated by the presence of capillary sprouts (Cliff, 1963; Schoefl, 1963) or by counting all anatomically present capillaries in tissue samples. Such counting can be based either on histochemical staining for various enzymes present only in capillary endothelium (such as alkaline phosphatase, acid-stable ATPase or PAS (periodic acid-Schiff reagent) in conjunction with amylase, see Hudlicka & Tyler, 1986) or by counting all capillary profiles in lowpower electron micrographs. These methods were used to demonstrate capillary growth induced in skeletal muscles by endurance training (Andersen & Henriksson, 1977; Ingjer, 1979) or long-term exposure to cold (Sillau, Aquin, Lechner & Bui, 1980), and in the heart by long-term exposure to high-altitude hypoxia in combination with cold (Banchero, Kayar & Lechner, 1985), or in endurance training (Tomanek, 1970; Unge, Carlsson, Ljungqvist, Tormling & Adolfsson, 1979; Anversa, Ricci & Olivetti, 1987). So far, the most extensive capillary proliferation was achieved in skeletal muscles by long-term electrical stimulation (Brown, Cotter, Hudlicka & Vrbova, 1976; Myrhage & Hudlicka, 1978; Joplin, Franchi & Salmons, 1987) and in the heart by long-term bradycardial pacing (Wright & Hudlicka, 1981).
WHAT MAKES BLOOD VESSELS GROW?
3
Capillary growth in chronically stimulated skeletal muscles It has been known for a long time that slowly contracting postural muscles have a higher capillary supply than fast muscles used in short-lasting phasic movements (Ranvier, 1874). I came to work in Birmingham shortly after Salmons & Vrbova
-1
E
1.2
~~~~~6001
.2 _; _
0.8E
1 1=1GraciIis stimulated
00
1.41
Li
ElGracilis EZJSoIeus
|
E 8500 3~~~~400 200
g 60 E 80
'>- 0
E 20
0-61~~~~~~~~~~~~~~~~~~~~Fig. 2. C/F ratio, capillary density (CD), and capillary length (CL) estimated from lowpower electron micrographs in cat gracilis (80 % glycolytic), soleus (100 % oxidative) and chronically stimulated gracilis at 10 Hz for 8 h/day (from Hudlicka et al. 1987).
(1969) demonstrated that long-term stimulation of fast contracting muscles at a frequency occurring naturally in nerves supplying slow muscles transformed the former into the latter. This transformation included changes in myosin isoforms as well as in enzyme patterns (see Pette & Vrbova, 1985) and I was interested to see whether it could also change the vascular supply. Fast muscles in rabbits, rats or cats were subsequently stimulated using electrodes implanted in the vicinity of the appropriate motor nerves and external - mainly portable - stimulators (Tyler & Wright, 1979). The extent of capillary supply was estimated either in frozen sections on the basis of histochemical staining for alkaline phosphatase or in electron micrographs and assessed as increase in capillary density (CD, the number of capillaries/mm2), capillary per fibre ratio, (C/F), or total capillary length (CL) (Hudlicka, Hoppeler & Uhlmann, 1987). Capillary growth occurred remarkably rapidly - after only 4 days of stimulation at 10 Hz for 8 h per day in rabbit extensor digitorum longus, and CD as well as C/F were doubled in muscles stimulated for 28 days (Brown et al. 1976; Hudlicka, Tyler, Wright & Ziada, 1984) (Fig. 1). A similar degree of growth was found in cat fast glycolytic gracilis muscle where the size of the capillary bed eventually equalled that in soleus (Fig. 2). The use of intravital microscopy - direct observation of the vascular bed in transilluminated or epiilluminated muscles - enabled us to identify growth as capillary sprouts. They were found in stimulated rat (extensor hallucis proprius, EHP) and rabbit (tenuissimus) muscles appearing usually at a bend in pre-existing capillaries (Myrhage & Hudlicka, 1978). Within a week, the capillary bed was transformed into one with more numerous and more tortuous capillaries forming loops (Fig. 3). Further experiments (Hudlicka et al. 1984) revealed that capillary growth can be achieved by other types of stimulation, such as trains of tetanic contractions, provided the total amount of
0. HUDLICKA
4
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Wo3
0
bD I-,
I4
Ca
o
I)
$411 bQ 4z;
'.
0
0
o
o
co
.,
0
0
_4
WHAT MAKES BLOOD VESSELS GROW? 5 stimuli was comparable to stimulation at 10 Hz and was carried out for at least 7 days (Fig. 4). This rapid onset and extent of capillary growth was very different from the increased capillarization described in human muscles where a very small increase in CD was described only after 5 weeks of vigorous endurance training (Andersen & Henriksson, 1977). E E2
40 Hz intermittent, EDL
10 Hz continuous, EDL *
Control
z Stimulated 40 Hz intermittent, soleus
t t
2-
*
Hz ~~~~~~~~~~~~~40 intermittent, FHL
cu
0~~~~~~~~~
3 times/mmn) with the same number of impulses (36000/h). Fast flexor hallucis longus (FHL) or slow soleus were stimulated at 40 Hz (5 s every 100 s) using a much lower total number of impulses (7200/h). *P < 0~01; tP < 0001.
Metabolic factors involved in capillary growth in stimulated muscles Endurance training activates predominantly oxidative fibres (Gollnick, Piehl &r Saltin, 1974) which already have a high capillary supply. In contrast, the type of stimulation used in the above experiments resulted in contraction of all fibres. It is thus possible that activation of glycolytic fibres with a relatively low capillary supply (Gray &; Renkin, 1978) could lead to severe hypoxia which may then act as a stimulus for capillary growth. Hypoxia has been considered as a stimulus for capillary growth since Valdivia (1958) found a considerable increase in capillarization in muscles of guinea-pigs bred at high altitude. Increased activity of oxidative enzymes described in ischaemic muscles was also attributed to hypoxia (Holm, Bjbrntorp & Scherst6n, 1972). Increased activity of cytochrome oxidase and succinate dehydrogenase (SDH) preceded capillary growth in muscles of endurancetrained athletes, and chronic stimulation significantly increased activity of oxidative enzymes at a relatively early stage (Pette, Smith, Staudte & Vrbova, 1973). If hypoxia is an important stimulus for capillary proliferation, growth should start in the vicinity of glycolytic fibres and oxygen tension in stimulated muscles should be low.
0. HUDLICKA The method of Gray & Renkin (1978) was used to localize the initial growth of capillaries. Rabbit fast muscles were stimulated for 8 h/day either continuously at 10 Hz for 2 or 4 days, or intermittently at 40 Hz for 4 or 7 days. Serial frozen sections were stained for myosin ATPase to identify slow or fast fibres, for succinate 6
H Control fl Stimulated at 10 Hz
E Stimulated B
at 40 Hz
7 days
4 days 16 1 4
0
1.2
UY
1.0
08 06 aW
a?
aR
BR
aW
a?
aR
BR
Fig. 5. A, cross-sections of rabbit EDL stained for ATPase after alkaline pre-incubation (a) to depict fast (black) and slow (white) muscle fibres, for succinate dehydrogenase (SDH) (b) to demonstrate darkly stained oxidative and pale glycolytic fibres (with some lightly stained fibres classified as intermediate) and for alkaline phosphatase (c) (black dots). Each capillary was classified as being surrounded by an estimated number of fast glycolytic (aW), fast intermediate (a?), fast oxidative (aR) or slow oxidative (BR) fibres as described by Gray & Renkin (1978). B, C/F ratio in muscles stimulated at 10 Hz for 4 days and those stimulated at 40 Hz for 4 and 7 days (from Hudlicka & Tyler, 1984). * Significantly different from control.
dehydrogenase (SDH) to differentiate oxidative and glycolytic fibres, and for alkaline phosphatase to depict capillaries (Fig. 5A). It was possible to demonstrate increased capillary density in the vicinity of glycolytic fibres in muscles stimulated for only 2 days at 10 Hz (Hudlicka, Dodd, Renkin & Gray, 1982); C/F ratio was also
7 WHAT MAKES BLOOD VESSELS GROW? increased in the vicinity of fast glycolytic or intermediate fibres, and the increase was found earlier in muscles stimulated at 10 than at 40 Hz (Fig. 5B, Hudlicka & Tyler, 1984). These findings would indicate that glycolytic fibres were indeed suffering from hypoxia. However, direct measurements of oxygen tension in the glycolytic part of A
Stimulation 10 Hz
2.016 -
20 fi
* +.
*.
Values at rest and 0-5 min g Postcontraction
~~~~~~~~~~~~~~~~Control
/
XX 0 e ° 1< 2
mfStimulated
0-4
B
4 days
501Stimulation
*2
14 days
4 days
14 days
A
1 0 '40Hzli*neir 30 day
4 days
0-4
-~~~~~~~~~4C
uce
ihra 0()o t4 eesiuae 14dys4das14dy 14 days
z()uigtesm
4 days l4 days
Fig. 6. Left side, oxygen tension (Po2) in control (contralateral) and stimulated rabbit tibialis anterior; muscles were stimulated either at 10 (A) or at 40 Hz (B) using the same number of impulses for 8 h/day for 4 and 14 days. PO was measured at rest and immediately after 5 min of muscle contractions at 4 Hz (lnked with resting values by interrupted lines). Right side, C/F ratio in contralateral and stimulated muscles at 10 Hz (A) or 40 Hz (B). * Significantly different from control.
rabbit tibialis anterior using oxygen electrodes (Hudlicka & Schroeder, 1978; Kanabus, Hudlicka & Tyler, 1980) did not show any changes that could explain capillary growth (Fig. 6): muscles stimulated for 4 days at 10 Hz had similar values of P02 as control muscles, while those stimulated at 40 Hz had higher values. C/F was increased with 10 Hz and not changed with 40 Hz. After 14 days of stimulation, C/F was increased to a similar extent with either frequency, but Po0 was increased in muscles stimulated at 10 Hz and decreased in those stimulated at 40 Hz. The possible role of hypoxia was also refuted by other experiments. In rat extensor digitorum longus, C/F ratio increased after 7 days of stimulation at 10 but not at 40 Hz (Hudlicka, Cotter & Cooper, 1986). Nevertheless, content of lactate (another indicator of muscle hypoxia) was higher in muscles stimulated at 40 Hz and lower in those stimulated at 10 Hz (Fig. 7). Furthermore, stimulation of tibialis anterior using a regime that activated glycolytic, but not oxidative fibres (very low voltage and short trains of tetanic contractions) caused capillary growth in the superficial glycolytic part of the muscle (and not in the oxidative core) that was not accompanied by increased activity of cytochrome oxidase (Egginton & Hudlicka, 1989).
0. HUDLICKA
8 A
30
Control
i
r
20 2
Control, rest
1 035±0 029 mean+S.E.M. n= 56
lo 1 0
-
30
-
C
0
4-
Control,
El
Stimulated at 40 Hz, contracting Stimulated at 10 Hz, contracting
7 days Stimulated at 40 Hz 1.10 - 0.027 ~~n=t 6U
