INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

CAPILLARIES PERFORM MANY FUNCTIONS in skeletal muscle: the delivery of oxygen and fuel sources as well as hormones and the removal of the byproducts of metabolism (e.g., heat, CO₂). Because the vascular supply to muscles is a major contributor to endurance capacity (17), capillarization has been determined in trained and untrained subjects (5, 6, 22) as well as after training (1, 7, 12, 14, 15, 17, 21) or detraining (17). It is well known that the number of capillaries is higher in endurance athletes than in sedentary subjects and increases with endurance training and that these increases parallel the changes in oxidative metabolism (7, 14, 15).

Most studies of the capillary supply of human skeletal muscle have examined the vastus lateralis. At present little is known about the capillarization of the tibialis anterior (TA) muscle, the primary ankle dorsiflexor. There is increased interest in studying the ankle dorsiflexors because of their important functions in gait and balance (29). In addition, the TA muscle is of interest because of the difference in fiber type composition compared with other human locomotor muscles and the striking differences with nonhuman mammalian TA muscle; whereas human TA muscle comprises about two-thirds type I fibers (10), nonhuman (e.g., rabbit) TA muscle comprises <10% type I fibers (19). To the best of our knowledge, only one study has determined the capillarization of the TA muscle in humans (16). These authors reported that the TA muscle of younger and older men and women had a higher capillary density than the vastus lateralis and the triceps brachii, but other indexes of capillarization were similar between the three muscle groups. In their study (16), men and women were combined within their age groups for comparisons, and potential gender differences were not examined. This makes it difficult to determine what the typical capillarization of the TA muscle would be for men or women of either age group.

Gender-related differences in muscle structure, function, and metabolism have received increased attention. Gender influences fiber type proportions and sizes (27), which are both known to affect capillary supply. In addition, gender differences in muscle metabolism (27) may result in differences in muscle capillarization. Only a few studies have examined the potential gender-related differences in capillary supply of muscles other than the TA muscle. These studies have found some gender-related differences, but the results are inconclusive. One study found greater capillarization in men (26), whereas another found no differences (2). Both of these studies examined a small number of subjects and one muscle biopsy from each subject, so sampling issues could explain part of the discrepancy. Furthermore, because differences in physical performance capacity influence capillary supply, it is important to control for training status in the study of capillarization. Thus, to adequately address the issue of gender differences in capillarization, several muscle biopsies from a sufficiently large sample of men and women with similar habitual physical activity patterns are needed.

To determine the capillary supply to any muscle, regardless of the subjects examined, a number of morphometric indexes have been derived (see Table 1). Morphometric indexes predominantly rely on histochemical or immunocytochemical staining and can be done at the global (whole sample) level or can be done such that capillaries per specific fibers are determined (see Ref. 18 for a review). To get the most complete picture of capillary supply of a given muscle, and to enable comparisons with data from previous studies, both global and individual fiber indexes should be used.

Given the paucity of information about capillary supply in the TA muscle, as well as on gender differences in capillarization, the purposes of the present study were 1) to determine the capillarization of the TA muscle in young healthy men and women and 2) to examine whether there are gender differences in the capillarization of this muscle. Thirty young healthy men and women who were similarly recreationally active were recruited. To reduce the effects of sampling variability, multiple biopsies were obtained from the TA muscle of each subject. A broad range of capillary indexes, both global as well as those that are directed toward individual fibers of a specific fiber type, were used to describe the capillarization of the TA muscle.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects. Fifteen men and fifteen women, between 20 and 31 yr of age, provided written, informed consent to participate in the study, which was approved by the Ethics Research Committee of Lund University. Subject characteristics are shown in Table 2. All subjects were healthy and reported no neuromusculoskeletal dysfunction in the tested leg within the past year. All subjects were physically active but were not specifically training for strength or an athletic event. According to the Grimby Scale of physical activity (9), 28 subjects had a score of 4, and 2 subjects had a score of 3. This meant that the subjects were homogeneous in their activity patterns because almost all subjects were moderately recreationally active two to three times per week.

Tissue preparation. Muscle biopsy samples were taken from the TA of the dominant leg (the right leg for all 30 subjects according to their self-reported preferred leg for kicking a ball) by using the conchotome biopsy technique. All muscle biopsy samples were taken from the superficial part of the TA ~12 cm below the fibular head. One to four biopsies (50-80 mg each) were taken from each subject with the average being 2.7 ± 0.7 for both the men and women (1 subject had one biopsy, 10 subjects had two biopsies each, 18 subjects had three biopsies, and 1 subject had four biopsies). Most investigators take one biopsy sample, but with only one biopsy the determination of fiber type composition (10, 20) as well as capillarity (8, 28) is affected. In the present study, the coefficient of variation in capillary density (CD), fiber density (FD), and capillaries-to-fiber ratio (C/F) for a subsample of 11 subjects was 11.4, 9.8, and 10.9%, respectively, within and between biopsies.

Immunocytochemical staining. The method of Qu et al. (25) for visualization of capillaries using a double-staining, avidin-biotinylated alkaline phosphatase method was implemented with slight modifications. Three types of antibodies were used as the primary antibodies (ulex europaeus lectin, rabbit anti-ulex europaeus agglutinin I pectin, and mouse anti-human collagen IV), and two were used as secondary antibodies (biotinylated goat anti-rabbit and biotinylated goat anti-mouse). The dilution buffer was Tris-buffered saline (TBS). The wash buffer, unless noted otherwise, was 1% BSA (Sigma A3912), diluted in TBS.

Enzyme histochemical staining. To visualize type I and type II fibers, sections were stained for myofibrillar ATPase (mATPase) after alkaline (pH 10.4) preincubation (4). In these 30 subjects, on average only 0.1 ± 0.3% (median = 0, range 0 to 1.4%) type IIx myosin, was found by SDS-PAGE (J. Lexell et al., unpublished observation). Differentiation of the type II subtypes was therefore not done, and all type II fibers can be assumed to be type IIa.

Capillary indexes. The indexes used in this study to measure capillarization are shown in Table 1. Some indexes were used primarily to compare the data with previous studies (CD, FD, C/F). Other variables provide a more direct depiction of capillarization specific to fiber sizes and types [capillary contacts (CC), fiber area per capillary (FA/C), and fiber perimeter per capillary (FP/C)]. Although CD is one of the most commonly reported global indexes of capillarization, it has many drawbacks related to tissue swelling or shrinking (3) and no accounting for fiber type (3) or size (23). The C/F attempts to account for some of these factors, although not fiber type, and therefore provides a better means to compare between studies (3). Indexes that involve counting capillaries per individual fiber and then measuring the individual fiber size provide a more direct measure of muscle capillarization. Plyley (23) suggests that the index FA/C "represents an ideal index of capillary supply" that accounts for the physical development of new capillaries and reflects differences in fiber dimensions that imply the potential effects on diffusion. Hepple (11) has further suggested that fiber perimeter may be influential in processes that rely on cell membrane transport-mediated events rather than diffusion. Therefore, the measures of most interest for making gender comparisons are the individual fiber indexes.

Image analysis. Both immunocytochemical (capillaries) and enzyme histochemical (muscle fiber type composition) slides (for examples, see Fig. 1) were examined with a Leitz Diaplan microscope (Leitz, Wetzlar, Germany) with a ×6.3 objective. Images were captured by a Kappa digital camera and then transferred to Image Pro Plus version 3.0 (Media Cybernetics, Silver Spring, MD) for analysis. Each rectangular image was 220 × 160 mm in size when viewed and analyzed on the computer screen, representing ~0.75 mm² of the actual biopsy at a magnification of ×220.

View larger version (123K): [in this window] [in a new window]   Fig. 1.   Microscopic images of a biopsy from the tibialis anterior muscle of a young man stained (A) immunocytochemically to visualize capillaries and (B) enzyme histochemically to visualize the two main fiber types, type I (lightly stained) and type II (darkly stained).

Statistical analysis. Gender differences for global capillary indexes (CD, FD, C/F) and fiber type proportion (% type I) were explored with independent sample t-tests (SigmaStat v2, SPSS, Chicago, IL). For the individual fiber capillary indexes (CC, FA/C, FP/C) and the muscle fiber size (area and perimeter) variables, separate repeated-measures ANOVAs were performed with gender and fiber type as factors and fiber type being the repeated factor, by using SigmaStat (v2, SPSS). Pairwise comparisons within gender and fiber type were done with Bonferroni t-tests. For all analyses, a significance level of P < 0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Muscle fiber type composition. Numerical data are presented in Table 3, and Fig. 2 gives a visual inspection of the repeated-measures ANOVA. There was no significant gender difference in the proportion of type I fibers (Table 3, P > 0.4). The repeated-measures ANOVA revealed significant gender, type, and interaction effects for both fiber area and perimeter (Table 4). Type II fibers were substantially larger than type I fibers for area and perimeter, respectively, in both men (80.4 and 32.0%) and women (50.8 and 21.4%; Table 3 and Fig. 2). There were significant gender differences in both fiber area and fiber perimeter. Type II fibers were proportionally larger in the men (Table 3 and Fig. 2), as exhibited by the percent differences in fiber area and perimeter between the men and women for type I (25.6 and 13.2%) and type II fibers (50.3 and 23.0%).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Type I and II muscle fiber sizes and individual fiber capillary indexes for men and women (means ± SE). Significant differences between gender and fiber type, as determined by pairwise comparisons of the repeated-measures ANOVA, are shown (**P < 0.01; ***P < 0.001).

Global capillary indexes. CD was not different between the men and women (P > 0.4; Table 3), but FD was significantly larger in the women (P < 0.001), so C/F was significantly lower in the women (P < 0.001). To further explore the effect of fiber size on C/F, an analysis of covariance was performed on C/F for the men and women, with type I and type II fiber area as covariates. Both type I and type II fiber area were significant, and there was no longer a difference between the men and women (P > 0.4). The model suggested that C/F was highly influenced by fiber size (R² = 0.72).

Indexes of capillary supply to individual fibers. Results of the repeated-measures ANOVA are shown in Table 4. CC were affected by gender but not fiber type, although there was an interaction between gender and fiber type. The men had more (P < 0.001) CC than the women for both type I and type II fibers (Table 3 and Fig. 2). In the women there was no difference between CC for type I and type II fibers, but in the men type II fibers had more CC than type I (P = 0.005). For FA/C, the only significant effect was type (Table 3), with type II fibers having a greater FA/C (Table 3); i.e., for each capillary around a type II fiber, on average a larger area was being supplied than for a type I fiber. There were no gender differences in FA/C, with the mean values being almost identical in the men and women for both type I and type II fibers (Tables 3 and 4, and Fig. 2). Type II fiber FP/C was also greater than that for type I, but there was no difference between the men and women for FP/C for type I fibers. There was a significant gender effect for type II FP/C (Table 3), in which the FP/C for the women was significantly greater than for the men (Table 3; P = 0.006); i.e., a larger perimeter was supplied by each capillary for type II fibers in women.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Morphologically, capillarization of skeletal muscle indicates the potential for exchange capacity between the vascular system and muscle fibers and has been linked to aerobic endurance performance. This is, to the best of our knowledge, the first study to examine gender differences in the capillarization of the TA muscle. It is also the most extensive examination of potential gender differences in capillarization for any muscle, because of the relatively large number of subjects with comparable physical activity levels, as well as the fact that multiple biopsies were taken. Our results demonstrate that, overall, there are no major gender differences in the capillarization of the TA.

The capillarization of the TA muscle in these young men and women with comparable activity levels generally falls within the range of other muscles in young subjects. It is difficult, though, to compare values between studies because of differences in the subjects and their physical activity patterns, muscle stains and other methods used, as well as fiber type composition of the muscles examined. Despite these difficulties, the human TA muscle, not surprisingly, falls somewhere between a muscle with a relatively high composition of type II fibers, like the triceps brachii muscle, and a muscle with a relatively high composition of type I fibers, like the soleus muscle. For example, the TA muscle in our study had a C/F of 2.20 in the male subjects. In a previous study the C/F for the triceps and soleus muscles have been reported to be 1.94 and 2.90, respectively, for young men (26). The vastus lateralis, a mixed muscle, which has most often been studied for capillarization in humans, has C/F values ranging from 1.13 in untrained male subjects (14) to 2.50 in endurance-trained men (26). In women, the values for the vastus lateralis range from 1.2 in sedentary subjects (22) to 1.79 after aerobic training (15).

When comparing our capillary indexes with the only other study examining the TA muscle (16), there are many similarities particularly with the male subjects in the present study. The comparison is somewhat clouded by the fact that the young subjects in the study of Jakobsson et al. (16) were both men and women, although predominantly men. The CD for the young subjects was 400 compared with our male value of 389. The C/F was 2.7 (16) compared with 2.2 for our male subjects, and the CC for type I fibers was also very similar: 5.8 vs. our value of 5.7. Because the fiber areas were larger in the study by Jakobsson et al. (16), the relative numbers of capillaries supplying a given area of type I and type II fibers were somewhat smaller. Some of the design differences between the two studies are that the former study had a combination of men (n = 13) and women (n = 2) who were older in their younger group and that there was no detailed information on the physical activity levels of the subjects (16). This previous study then provided no data on whether there are gender differences in the capillarization of the TA muscle.

The men and women in the present study had similar CD, but the women had a higher FD, so the C/F was higher in the men than the women. At face value this would suggest that the men had a higher level of capillarization than the women. When we examined whether the difference in fiber size was truly taken into account by C/F, we found that analysis of covariance for fiber area eliminated the difference in C/F between the men and women. Although C/F may be slightly better than CD to compare results from different studies, it is still limited in its ability to account for differences in fiber sizes.

Although type II fibers had higher numbers of CC compared with type I fibers, the fact that type II fibers were larger than type I fibers for both men and women resulted in a higher FA/C and FP/C for type II fibers compared with type I fibers. This indicates that a larger area was being supplied by each capillary, and this reflects the lower level of capillarization of type II fibers. Data from the vastus lateralis muscle similarly indicate that type I fibers generally have a higher level of capillary supply than type II fibers (15, 26) and that sometimes larger muscle fibers are surrounded by more capillaries than smaller muscle fibers (3).

In this study, the men had larger fibers, particularly type II fibers, compared with the women. The men also had a higher number of capillary contacts for both fiber types than the women, so there were no gender differences in FA/C for either fiber type or FP/C for type I fibers. However, type II FP/C was greater in the women compared with the men, suggesting lower capillarization of type II fibers in the women. Therefore, even though the women did not seem to adapt by increasing the number of capillaries surrounding their larger type II fibers, as the men did, FA/C remained similar between the men and women because the type II fibers were not enlarged as much in the women as in the men. However, the lower relative number of CC for the type II fibers in the women resulted in an increased perimeter supplied by each capillary for type II fibers. Physiologically the consequences of this latter finding remain to be determined, particularly because there are relatively few type II fibers in the TA muscle. It has been suggested that muscle perimeter measures relative to capillarization may be more indicative of oxygen delivery to the muscle (13), whereas the area of the fiber relative to capillary contacts may be more important for processes that require diffusion, such as fuel delivery, waste removal, and heat dissipation. In these subjects, though, it seems that there are no major gender differences in capillarization, beyond the perimeter supplied per capillary for type II fibers. Further studies comparing capillary indexes with physiological measures of endurance and biochemical markers of metabolism are needed to fully understand the significance of the differences in capillary supply between the different fiber types and between the genders.

Other studies that have investigated gender effects on capillarization have provided mixed results (2, 26). Using FA/C, Sjogaard (26) found greater capillarization of the vastus lateralis and the soleus muscles in moderate to well-trained young men compared with young women for type I fibers only. The sample size was rather small in their study (n = 6 per group), and it is unknown whether the male and female subjects had the same physical activity patterns. Because endurance exercise is known to increase capillarization (7, 12, 14, 15, 17, 21), it is important to ensure that physical activity levels are similar when making group comparisons. The men in this study (26) had maximal oxygen uptake values of 51-62 ml · kg¹ · min¹, but this parameter did not appear to have been measured in the women. In the present study, young men and women with comparable physical activity patterns were recruited, allowing us to make more detailed inferences regarding muscle capillarization and gender per se. Bell and Jacobs (2) also studied gender effects on capillarization of the vastus lateralis in young body builders as well as control subjects. The body builders had a greater capillarization than the control subjects, but there were no gender effects. The limitations of the latter study (2) are that only global indexes were used and CD was based on a sample of ~35 fibers per subject. As with most human studies of muscle morphology, only one biopsy sample per subject was examined in both of the above studies (2, 26).

In conclusion, in this study we have shown that the TA muscle of young active men and women has a level of capillarization that reflects the muscle fiber composition of this muscle. There were no gender differences found for CD, FA/C, or FA/P for type I fibers or FA/C for type II fibers. C/F was significantly different between the men and women, but the difference in C/F could be explained by differences in fiber size. FA/P for type II fibers was significantly different between the men and women, suggesting lower capillarization of type II fibers in the women, but the physiological implications of this difference remain to be determined. Thus, despite gender differences in TA muscle fiber size, overall, capillarization was similar between the men and women.