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J Appl Physiol 91: 2150-2156, 2001;
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Vol. 91, Issue 5, 2150-2156, November 2001

Estimating the size of the capillary-to-fiber interface in skeletal muscle: a comparison of methods

Russell T. Hepple and Odile Mathieu-Costello

Department of Medicine, University of California, San Diego, La Jolla, California 92093


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Current evidence suggests that the size of the capillary-to-fiber (C/F) interface is a major determinant of O2 flux into muscle fibers, and methods have been developed for estimating the size of this region via the C/F perimeter ratio in perfusion-fixed material (Mathieu-Costello O, Ellis CG, Potter RF, MacDonald IC, and Groom AC. Am J Physiol Heart Circ Physiol 261: H1617-H1625, 1991) and the quotient of the individual, fiber-based C/F number ratio and fiber perimeter (C/F perimeter exchange index) in muscle biopsies (Hepple RT. Can J Appl Physiol 22: 11-22, 1997). The purpose of this study was to compare the two methods and examine how differences in muscle tissue preparation between perfusion fixation and frozen biopsy can influence the estimate of the size of the C/F interface. The left medial gastrocnemius muscle of nine purpose-bred dogs was perfusion fixed in situ, and a sample from the midportion of the midbelly was processed for microscopy. A corresponding sample from the right gastrocnemius muscle obtained by open biopsy in six of the nine animals was frozen for histochemistry. A significant correlation was found between the two estimates of the size of the C/F interface in the same sections of perfusion-fixed material (r = 0.75, P < 0.05). However, estimates of the size of the C/F interface were smaller in biopsies than perfusion-fixed material, and there was no significant relationship between the estimates in the two preparations. This was due to differences in fiber size (33% larger fiber cross-sectional area in biopsy material after normalization for sarcomere length; P < 0.05) and muscle sampling between the two tissue preparations.

capillaries; morphometry; blood-tissue exchange; oxygen diffusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

STRUCTURAL EVIDENCE ACROSS several species (e.g., Refs. 14, 15, 18, 19), including humans (8), supports the notion that the size of the capillary-to-fiber (C/F) interface is an important determinant of maximal O2 flux capacity in skeletal muscle (for a review, see Ref. 11). In this respect, a method of choice to quantify the size of the C/F interface in muscles is to measure it directly, via the estimation of the C/F perimeter ratio (16), by light microscopy in perfusion-fixed material. However, such measurement cannot be made in histochemically stained biopsies because of the collapse of capillaries (and unreliability of their perimeter measurement by light microscopy) in nonperfusion-fixed tissue. Thus the quotient of individual, fiber-based C/F number ratio and fiber perimeter [C/F perimeter exchange (CFPE) index] was introduced as an alternative to estimate the size of the C/F interface in muscle biopsies (6). Differences between the methods relate to both methodology and tissue preparation. Unlike the measurement of C/F perimeter ratio (16), using the C/F number per fiber perimeter (CFPE index) does not account for capillary geometry, i.e., it does not incorporate the differential perimeter of oblique sections of branches and tortuous capillaries vs. vessel cross-perimeters into the estimate of the size of the C/F interface in a muscle transverse section. In addition, the degree of muscle contraction, reflected by sarcomere length, influences both capillary geometry (13) and fiber size (3, 13, 17). Whereas sarcomere length can be readily measured in longitudinal sections of perfusion-fixed material, allowing comparison of capillary geometry and normalization of fiber cross-sectional area among samples (13), it is rarely measured in muscle biopsies because of technical difficulties (27). Fiber size is also known to differ between perfusion-fixed and fresh-frozen muscle examined at similar muscle lengths (21). Last, because different histochemical staining methods are known to yield differences in the number of capillaries visualized (24), differences in the number of capillaries seen by histochemistry compared with perfusion fixation could also affect results between the two methods.

To evaluate the effect of the above factors, we first compared the two measurements of the size of the C/F interface [i.e., via the C/F perimeter ratio (16) and the quotient of the individual C/F ratio and fiber perimeter, CFPE index (6)] in the same sections of vascular perfusion-fixed muscle, i.e., where sarcomere length, fiber size, and identification of capillaries are the same in the two methods. We hypothesized that there would be a significant correlation between the two independent estimates of the size of the C/F interface measured in the same sections. Then we compared the estimates of the size of the C/F interface in fresh-frozen muscle (obtained by open biopsy and processed for histochemistry) with those made in perfusion-fixed material using the same method (CFPE index). Because of the confounding differences in fiber size, sarcomere length, and capillary visualization in perfusion-fixed vs. histochemically stained material, we hypothesized that there would be a significant difference in the magnitude of the size of the C/F interface estimated by the CFPE index in the two preparations.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. The animals used in this study were part of a study published previously (7), where neither measurements of the C/F interface nor histochemical analyses were reported. In compliance with University of Califronia San Diego Animal Subjects Committee approval, nine purpose-bred dogs were used. As described previously (7), the animals were prepared for study of electrically stimulated muscle contractions in the gastrocnemius and superficial digital flexor muscle complex (data not reported here). Briefly, the animals were anesthetized with 30 mg/kg pentobarbital, intubated with a cuffed endotracheal tube, and ventilated with a Harvard 613 ventilator. The left gastrocnemius, superficial digital flexor muscle complex, and accompanying vasculature were then surgically isolated (10). After the muscle contraction studies, the animals were euthanized with a lethal dose of pentobarbital, and the entire left gastrocnemius-superficial digital flexor muscle complex was perfusion fixed in situ at a nonpulsatile pressure of 80-100 Torr, according to well-established methods (13). Briefly, the vasculature was first perfused with saline until the venous drainage was clear of blood and then perfused with glutaraldehyde fixative (6.25% glutaraldehyde solution in 0.1 M sodium cacodylate buffer; total osmolarity of 1,100 mosM; pH 7.4). Muscle samples were obtained from the midportion of the midbelly of the left medial gastrocnemius and processed for microscopy (13). Eight blocks were cut from each muscle into four transverse and four longitudinal 1-µm-thick sections using an LKB Ultratome III and stained with a 0.1% toluidine blue aqueous solution. Sarcomere length was measured in each longitudinal section at ×1,000 magnification (13).

The contralateral gastrocnemius-superficial digital flexor complex was sampled in six out of the nine animals by open biopsy. Before perfusion fixation on the left side, the right gastronemius-superficial digital flexor complex was excised, and samples were taken from the midportion of the midbelly of the medial gastrocnemius (i.e., at a similar site as in the perfusion-fixed muscle), mounted on cork blocks in tissue freezing medium (Triangle Biomedical Sciences), and frozen in liquid isopentane cooled to -140°C in liquid nitrogen. Samples were stored at -80°C until sectioning. Eight-micrometer-thick transverse sections (one per muscle sample) were cut at -20°C on a cryostat (Jung-Reichert Cryocut 1800) and kept at -20°C until histochemical processing (performed within 1 wk of sectioning). The sections were first fixed in a Guth and Samaha fixative (5) and then incubated at 37°C for 1 h in a lead (Pb)-ATPase staining medium to simultaneously stain for fiber types and capillaries in the same sections, according to the methods described by Rosenblatt et al. (25).

Five-micrometer-thick longitudinal sections were cut at -20°C and stained with a 0.1% toluidine blue aqueous solution, in which sarcomere length was measured at ×1,000 magnification. As noted previously (27), it is difficult to yield truly longitudinal sections with frozen muscle biopsy material. To facilitate this process, the longitudinal sections were cut from the same muscle blocks used for obtaining transverse sections by bisecting the block through the middle (90° to the transverse axis) and remounting the sample in tissue-freezing medium for longitudinal section orientation. Even with this approach, portions of longitudinal sections contained obliquely cut material. Thus sarcomere length in a given longitudinal section was obtained from the minimum average distance among 10 consecutive sarcomeres measured only in regions of longitudinally sectioned fibers. An average of 8.6 ± 0.6 groups of 10 consecutive sarcomeres were measured per longitudinal section in five samples.

Morphometry in perfusion-fixed material. Morphometry was performed by a single observer, blinded to the identity of the samples, according to well-established methods (13, 19). Capillary density was estimated in each of the four transverse sections by point counting with a 100-point eyepiece grid test at a magnification of ×400, using the fibers as the reference space (2, 13). Each section was systematically subsampled to yield as many nonoverlapping frames as possible (200-300 fibers/muscle), with an average of 14 ± 1 (SE) frames measured in each muscle. Fiber cross-sectional area and perimeter, as well as the number of capillaries around a fiber, were measured using an image analyzer (Videometric V150) at ×400 on an average of 55 ± 2 fiber profiles per muscle. C/F number ratio was estimated as the product of capillary density and fiber cross-sectional area (18). C/F perimeter ratio was measured by intersection counting at ×1,000 using a 100-point eyepiece grid test in each transverse section (16). Independent measurements of fiber size (cross-sectional area and perimeter) and capillary number (number of capillaries around a fiber and the individual, fiber-based C/F ratio; Ref. 9) were made using a separate image analysis system (Sigmascan Pro, version 4.0) on an average of 99 ± 7 fibers/muscle. The quotient of the individual C/F ratio and fiber cross-sectional perimeter (CFPE index; Ref. 6) was then calculated.

Morphometry in histochemically stained material. All analyses were performed by a single observer blinded to the identity of the samples. Fiber-type proportion was assessed by point-counting using a 100-point eyepiece grid test at a magnification of ×250. Each section was systematically subsampled to yield 20 frames/muscle. Muscle fiber cross-sectional area and perimeter, the number of capillaries around a fiber, and the individual, fiber-based C/F ratio were estimated at a magnification of ×250 using an image analyzer (Sigmascan Pro 4.0, SPSS Science), in the same sections used to determine fiber-type proportion. An average of 4 ± 0 fields were examined in each section, yielding 126 ± 6 fiber profiles per muscle. The size of the C/F interface in biopsy material was estimated using the CFPE index (6). Briefly, this involved first determining the individual (i.e., fiber-based) C/F ratio (9), which is the sum of the fractional contribution of each capillary around a fiber, based on the number of fibers sharing each capillary [i.e., the sharing factor (22)], for all fibers in a given field. The CFPE index was then calculated as the quotient of individual fiber-based C/F ratio and fiber perimeter for each fiber.

Normalization for sarcomere length. To permit comparison of fiber size and estimates of the size of the C/F interface at the same sarcomere length between tissues, fiber size (area and perimeter) in muscle biopsy material was normalized to the sarcomere length measured in the perfusion-fixed samples, based on the inverse relationship between fiber cross-sectional area and sarcomere length (13, 17) and between fiber perimeter and the square root of sarcomere length (16).

Comparison between morphometric estimates of the size of the C/F interface. While it is measured directly by intersection counting (see above), the C/F perimeter ratio can also be expressed mathematically by the following equation
B<SUB>B</SUB>(c<IT>,</IT>f)<IT>=N</IT><SUB>N</SUB>(c<IT>,</IT>f)<IT>×<A><AC>b</AC><AC>&cjs1171;</AC></A></IT>(c)<IT>/<A><AC>b</AC><AC>&cjs1171;</AC></A></IT>(f) (1)
where BB(c,f) is the C/F perimeter ratio, NN(c,f) is the C/F number ratio, <A><AC>b</AC><AC>&cjs1171;</AC></A>(c) is the average capillary perimeter, and <A><AC>b</AC><AC>&cjs1171;</AC></A>(f) is the average fiber perimeter. In contrast, the CFPE index is the quotient of the individual C/F number ratio NN(c,f)i and fiber perimeter b(f)
CFPE index<IT>=N</IT><SUB>N</SUB>(c<IT>, </IT>f)<SUB><IT>i</IT></SUB><IT>/b</IT>(f) (2)
Thus the mathematical difference between these two estimates of the size of the C/F interface is average capillary sectional perimeter.

Statistics. Values are reported as means ± SE. Differences between the same measurements made by independent methods in the same samples, or by the same method in biopsy vs. perfusion-fixed material, were assessed by paired Student's t-test. Pearson correlation analysis was used to assess the relationship between measurements made independently in the same samples and between measurements made in the left vs. right gastrocnemius muscle. The level of significance was set as alpha  = 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Comparison of methods for estimating C/F ratio. Table 1 shows the results of the comparison of methods in the same sections. We found no difference between estimates of the C/F ratio calculated on a fiber-by-fiber basis (individual C/F ratio, 2.46 ± 0.07) and as the product of capillary density and fiber cross-sectional area (2.39 ± 0.06) in vascular perfusion-fixed samples.

                              
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  		Table 1.   Comparison of independent measurements made in the same perfusion-fixed sections

Comparison of vascular perfusion-fixed muscle and histochemically stained biopsy material. Figure 1 shows micrographs of tissue fixed by vascular perfusion with glutaraldehyde and stained with 0.1% toluidine blue (Fig. 1A) and biopsy material stained with Pb-ATPase staining medium (25) to demonstrate fiber types and capillaries (Fig. 1B). Note the marked difference in muscle fiber size between the two preparations, with fiber cross-sectional area being 45% less (P < 0.05) in the vascular perfusion-fixed (1,708 ± 63 µm2; Table 2) than in biopsy material (3,084 ± 109 µm2; Table 3). Similarly, fiber perimeter was 23% lower in perfusion-fixed material (179 ± 3 µm) than in biopsy material (231 ± 4 µm). The greater fiber size in the biopsies was partly due to greater muscle contraction, with sarcomere length being 1.58 ± 0.03 µm (n = 5) in biopsy samples compared with 1.87 ± 0.00 µm (n = 6) in perfusion-fixed material (P < 0.05). However, taking this difference into account by normalizing fiber size to 1.9-µm sarcomere length did not completely eliminate the differences in fiber size, with a 33 and 15% difference remaining in fiber cross-sectional area (perfusion fixed, 1,682 ± 76 µm2; biopsy, 2,486 ± 85 µm2) and perimeter (perfusion fixed, 178 ± 3 µm; biopsy, 206 ± 3 µm), respectively, between the two preparations. As shown in Tables 2 and 3, no difference was seen in the individual (fiber-based) C/F ratio between perfusion-fixed (2.55 ± 0.03) and biopsy material (2.38 ± 0.13). Similarly, there was no difference in the number of capillaries around a fiber between perfusion-fixed (6.3 ± 0.1) and biopsy material (6.1 ± 0.3), demonstrating that the number of capillaries identified was independent of tissue processing. Because of the greater fiber size in biopsies, CFPE index was significantly greater in perfusion-fixed than biopsy material (Tables 2 and 3).


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  		Fig. 1.   Photomicrographs of portions of muscle bundles in plastic-embedded sections of perfusion-fixed dog gastrocnemius muscle (A) and histochemically stained frozen sections of muscle biopsy taken in a similar anatomic site in the contralateral hindlimb (B). Note the large difference in fiber size between the 2 preparations, in part because of the greater degree of contraction in biopsy (sarcomere length, 1.63 µm) than perfusion-fixed tissue (1.87 µm) and expansion with thawing in biopsies vs. shrinkage in perfusion-fixed material. Bar = 50 µm.


                              
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  		Table 2.   Measurements made in individual vascular perfusion-fixed muscles


                              
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  		Table 3.   Measurements made in individual histochemically stained biopsies

Correlations between estimates of the size of the C/F interface. There was a significant correlation between the two independent estimates of the size of the C/F interface (C/F perimeter ratio and CFPE index) in the same sections of perfusion-fixed material (r = 0.75, P < 0.05; Fig. 2). Despite this finding, there was no significant relationship between the CFPE index measured in perfusion-fixed vs. biopsy material (r = 0.06, P = 0.91). Accounting for differences in sarcomere length had a small effect on the correlation but did not yield significance (r = 0.27, P = 0.66, n = 5; Fig. 3). Similarly, there was no significant relationship between the C/F perimeter ratio measured in perfusion-fixed material and CFPE index in histochemically stained biopsies, with or without normalization for sarcomere length (r < 0.06, P > 0.9).


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  		Fig. 2.   Plot of the quotient of the individual capillary-to-fiber (C/F) ratio and fiber perimeter [C/F perimeter exchange (CFPE) index] vs. the C/F perimeter ratio estimated in the same perfusion-fixed sections.



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  		Fig. 3.   Plot of the quotient of the individual, fiber-based C/F ratio and fiber perimeter (CFPE index) in sections of perfusion-fixed vs. histochemically stained biopsy material at the same sarcomere length. There is no significant relationship (r = 0.27, P = 0.66).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results show a significant correlation between two independent estimates of the size of the C/F interface, C/F perimeter ratio and CFPE index, when both measurements are made in the same sections of perfusion-fixed muscle. However, when comparison is made of one estimate (CFPE index) between plastic-embedded sections of perfusion-fixed material and frozen sections of biopsies from a similar anatomic site in contralateral limbs, significantly smaller CFPE index values are found in the biopsies, and no correlation is seen between CFPE index estimates in the two preparations. Similarly, there is no correlation between CFPE index measured in biopsies and C/F perimeter ratio in perfusion-fixed tissue. As discussed below, these latter observations reflect the confounding influence of several factors in histochemically stained frozen biopsies vs. perfusion-fixed material, including differences in fiber size, sarcomere length, and muscle sampling between the two tissue preparations.

Comparison of C/F perimeter ratio and CFPE index in the same sections of perfusion-fixed muscle. A significant correlation was found between the two methods when they were applied to the same sections (r = 0.75, P < 0.05). The close agreement between the estimates of the C/F number ratio on a fiber-by-fiber basis (the individual C/F ratio; Ref. 9) and those calculated from capillary density and fiber cross-sectional area (18) shows that differences in the approach used to quantify C/F number ratio did not adversely affect our comparison. In addition, there was a similar degree of intramuscle sample variability using each method. Specifically, the average coefficient of variation between frames was 28 ± 1 and 25 ± 1% for CFPE index and C/F perimeter ratio, respectively, yielding relative standard errors of 3-5% for both estimates in each muscle. Therefore, as expected, because the difference between CFPE index and C/F perimeter ratio is in capillary perimeter (not measured in CFPE index; see METHODS), calculating C/F perimeter ratio from the product of capillary perimeter (24.4 ± 0.7 µm; range: 21.9-28.1 µm) and CFPE index reveals no difference with measured C/F perimeter ratio (0.33 ± 0.01 measured vs. 0.34 ± 0.02 calculated; P > 0.05) and improves the correlation between the two measurements (r = 0.95, P < 0.01). In other words, when made in the same muscle samples, the two independent estimates of the size of the C/F interface correspond closely and differ primarily in proportion to capillary perimeter. As this comparison demonstrates, a major advantage of the C/F perimeter ratio is that, by incorporating capillary perimeter, it accounts for capillary geometry (i.e., the degree of capillary tortuosity and branching), because more tortuous and branched capillaries have a greater probability of being sectioned obliquely and, therefore, yield larger perimeters in muscle transverse sections (16). Accounting for capillary perimeter becomes of further importance when the size of the C/F interface is compared across species or muscles that may differ in capillary geometry (e.g., bat flight muscle vs. bat and rat hindlimb muscles; Ref. 19) at a given sarcomere length. In this type of comparison, differences in capillary perimeter (because of the differences in capillary geometry among muscles and/or species) would result in the CFPE index systematically underestimating the difference in size of the C/F interface between the muscles compared with that detected via the C/F perimeter ratio.

Comparison between perfusion-fixed and histochemically stained material. In contrast to the significant relationship between the two independent estimates of the size of the C/F interface in the same sections of perfusion-fixed material, we found significantly smaller values in biopsies and no significant correlation when the same estimate (CFPE index) was compared in perfusion-fixed and biopsy material at the same sarcomere length. In accounting for these results, several significant differences were found when the same measurements were made in perfusion-fixed and biopsy material. Perhaps the most striking difference was that fiber cross-sectional area was 45% less and fiber perimeter 23% less in perfusion-fixed tissue than in frozen muscle biopsies. Because fiber size is inversely related to sarcomere length (13, 17), it is important to account for sarcomere length in each tissue (frozen muscle biopsies, 1.58 ± 0.03 µm; perfusion-fixed samples, 1.87 ± 0.00 µm) when fiber cross-sectional area and perimeter are compared in the two preparations. However, normalizing fiber size to sarcomere length did not eliminate the difference in fiber size, with a 33% difference in fiber cross-sectional area and 15% in fiber perimeter remaining between the tissues. This difference is the same as reported previously in perfusion-fixed rat soleus muscle vs. contralateral muscle stretched to the same muscle length and frozen for histochemistry (21). In this latter study, approximately one-third of the difference in fiber cross-sectional area between perfusion-fixed and histochemically stained material was attributed to fiber swelling during thawing of frozen muscle sections on glass slides. Specifically, sections not fixed before thawing on the glass slides before histochemical staining were 10% larger than those fixed with formalin vapor before thawing (21). Identifying the reason(s) for the remaining difference in fiber size between perfusion-fixed muscle tissue and frozen biopsies, such as specific shrinkage during fixation and tissue processing, is beyond the scope of this investigation. The main point to be made here is that a portion of the difference in fiber size between histochemically stained sections of frozen biopsies and plastic-embedded sections of perfusion-fixed material can be attributed to the expected greater degree of muscle contraction in the biopsies than in muscle perfusion fixed in situ, as was the case in this study. However, significant differences in fiber size between the two preparations remain after accounting for differences in sarcomere length; i.e., all other things being equal, estimates of the size of the C/F interface will be larger in perfusion-fixed than biopsy material because of the smaller fiber size in fixed than frozen material. Indeed, this is precisely what we see in the present investigation when we compare the CFPE index between perfusion-fixed and histochemically stained material. Specifically, the quotient of the C/F ratio and fiber perimeter was 22% smaller in biopsy than perfusion-fixed muscle (11.11 vs. 14.28 capillaries per 1,000-µm fiber perimeter) at the same sarcomere length.

Importantly, there was no difference in the estimates of capillary number between perfusion-fixed and histochemically stained biopsy material. This was true regardless of whether the number of capillaries around a fiber or the individual (fiber-based) C/F ratio were compared. It shows that this particular histochemical assay, which uses fixation of the thawed sections in formalin fixative followed by incubation in a Pb-ATPase medium to simultaneously reveal fiber types and capillaries in a single section (25), allows identification of the same number of capillaries as in perfusion-fixed tissue, where presumably all capillaries are visible and easily identified. However, despite the similarity in average numbers of capillaries identified and similar intramuscle variability in capillary counts in biopsies (between-frame coefficient of variation, 12 ± 4%) and perfusion-fixed material (between-block coefficient of variation, 12 ± 2%), there was no correlation between estimates of the individual C/F ratio in the two preparations (r = 0.05, P = 0.93). This lack of correlation certainly contributes to our results, as discussed below.

Sources of variability in comparing histochemically stained to perfusion-fixed material from contralateral muscles. Despite the differences in fiber size, one might still expect a correlation in the CFPE index between perfusion-fixed and biopsy material, if reduction in fiber size due to tissue preparation systematically affected samples from one of the two hindlimbs of the same animals. However, the expected correlation between measurements of fiber cross-sectional area normalized to sarcomere length in each tissue was not significant (r = 0.60, P = 0.29, n = 5). This observation is similar to that reported previously in a comparison of biopsies taken from the left and right vastus lateralis in human subjects (12). Specifically, in biopsies sampled from similar sites in the left and right legs, there was up to a 25% difference in fiber size (12) and no correlation between fiber-size estimates between contralateral vastus lateralis muscles. This random variation between contralateral limbs likely contributes to the lack of correlation that we observed between estimates of fiber size and capillary counts and thus obscures correlation between CFPE index estimates in perfusion-fixed vs. histochemically stained tissue. Note that, although the poor correlation between CFPE index estimates in perfusion-fixed vs. histochemically stained material appears strongly influenced by one data point that could be considered an outlier (Fig. 3), there is no statistical or scientific basis for excluding this data point. Also, the lack of correlation observed between fiber size and capillary counts between contralateral muscles suggests that the lack of correlation in CFPE index estimates is not an artifact.

In addition to variation between contralateral limbs, it is also possible that the regions of the gastrocnemius muscle that were sampled were not rigorously identical between the two hindlimbs, i.e., that regional heterogeneity within the gastrocnemius is contributing to obscure relationships in fiber size and capillarization between opposing limbs, even if group averages in C/F numbers did not differ between the two sides. Two observations support this interpretation. First, there was a greater intermuscle variation in capillary counts in the biopsies (coefficient of variation for the individual C/F ratio, 13%) than in perfusion-fixed muscles (3%), despite the similar intramuscle variation noted above. Second, there was no correlation in fiber size (r = 0.40, P = 0.43, n = 6; R. T. Hepple, unpublished results) seen between samples taken from the superficial vs. deep midportion of the medial gastrocnemius in the same animals used in this study. The small variation in C/F number between muscles, coupled with the above considerations, precludes further assessment of the potential relationship between our measurements of the size of the C/F interface in perfusion-fixed and biopsy material. Thus a valuable future investigation would be to compare estimates in muscles representing a broader range of fiber capillarization. Given the high degree of capillarization seen in the canine gastrocnemius muscle (present results and Ref. 20) and other canine hindlimb muscles (20, 26), inclusion of less oxidative muscles, such as plantaris (1, 23) and the white region of gastrocnemius (1, 4) in rat, would be appropriate. Nonetheless, the present results underscore some of the significant differences that exist between perfusion-fixed and histochemically stained muscle and how they can influence estimates of the size of the C/F interface.

In summary, the present results show that the two methods available to assess the size of the C/F interface in perfusion-fixed muscle (C/F perimeter ratio) and histochemically stained biopsy material (CFPE index) are significantly correlated and only differ in proportion to capillary perimeter (not measured in CFPE index) when they are applied to the same sections of perfusion-fixed muscle. However, smaller CFPE index estimates are found in biopsy than in perfusion-fixed material, with no significant correlation between CFPE index values normalized to a common sarcomere length in the two preparations. Similarly, there is no significant correlation between CFPE index and C/F perimeter ratio measured in biopsy and perfusion-fixed tissue, respectively. The smaller CFPE index estimates in biopsies is due to greater fiber cross-sectional area in histochemically stained thawed sections than in perfusion-fixed sections of muscle, even after accounting for differences in sarcomere length between tissue preparations. Furthermore, the lack of correlation between estimates of the size of the C/F interface in the two preparations relates to the confounding influence of random variability between contralateral muscles and potential sampling differences between contralateral limbs.


    ACKNOWLEDGEMENTS

We thank Peter Agey for technical assistance with this study.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-PO1-17731.

Address for reprint requests and other correspondence: R. T. Hepple, Faculty of Kinesiology, Univ. of Calgary, 2500 Univ. Dr. NW, Calgary, Alberta, Canada T2N 1N4 (E-mail: hepple{at}ucalgary.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 March 2001; accepted in final form 10 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Armstrong, RB, and Phelps RO. Muscle fiber type composition of the rat hindlimb. Am J Anat 171: 259-272, 1984[ISI][Medline].

2.   Eisenberg, BR, Kuda AM, and Peter JB. Stereological analysis of mammalian skeletal muscle. J Cell Biol 60: 732-754, 1974[Abstract/Free Full Text].

3.   Gray, SD, and Renkin EM. Microvascular supply in relation to fiber metabolic type in mixed skeletal muscles of rabbits. Microvasc Res 16: 406-425, 1978[ISI][Medline].

4.   Gute, D, Laughlin MH, and Amann JF. Regional changes in capillary supply in skeletal muscle of interval-sprint and low-intensity, endurance-trained rats. Microcirculation 1: 183-193, 1994[Medline].

5.   Guth, L, and Samaha FJ. Procedure for the histochemical demonstration of actomyosin ATPase. Exp Neurol 28: 365-367, 1970[ISI][Medline].

6.   Hepple, RT. A new measurement of tissue capillarity: the capillary-to-fibre perimeter exchange index. Can J Appl Physiol 22: 11-22, 1997[ISI][Medline].

7.   Hepple, RT, Hogan MC, Stary C, Bebout DE, Mathieu-Costello O, and Wagner PD. Structural basis of muscle O2 diffusing capacity: evidence from muscle function in situ. J Appl Physiol 88: 560-566, 2000[Abstract/Free Full Text].

8.   Hepple, RT, MacKinnon SLM, Goodman JM, Thomas SG, and Plyley MJ. Resistance and aerobic training in older men: effects on VO2 peak and the capillary supply to skeletal muscle. J Appl Physiol 82: 1305-1310, 1997[Abstract/Free Full Text].

9.   Hepple, RT, MacKinnon SLM, Thomas SG, Goodman JM, and Plyley MJ. Quantitating the capillary supply and the response to resistance training in older men. Pflügers Arch 433: 238-244, 1997[ISI][Medline].

10.   Hogan, MC, Roca J, West JB, and Wagner PD. Dissociation of maximal O2 uptake from O2 delivery in canine gastrocnemius in situ. J Appl Physiol 66: 1219-1226, 1989[Abstract/Free Full Text].

11.   Honig, CR, Gayeski TEJ, and Groebe K. Myoglobin and oxygen gradients. In: The Lung: Scientific Foundations, edited by Crystal RG, and West JB.. New York: Raven, 1991, p. 1489-1496.

12.   Lexell, J, and Taylor CC. A morphometrical comparison of right and left whole human vastus lateralis muscle: how to reduce sampling errors in biopsy techniques. Clin Physiol 11: 271-276, 1991[ISI][Medline].

13.   Mathieu-Costello, O. Capillary tortuosity and the degree of contraction or extension of skeletal muscles. Microvasc Res 33: 98-117, 1987[ISI][Medline].

14.   Mathieu-Costello, O, Agey PJ, Wu L, Hang J, and Adair TH. Capillary-to-fiber surface ratio in rat fast-twitch hindlimb muscles after chronic electrical stimulation. J Appl Physiol 80: 904-909, 1996[Abstract/Free Full Text].

15.   Mathieu-Costello, O, Agey PJ, Wu L, Szewczak JM, and MacMillen RE. Increased fiber capillarization in flight muscle of finch at altitude. Respir Physiol 111: 189-199, 1998[ISI][Medline].

16.   Mathieu-Costello, O, Ellis CG, Potter RF, Macdonald IC, and Groom AC. Muscle capillary-to-fiber perimeter ratio: morphometry. Am J Physiol Heart Circ Physiol 261: H1617-H1625, 1991[Abstract/Free Full Text].

17.   Mathieu-Costello, O, Poole DC, and Logemann RB. Muscle fiber size and chronic exposure to hypoxia. Adv Exp Med Biol 248: 305-311, 1989[Medline].

18.   Mathieu-Costello, O, Suarez RK, and Hochachka PW. Capillary-to-fiber geometry and mitochondrial density in hummingbird flight muscle. Respir Physiol 89: 113-132, 1992[ISI][Medline].

19.   Mathieu-Costello, O, Szewczak JM, Logemann RB, and Agey PJ. Geometry of blood-tissue exchange in bat flight muscle compared with bat hindlimb and rat soleus muscle. Am J Physiol Regulatory Integrative Comp Physiol 262: R955-R965, 1992[Abstract/Free Full Text].

20.   Maxwell, LC, Barclay JK, Mohrman DE, and Faulkner JA. Physiological characteristics of skeletal muscles of dogs and cats. Am J Physiol Cell Physiol 233: C14-C18, 1977[Abstract/Free Full Text].

21.   Ogilvie, RW, Agey PJ, Hansens C, and Mathieu-Costello O. Comparison of fiber size and capillarity in perfusion fixed and frozen rat soleus muscle (Abstract). Physiologist 35: 198, 1992.

22.   Plyley, MJ, and Groom AC. Geometrical distribution of capillaries in mammalian striated muscle. Am J Physiol 228: 1376-1383, 1975.

23.   Poole, DC, and Mathieu-Costello O. Relationship between fiber capillarization and mitochondrial volume density in control and trained rat soleus and plantaris muscles. Microcirculation 3: 175-186, 1996[Medline].

24.   Qu, Z, Andersen JL, and Zhou S. Visualization of capillaries in human skeletal muscle. Histochem Cell Biol 107: 169-174, 1997[ISI][Medline].

25.   Rosenblatt, JD, Kuzon WM, Plyley MJ, Pynn BR, and McKee NH. A histochemical method for the simultaneous demonstration of capillaries and fiber types in skeletal muscle. Stain Technol 62: 85-92, 1987[ISI][Medline].

26.   Rosenblatt, JD, Kuzon WM, Pynn BR, Plyley MJ, and McKee NH. Fiber type, fiber size, and capillary geometric features of the semitendinosus muscle in three types of dogs. Am J Vet Res 49: 1573-1576, 1988[Medline].

27.   Zumstein, A, Mathieu O, Howald H, and Hoppeler H. Morphometric analysis of the capillary supply in skeletal muscles of trained and untrained subjects-its limitations in muscle biopsies. Pflügers Arch 397: 277-283, 1983[ISI][Medline].

J APPL PHYSIOL 91(5):2150-2156
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


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