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1 Laboratory of Physiology, GIP Exercice-Sport-Santé, Research Group Physiology, Physiopathology of Exercise and Handicap, University Jean Monnet, Saint-Etienne, France2 Department of Physical Education and Health, Örebro University, Örebro, Sweden
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Abstract |
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(Received 12 May 2003;
accepted after revision 21 October 2003;
first published online 27 January 2004)
Corresponding author N. Charifi: Laboratory of Physiology, GIP Exercice-Sport-Santé, Research Group Physiology, Physiopathology of Exercise and Handicap, University Jean Monnet, Saint-Etienne, France. Email: nadia.charifi{at}univ-st-etienne.fr
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Introduction |
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In electrically stimulated rat skeletal muscle, angiogenesis starts with the proliferation of capillary endothelial cells and results in the formation of sprouts (Mathieu-Costello, 1993; Hudlicka, 1998). The new sprouts form loops and cross-connections, but not necessarily more numerous single elongated vessels running parallel to the muscle fibres, thus contributing to a more tortuous capillary network architecture (Hansen-Smith et al. 1996). At the moment, the role of enhanced capillary tortuosity in the improvement of O2 diffusing capacity is still under debate. It has been shown that there were no changes in capillary tortuosity in skeletal muscles of rats in response to treadmill running or to chronic electrical stimulation (Poole et al. 1989; Mathieu-Costello et al. 1996). In contrast, in chronically stimulated skeletal muscles of cats, an increase in the number and in the length of the capillaries was observed (Hudlicka et al. 1987; Hudlicka, 1991). In humans, only one study showed that the percentage of capillaries cut longitudinally in muscle cross-sections was higher in trained than in untrained young adult subjects (Parsons et al. 1993). The effect of the degree of capillary tortuosity on the improvement of muscle tissue oxygenation received recent support from a theoretical mathematical computational model (Goldman & Popel, 2000). However, the question of whether capillary tortuosity is altered in old subjects remains unanswered.
The morphometrical strategies used to quantify the training-induced changes in muscle capillary network are of great importance (Lexell, 1997). The capillary supply is usually assessed by counting the number of capillaries around each fibre (CAF) or by computing the ratio between the number of capillaries present in an area and the number of fibres in the same area (C/F). Other indices derived from Krogh's hypothesis (Krogh, 1919) and based on the role of the O2 diffusion distance between each capillary and the centre of the fibre have been used: capillary density (CD) and CAF related to the fibre area (CAFA). Other studies have shown that the muscle–capillary interface is the most important factor involved in the resistance to O2 diffusion (Gayeski & Honig, 1986; Honig et al. 1992). For this reason, precise stereological procedures, for instance, capillary-to-fibre perimeter ratio, i.e. capillary perimeter divided by fibre perimeter based on the analysis of perfused muscles, were used to assess the capillary-to-fibre interface, including the tortuosity of the capillary network (Mathieu-Costello et al. 1991). However, these indices cannot be used in human studies because skeletal muscles need to be perfused in order to prevent capillary collapse. To overcome this methodological issue, the capillary-to-fibre perimeter exchange index (CFPE) has been used in the study of human tissue (Hepple, 1997). CFPE represents the ratio between the capillary-to-fibre ratio calculated for each individual fibre (C/Fi) and the perimeter of the fibre (PF). In this respect, CFPE index and capillary-to-fibre perimeter ratio were found to be correlated (Hepple & Mathieu-Costello, 2001). However, CFPE index does not take into account the orientation of the capillaries in a transverse section. Therefore, in order to identify the size of the muscle–capillary interface, it would be necessary to assess the length of the capillary-to-fibre contact. In this respect, the percentage of muscle fibre perimeter in contact with the capillary wall in transverse sections (LC/PF) can be used (Sullivan & Pittman, 1987). Capillary tortuosity can thus be indirectly determined using LC/PF in repeated muscle samples from a longitudinal study (i.e. exercise training programme), with the condition that the length of sarcomeres is equal in each sample (Mathieu-Costello et al. 1989).
The aim of the present study was to investigate whether endurance training induces an increase in capillary tortuosity of the vastus lateralis muscle in old men. For this purpose the capillary supply was assessed using the CD31 antibody (Horak et al. 1992) and different capillary indices including CFPE and LC/PF were determined. In parallel, muscle oxidative capacity was quantified by the study of citrate synthase activity.
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Methods |
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Eleven healthy men (age, 73 ± 3 years; body mass, 80 ± 12 kg; height, 171 ± 5 cm) participated in the study. All subjects were fully informed of the nature and possible risks of the various procedures before giving their written consent. This investigation was approved by the Consultant Committee on Human Protection from Medical Research of Rhône-Alpes-Loire Region in accordance with the French Law and with the Declaration of Helsinki.
Training procedure
The subjects trained for 14 weeks by pedalling a mechanically braked Monark bicycle ergometer (Varberg, Sweden) for 45 min daily on 4 days per week. Each session was performed with a 5 min intermittent mode, i.e. 4 min at a workload corresponding to 65–75% of peak oxygen consumption 
and the subsequent 5th minute at 85–95%
. This sequence was repeated 8 times without interruption until the 45th minute. Heart rate was used as the criterion to maintain an adequate relative workload and to adjust the absolute workloads every week because of the training effect. All training sessions were supervised by one of the authors.
Determination of peak oxygen consumption
Before and after the training period, subjects performed an exercise test for 
determination. Workload increments of 20 W were imposed every 2 min on the bicycle ergometer (beginning at 0 W) until voluntary exhaustion. Oxygen uptake was measured during the last 30 s of each separate exercise level. The subjects breathed through a Hans Rudolph respiratory valve 2700 and the expired gas was collected in polyethylene Douglas bags. The gas fractions were measured with an infrared CO2 analyser (Normocap, Datex, Helsinki, Finland) and a paramagnetic O2 analyser (Servomex 1440, Crowborough, England) calibrated with gas mixtures determined by Scholander's method (Scholander, 1947). The ventilatory volumes were determined with a Tissot spirometer. Heart rate was monitored and plotted from the electrocardiogram signal. Three minutes after cessation of exercise, a fingertip blood sample was taken to be analysed for lactate concentration (YSI 2300, Yellow Springs Instruments, Yellow Springs, OH, USA). Heart rate, respiratory exchange ratio and blood lactate concentration were used as criteria for acceptance of 
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Muscle biopsy
Muscle biopsies were taken before and after the training period from the vastus lateralis (at a level corresponding to one third of the distance from the upper margin of the patella to the anterior superior iliac spine) using Weil-Blakesley forceps. The muscle biopsies were taken from the same leg pre- and post-training. The post-training biopsy was taken 2 cm away from the pretraining biopsy site. The weight of the biopsy sample averaged 80 mg. The sample was divided into four equal parts for different analyses. A part of the sample containing well-identified fascicles was orientated under a stereo microscope, included in an embedding medium (Cryomount; Histolab, Göteborg, Sweden), frozen in isopentane cooled to its freezing point in liquid nitrogen, and stored in liquid nitrogen until further cryostat sectioning. The remainder of the sample was rapidly frozen and stored in liquid nitrogen until enzyme activity assays were performed.
Enzyme activity assays
Muscle samples were freeze dried (Lyovac GT2, Leybold-Heraeus, Köln, Germany), dissected free from connective tissue and blood, and powdered in a chamber of controlled humidity (<40% relative humidity). The muscle powder was weighed in the same chamber, homogenized at 4°C in 0.1 M phosphate buffer (pH 8.2) containing 5 mM 2-mercaptoethanol, 30 mM NaF, 5 mM MgCl2 and 0.5 mM ATP. This tissue suspension was used to measure spectrophotometrically the activity of phosphofructokinase (PFK; Essen-Gustavsson & Henriksson, 1984) as a cytosolic glycolytic marquer of metabolism. The activity of citrate synthase (CS) was determined fluorometrically (Mansour, 1966) to attest the oxidative mitochondrial capacity. All the enzyme activities were measured at 25°C and expressed in micromoles per minute per gram dry weight (UdW).
Immunocytochemical and histochemical assays
Serial transverse sections, 10 µm thick, were cut with a microtome at –20°C (Tissue Tek II, Miles Laboratories, Elkhart, IN, USA). The identification of capillaries was performed using the monoclonal antibody CD31 (Dako, Glostrup, Denmark; M0823) which recognizes PECAM-1 (platelet endothelial cell adhesion molecule), a transmembranous glycoprotein strongly expressed by vascular endothelial cells. CD31 has been used successfully to identify vascular endothelium in tumour tissue (Horak et al. 1992; Vermeulen et al. 1996) and in normal muscle (Brey et al. 2002). In a pilot study on serial sections from human muscle biopsy material (n= 5) we found no statistical difference between the number of capillaries determined using 
-amylase-PAS (Andersen, 1975) and CD31. We noted that visualization of the capillaries was easier using CD31. The slides were incubated in an atmosphere saturated with water vapour, for 1 h with CD31 antibody (mouse-antihuman), for 30 min with the secondary antibody (rabbit-antimouse, Dako P0260), and for 30 min with the tertiary antibody (swine-antirabbit, Dako P0217). All the incubations were performed at room temperature, and the slides were rinsed between each incubation with a phosphate-buffered saline solution. Peroxidase labelling was performed using a DAB substrate kit (Vector, Burlingame, USA; SK-4100), that yields a brown reaction end-product at the site of the target antigen (endothelial cell, Fig. 1).
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Longitudinal cryo-sections, 10 µm thick, were obtained by rotating at –20°C each block of biopsy previously cross-sectioned. They were stained using Haematoxylin, Eosin and Safran in order to measure the mean sarcomere length.
Morphological assessments
Muscle sections were viewed under a light microscope (Eclipse E400, Nikon, Badhoevedorp, the Netherlands) connected to a digital camera (Coolpix 990, Nikon). All the following measurements were made under blinding protocols. Photographs were taken at x400 magnification and analysed using a free image section software (Scion Image). An average of eight fields were examined in each section, to give an analysis of an average of 70 fibres per individual sample.
Fibre area and perimeter were measured. Obliquity in fibre sectioning was assessed using the form factor that represents: (4
x fibre area)/(fibre perimeter)2. The less elliptic the cross-section, the nearer the form factor is to 1. If a given fibre has a cylindrical shape, any perpendicular section will have a circular area (r= radius) and consequently the form factor is 1[(4xr2)/[2r]2= 1]. An oblique section will result in an elliptic slice (r and R as minor and major radius) with a form factor 
. For example, if R= 2r, then the form factor is 0.8.
Assessment of capillary network
Global indices. In a given area, capillary density (CD) was expressed as the number of capillaries counted per square millimetre (cap mm-2).
Capillary-to-fibre ratio (C/F), the ratio between the number of capillaries present in an area and the number of fibres in the same area, was calculated. In this calculation, the number of capillaries is corrected by subtracting half the number of the capillaries in the periphery of the area (Andersen & Henriksson, 1977; Brodal et al. 1977).
Individual fibre indices. The mean number of capillaries around a single fibre (CAF) and CAF relative to the area of the fibre (CAFA) were calculated.
To assess the capillary-to-fibre interface we calculated the CFPE index (capillary-to-fibre perimeter exchange; Hepple, 1997). We first determined the capillary-to-fibre ratio for each individual fibre (C/Fi): for each fibre, capillaries in contact with that fibre were counted by taking into account their sharing factor. The sharing factor represents the inverse number of fibres in contact with the same capillary. The CFPE index was then calculated as the ratio between C/Fi and fibre perimeter (Hepple, 1997).
In transverse sections, several capillaries are cut longitudinally, owing to their tortuous arrangement around muscle fibres. Capillaries were identified by the size of their cross-section: transverse or longitudinal profiles with a diameter less than 15 µm were counted. This helped to exclude vessels larger than capillaries (arterioles or venules). In the present study, most of the vessels considered in the endomysium had a diameter <10 µm. However, even within this range (diameter <10 µm), some microvessels might well represent terminal arterioles (Hansen-Smith et al. 1998). Thus, the criterion of size is important but not sufficient for the distinction between a capillary and a microarteriole. For this reason, it seems justified to use the term ‘microvessel’ and not ‘capillary’ in the interpretation of our results. This will be done in the following text. However, indices computed in this study have been termed as described in previous studies.
The immunostaining of microvessel walls allows visualization of all portions of transversally or longitudinally running microvessels and measurement on transverse sections of the length of the contact (LC) between the microvessels and the fibres. The index of tortuosity was calculated as follows: LC/PF (perimeter of fibre). LC/PF, originally called ‘length of capillary-fibre contact’ (Sullivan & Pittman, 1987), is expressed as a percentage of muscle fibre perimeter in contact with the capillary wall. In a muscle cross-section, the profile of the microvessels will depend on their orientation in relation with the fibres. Microvessels were classified into three classes using as a criterion the length of their contact with fibres (LC): structures with LC 
10 µm were considered as parallel microvessels, structures with 10 µm LC = 30 µm were considered as oblique microvessels, and structures with LC > 30 µm were considered as perpendicular microvessels. The percentages of microvessels with either of these profiles were calculated before and after training.
Mean sarcomere length. Mean sarcomere length was measured on longitudinal sections. Well orientated fibre portions with regular striations were selected on approximately 20 different zones and photographed. The length occupied by 10 consecutive sarcomeres was measured with the software previously described. Consequently, an average of 200 sarcomeres per longitudinal section were measured.
Statistical analysis
Data are presented as means and standard deviations. The statistical significance of the differences between the pre- and post-training period was determined using a non-parametric Wilcoxon signed rank test. A two-way analyse of variance (ANOVA) was performed in order to study whether there were differences in C/F and C/Fi (method and training as independent variables). Linear regression and correlation coefficients (R) were used to determine the degree of relationship between two variables. The effect of the training programme on a given relationship between two variables was tested using multiple regression analysis with the introduction of a qualitative variable (0 and 1 corresponding to pre- and post-training data, respectively) as described by Daniel (1995). P values below the 0.05 level were considered statistically significant.
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Results |
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A main outcome in the present study was the significant increase in LC/PF index (+56%; P < 0.001; Fig. 1). LC/PF increases reached 39% in type I fibres (P < 0.05) and 69% in type IIa fibres (P < 0.05). The measurement of the mean sarcomere length showed no significant changes between muscle biopsies taken before and after training (Table 3). Thus the absence of any change either in the contraction state of the postbiopsy sample or in the form factor suggests that the training-induced increase in LC/PF was the consequence of a remodelling in microvessel tortuosity.
The percentages of microvessels classified according to their length of contact with fibres are presented in Table 4. The training programme induced a significant decrease in transverse section microvessel profile and significant increases in oblique and longitudinal microvessel profiles.
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Discussion |
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Immunohistochemical preparation to identify microvessels on muscle cross-sections
In the present study, most of the vessels considered had a diameter <10 µm. However, as discussed in the Methods, the criterion of size is not sufficient for the distinction between a capillary and a terminal arteriole. A labelling of the vessel wall revealing the presence of smooth muscle actin would represent a reliable method to separate capillaries from arterioles (Hansen-Smith et al. 1998). For this reason, we chose to use the term ‘microvessel’ and not ‘capillary’ in the interpretation of our results. Moreover, it has been shown that oxygen exchange occurs not only across the wall of capillaries but also arterioles. Thus, the arteriolar network also participates in oxygen diffusion between blood and muscle cells (Pittman, 2000).
In the field of exercise physiology, the effects of endurance exercises on the capillary network have been mostly assessed using the -amylase PAS reaction (Andersen, 1975). During the last decade, other approaches based on immunohistochemistry have been proposed. Antibodies against Ulex europaeus agglutinin I lectin (UEA-I) as an endothelial marker (Parsons et al. 1993; Lexell, 1997; Qu et al. 1997; Porter et al. 2002) and against laminin as a basal lamina marker (Kadi et al. 1998, 1999) were used. While frequently used in the identification of tumoral neoangiogenesis in human anatomical specimens, CD31 antibody has not been extensively used to identify capillaries and microvessels in normal human muscles. Recently, CD31 has been successfully used in three-dimensional analysis of the microvascular network in rat skeletal muscle (Brey et al. 2002). Our results showed that CD31 allows a clear and reliable identification of microvessels. As illustrated in Fig. 1, microvessels can be seen as spots (when a microvessel runs parallel to the fibre) or as segments (if a microvessel runs obliquely or perpendicularly to the length of the fibre) on transverse sections. Thus, it is possible to measure a given length of microvessel wall in contact with fibre perimeter. For a given fibre, the sum of the different segments (Fig. 3) gives a quantitative indicator of the microvessel-to-fibre interface, while the length of this interface cannot be measured using a stereological approach.
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An increase in the number of capillaries around fibres has been reported in response to endurance training in young (Saltin & Gollnick, 1983; Hudlicka et al. 1992 for reviews) and in elderly subjects (Coggan et al. 1992; Freyssenet et al. 1996; Hepple et al. 1997). By relating the changes in the number of capillaries to the size of muscle fibres it was possible to investigate whether the capillary supply of a given fibre was altered. In the elderly, previous studies have shown an increase in the number of capillaries (Coggan et al. 1992; Freyssenet et al. 1996; Hepple et al. 1997), while in one study the number of capillaries was maintained (Denis et al. 1986). However, in all these studies, the capillary number in relation to fibre area was increased because of different modulations in fibre area. In the present study, an increase in CAF occurred but did not lead to an increase in CD or in CAFA due to the simultaneous increase in fibre area.
Training-induced changes in the capillary-to-fibre interface
Sullivan & Pittman (1987) proposed an index that accounts for capillary geometry in rat skeletal muscles. As they observed a high incidence of capillaries cut longitudinally on transverse sections, they measured the percentage of muscle fibre perimeter in contact with capillary wall (capillary–fibre contact, which is presented as LC/PF in our study). This parameter was considered to give a better assessment of the vascular supply of O2 to the fibre than the number of capillaries around a fibre. Based on calculations predicting a rapid drop of O2 over the short distance between the interior of the red cell and the surface of the surrounding tissue, Gayeski & Honig (1986) showed that capillaries and their immediate surrounding represent the principal site of resistance to O2 diffusion. The capillary-to-fibre interface appeared to be an important determinant of the oxygen flux rates. In this perspective, Mathieu-Costello et al. (1991) proposed an index close to the capillary–fibre contact index, i.e. the ratio between the capillary perimeter and the fibre perimeter (capillary-to-fibre perimeter ratio index). They also showed that this index was independent of sarcomere length. In consequence, this index would allow the comparison of the size of the capillary-to-fibre interface between muscles of different fibre size or different sarcomere length.
The major inconvenience of the indices proposed by Sullivan & Pittman (1987) and Mathieu-Costello et al. (1991) is related to the fact that muscles have to be perfused and fixed. In this respect, Hepple (1997) proposed the measurement of an index that can be computed on transverse sections of non-perfused muscles (i.e. muscle samples obtained from a biopsy procedure in humans and histochemically assayed). This index was defined as the capillary-to-fibre perimeter exchange index (CFPE). Hepple et al. (1997) described a relation between the capillary supply and 
in old subjects. It was also shown that when CFPE and CD were both significantly increased after aerobic training, CFPE would explain a greater part of the variance in 
than would CD. By taking into account the fibre perimeter, the CFPE index introduces the role of the capillary-to-fibre interface. However, a major disadvantage of CFPE index is its insensitivity to the length of capillaries in contact with the fibre. Hepple & Mathieu-Costello (2001) assumed that CFPE index does not take into account capillary geometry. However, they considered that CFPE index remains a reliable tool to estimate the capillary-to-fibre interface in muscles when capillary tortuosity is unchanged. Indeed, by reference to Poole et al. (1989), who showed no increase in capillary tortuosity in muscles of rats after endurance training, they concluded that the limitation of CFPE index in studies of capillary supply in response to exercise should not be significant (Mathieu-Costello & Hepple, 2002).
Our study showed an increase in capillary-to-fibre interface with training (LC/PF). The fact that CAF and LC/PF increased, respectively, by 19 and 56% indicates that the mean length of microvessel segments increased per se. The mean length of sarcomeres was similar between pre- and post-training muscle samples. This allows us to conclude that the degree of contraction in pre- and post biopsy muscles was similar. Altogether these data indicate that the significant increase in LC/PF is a strong indication of the enhancement of microvessel tortuosity. This conclusion is supported by the significant increase in the proportion of oblique and longitudinal microvessels observed in muscle cross-sections (Table 4). These results are also in line with those reported in trained cross-country skiers and sedentary subjects (Parson et al. 1993).
Unexpectedly, the change in CFPE index was not significant. These conflicting results between LC/PF and CFPE changes are further analysed in Fig. 3. It appears that microvessel-to-fibre interface is dependent on three main morphological factors, i.e. microvessel number, length of the microvessel-to-fibre contacts and the perimeter of the fibre (PF). Any increase in the microvessel number (the two other factors remaining constant) is linked to a corresponding increase in microvessel-to-fibre contacts. In this case, all the indices are altered with the same amplitude (Fig. 3B). Any increase in the length of the contacts (due to tortuosity, microvessel number and PF remaining constant) only affects LC/PF (Fig. 3C). When both the number and the length of microvessels increase (PF constant), the amplitude of LC/PF change is twice that of the other indices (Fig. 3D). According to the results of the present study, Fig. 3E illustrates the changes observed in the three morphological factors (increase in CAF, tortuosity and PF): the slight increase in PF induces a decrease in both CFPE and LC/PF. Owing to the amplitude of the response, only LC/PF change remains statistically significant. Consequently, when changes in microvessel geometry result from an increase in both number and tortuosity, LC/PF seems to be the most robust index, even in the event of modest muscle hypertrophy.
Interrelationship between microvascular supply and oxidative capacity
In the present study the training-induced increase in microvascularization was accompanied by an enhancement of oxidative capacity. Moreover, a strong correlation was observed between the index of microvessel tortuosity and citrate synthase activity. In animal models, a few studies showed contradictory results (Maxwell et al. 1980; Egginton & Hudlicka, 2000). In humans, while a few studies have investigated the relationship between oxidative capacity or mitochondrial content and capillary supply (Romanul, 1965; Sullivan & Pittman, 1987; Poole & Mathieu-Costello, 1996), many have shown concomitant increases in capillarization and oxidative capacity in response to endurance training, both in young (Andersen & Henriksson, 1977; Ingjer, 1979; Coggan et al. 1992; Proctor et al. 1995) and in old subjects (Coggan et al. 1992; Proctor et al. 1995). Consequently, it is generally accepted that the increase in capillary supply would allow an enhancement of oxygen delivery to muscle fibres due to the increased mitochondrial metabolic demand.
We used different indices in our study and, as shown in Fig. 2, the results concerning the relation between microvascular supply and oxidative capacity depended largely on how microvascularization was expressed. Indeed, LC/PF is the only index that exhibited a strong relationship with CS activity before and after training (Fig. 2A). We also found a correlation between the number of microvessels and CS activity (Fig. 2B). Fig. 2C and D show that fibre dimensions can affect the relationship between CS activity and the indices of microvascularization such as CFPE and CAFA. Finally, LC/PF, which also takes into account the fibre perimeter, seems to be the most sensitive index because the correlation with CS activity was not affected by the slight changes in fibre size after training. Taken together these results suggest that the entire adaptive response to increased oxidative metabolism cannot be explained solely by increased microvascularization and reduced distance diffusion. Increased surface area available for microvessel–muscle fibre exchange seems to be another important cellular strategy in the adaptive response of the microvascular bed to the increase in the oxidative demand of the fibres. These conclusions are in line with a recent demonstration proposed by Goldman & Popel (2000) using a computational model.
Training-induced increase in muscle microvessel tortuosity in the elderly: a specificity?
Most of the earlier studies on muscle transverse sections from humans were performed with -amylase-PAS histochemical assay. Using electron microscopy, Brodal et al. (1977) and Zumstein et al. (1983) compared the capillary supply in skeletal muscle of young trained and untrained subjects. However, in these studies, neither a clear identification nor a counting of segments of longitudinally cut capillaries has been mentioned. Consequently, a comparison of the present findings observed in elderly men with results in younger adults has yet to be performed. Younger muscle may exhibit cellular mechanisms more readily facilitating the formation of new microvessels while older muscle may rely more on a compensatory increase in the length of pre-existing capillaries. Further studies would be required to verify these hypotheses. The results of the present study do, however, support such a speculation, since training-induced increase of LC/PF expressed in as a percentage was more pronounced than the increase in CS activity (see the shift of the relation between LC/PF and CS activity). In an earlier study on young adults (Andersen & Henriksson, 1977), endurance training was found to have a greater influence on oxidative capacity than on the capillary network.
In conclusion, our findings clearly demonstrate an increase in microvessel tortuosity in skeletal muscle of elderly men in response to endurance training. Moreover, there is a significant relation between microvessel tortuosity and CS activity both before and after training.
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) and after (
) training
; PF, perimeter of the fibre.