THE AMERICAN JOURNAL OF ANATOMY 191:74-84 (1991)
Morphometric Analysis of Capillary Geometry in Pigeon Pectoralis Muscle ODILE MATHIEU-COSTELLO Department of Medicine, M-023A, University of California, San Diego, La Jolla, California 92093-0623
crease in oxygen consumption during flight (5 to 10 times that of resting; Butler et al., 19771, remarkable endurance has been reported in pigeons, far beyond the capacity of the most athletic mammals (Pennycuick, 1968). Such endurance has been related to the presence of a large proportion of highly aerobic fibers, which in the pigeon can represent more than 80% of the area of M. pectoralis (reviewed by Rosser and George, 1986). Although the characterization and the distribution of fiber types in the pectoralis muscle of birds are well known, there have been only a few reports on the capillary supply of such highly aerobic skeletal muscles (Rakusan et al., 1971; Khan, 1979; Lundgren and Kiessling, 1988). To our knowledge, the geometry of the capillary network in bird flight muscle has never been analyzed. The importance of considering capillary geometry when estimating muscle capillary supply from histological sections was recognized earlier (Andersen and Kroese, 1978; Appell and Hammersen, 1978). Ellis e t al. (1983) showed that capillary configuration can substantially affect the predicted efficiency of 0, transfer in muscles. We have previously demonstrated that fiber shortening is a major determinant of capillary tortuosity in skeletal muscles of mammals (Mathieu-Costello, 1987; Mathieu-Costello e t al., 1988, 1989a). In mammalian locomotory muscles with large differences in capillarity (capillary countdfiber crosssection range, 450-2,500 mm-,), we found that capillary tortuosity was related to sarcomere length rather than to muscle aerobic capacity (Mathieu-Costello et al., 1989a). The purpose of this study was to examine the relationship(s) between capillary density and geometry in pigeon M. pectoralis. Using samples with large differences in capillary density, we specifically addressed the question of 1) whether or not capillary tortuosity depends on sarcomere length rather muscle aerobic capacity by using samples with larger 0, demands and capillary densities than the mammalian hindlimb muscles previously examined and 2) whether a systematic difference(s) exists in the configuration of the capillary network in the pigeon flight muscle that may contribute to its considerably greater endurance comINTRODUCTION pared to mammalian locomotory muscles. A prelimiThe avian pectoralis muscle is of particular interest nary report has been published (Mathieu-Costello, for the study of structure-function correlations in 1988). blood-tissue gas exchange because of its considerable energy requirements during sustained flight. Except for hovering birds (hummingbirds), practically all the power for flight is generated by one muscle, the downstroke wing depressor, M. pectoralis. In pigeons, the pectoralis muscle constitutes as much as 20% of body Received August 31, 1990 Accepted December 26, 1990. weight (Hartman, 1961). Besides a substantial in-
ABSTRACT The objective of the study was to examine the relationship(s) between the size and the geometry of the capillary network in the flight muscle of pigeon (Columbia Ziuia). To this end, we used morphometry to analyze the degree of anisotropy (i.e., orientation) of capillaries with respect to the axis of the muscle fibers in perfusion-fixed samples of pigeon pectoralis muscles with large difference in capillary density. Capillary number per fiber cross-sectionalarea (range, 1,4914,680 mm-2) depended on fiber size (aerobic fibers, 304-782 pm2; glycolytic, 1,7852,444 pm2), as well as sarcomere length (1.69-2.20 pm), and the relative sectional area of aerobic and glycolytic fibers (aerobic, 4244%of total fiber area). The degree of tortuosity of capillaries, i.e., their bending or sinuosity relative to the muscle fiber axis, was primarily a function of sarcomere length. In spite of largc differences in capillary density, capillary orientation at a given sarcomere length was remarkably similar among samples. In addition to capillaries running parallel to the muscle fiber axis, a unique arrangement of branches running perpendicular to the muscle fiber axis was found in all samples. This arrangement yielded a large circumferential distribution of capillary surface around the muscle fibers. Compared to mammalian limb muscles examined over a 10-foldrange of capillary density (range, 4504,670 rnmp2), the degree of anisotropy of capillaries was greater in all samples of pigeon M. pectoralis. In the pigeon, there was no increase in the amount of capillary surface area available for exchange per microvessel as a result of a greater degree of capillary tortuosity in samples with larger capillary density (capillary number per fiber cross-sectional area > 4,000 mm-2), as compared to samples with a capillary density < 4,000 mrnp2.
0 1991 WILEY-LISS, INC.
75
CAPILLARY GEOMETRY IN PIGEON PECTORALIS
TABLE 1. Measurements (mean pigeon pectoralis muscle Animal No. Weight (gm) Show Racer 1 490 1 490 2 560 2 560 White Carneau 3 50 1 3 501 White King 4 622 4 622 5 617 5 617 6 554 6 554
* SE) of sarcomere length, fiber size and distribution, and capillary density in
Muscle No. Site
Aerobic Glycolytic pm2 ~ ~ (% 0 )g(fg)z.12 ~
1', Pm
~(fa)z.,zkm2
1 2 3 4
sup deep sup deep
1.80 t 0.03 1.83 2 0.10 1.79 t 0.04 1.79 i 0.02
379 i 31 719 t 59 362 2 17 581 t 71
84 68 82 74
5 6
sup deep
1.93 i 0.04 1.87 t 0.12
7 8 9 10 11 12
sup deep sup deep sup deep
2.12 i 0.01 2.20 t 0.01 1.96 i 0.01 1.97 i 0.01 1.69 t 0.03 1.79 t 0.02
'Sarcomere length. 2Cross-sectional area at I , = 2.1 pm. 3Relative cross-sectional area of aerobic fibersitotal fiber area. 4Capillary number per mm2 of muscle fiber cross-sectional area at 1,
MATERIALS AND METHODS
Twelve perfusion-fixed muscles from a total of six pigeons (Columba liuia; body mass Mb, 490-622 gm; Table 1) were used in the study. Three breeds were used in a n attempt to obtain samples with larger differences in capillary density. Although it was not the main purpose of the study, we were interested in possible genetic difference(s1 in flight muscle capillary density and geometry. Show Racers (SR) and White Carneau (WC), both 9 months old, were derived from animals obtained from the Palmetto pigeon plant. They were kept in a barn with flying coops (approx. 10 x 20 x 8 feet). White Kings (WK; 6 months old) were obtained from a local squab ranch where they had been kept in a small aviary (approx. 7 x 6 x 10 feet). All animals were anesthetized by intravenous injection of sodium pentobarbital (3 mg/100 gm). The vascular perfusion of the muscles in situ was performed as described elsewhere in detail (Mathieu-Costello, 1987). Briefly, the entire vasculature was perfused with saline (11.06 gm NaCl/liter; 350 mOsm; 20,000 USP units heparidliter) via a cannula inserted directly into the left ventricle, while the right atrium was cut open to secure outflow. Perfusion fixation followed with a 6.25% solution of glutaraldehyde in 0.1M sodium cacodylate buffer (total osmolarity of the fixative: 1,100 mOsm; pH 7.4) as described previously (Mathieu-Costello, 1987). All perfusions were performed at a nonpulsatile pressure of 150-170 mm Hg. Muscles samples (approx. 1 cm x 4 mm x 1 mm) were taken in the superficial (ventral) and deep portions (dorsal) of the pectoralis muscle a t one sampling site, approximately halfway along the cranial-caudal and the lateral axis of either right or left pectoralis major muscle in each bird. In that site, the thickness of the pectoralis muscle ranged from 5-15 mm (WK) to 20 mm (SR). All samples were cut into thin longitudinal strips, stored in glutaraldehyde fixative, and processed for electron mi-
=
t3 t2 t5
2
1,785 t 96 1,853i 125 2,051 t 225 2,263 i 248
3,194 i 141 2,615 t 189 4,108 2 317 2,491 t 99
428 i 27 782 5 94
62 i 4 48 2 4
2,444 t 298 2,186 2 244
3,229 i 221 1,979 5 164
382 i 27 367 2 22 334 t 22 304 i 19 348 t 27 441 2 38
73 i 4 76 t 3 42 i 2 74 i 3 61 t 5 54 i 4
2,257 t 277 2,078 t 79 1,909 i 219 2,256 t 177 2,284 ? 129 2.045 i 146
4,143 i 187 3,974 t 133 3,490 t 150 6,055 i 283 5,163 t 402 1.749 t 102
2
2.1 pm
croscopy as previously described (Mathieu-Costello, 1987). Tissue Sectioning
One-micrometer sections were cut on a LKB Ultrotome I11 and stained with 0.1% aqueous toluidine blue solution. From each muscle sample, 4-8 blocks were cut into 4 transverse sections (angle between normal to section and fiber axis, a = O O ) , and 4 longitudinal sections ( a= d 2 ) , following a procedure described elsewhere in detail (Mathieu-Costello, 1987). In one sample, we also evaluated capillary orientation distribution as described previously (Mathieu-Costello, 1987) by using a series of sections taken at 0" to 90" to the fiber axis by steps of 5 to 10"from 4 blocks. Sarcomere length (1,) was measured in each longitudinal section (average of 10 measurements of groups of consecutive sarcomeres, systematically sampled over the entire area of each section examined a t magnification of x 630; Mathieu-Costello, 1987). Morphometry
The 1-pm sections were subsampled by a s many systematic, non-overlapping quadrats (i.e., micrographs) a s technically possible from the section area. The number of micrographs per section averaged 6 to 14, yielding about 240-560 fiber profiles in transverse sections for each sample. An average of 8 to 16 micrographs were obtained from each longitudinal section, yielding about 60-110 portions of fiber profiles for each sample. The micrographs were taken on 35-mm direct positive films (Kodak Direct MP 5360) at magnification x 400 by using a Leitz Ortholux light microscope equipped with a Leica camera. A micrograph of a stage micrometer was recorded for calibration. The 35-mm films were projected on a square grid test A 144 (see Weibel, 1979, Appendix 31, a t a final magnification of x 2,060 by using a microfilm reader (Documator DL 2, Jenoptic,
76
0 . MATHIEU-COSTELLO
Jena). The point counts of numerical and volume densities of capillary profiles were collected, stored, and processed by using a n Apple computer. As in previous studies (see Mathieu-Costello, 1987), capillary density estimates were related to the muscle fibers as a reference space in all samples in order to avoid variations due to the unreliable preservation of the intercellular spaces by the preparation procedures in different samples. As discussed previously, it is important to assess the effect of tissue preparation on morphometric estimates of fiber size and sarcomere length (Mathieu-Costello, 1987). In tissues prepared with the same fixatives and procedures as in this study, we found fiber sizes similar to those reported by others in comparable material processed for histochemistry (Zumstein et al., 1983; Mathieu-Costello, 1987). Because histochemical preparation yields a minimum amount of tissue shrinkage, this indicated no major bias in our estimates of fiber size in our glutaraldehyde-fixed material. In the same study, measurements of filament length revealed less than 10% shrinkage compared to filament length in living muscle and compared with data obtained by others (Mathieu-Costello, 1987). The method used to estimate capillary anisotropy in each sample has been described elsewhere in detail (Mathieu et al., 1983).We first estimated the ratio between capillary counts per sectional area of transverse and longitudinal sections of muscle fibers, QA(0) and QA(d2),respectively. This ratio was then used to calculate, via a table or graph of known coefficients, l)the capillary orientation concentration parameter, I 4,000 mm-' (range, 4,342-6,021 mm-2; muscles 3,7,8,10, and 11; Table 3), com ared to samples with Jv(c,f) values < 4,000 mm-'(range 1,834-3,485 mm-'; other muscles, Table 3). Figure 9 shows the plot of the numerical density of capillaries per fiber cross-sectional area, Q A ( a ) , as a function of the sectioning angle, a,on the basis of the Fisher axial distribution model of capillary segments. In the sample considered (muscle lo), capillary number per fiber sectional area was 5,680 2 264 mm-' in transverse sections and 1,090 2 93 mm-2 in longitudinal sections (Table 2). Experimental data were adequately fitted by the Fisher axial distribution model (Fig. 9). Note that the solid line in Figure 9 is not the regression line but the computed value of Q A ( a ) under the Fisher axial distribution model, based on the value of Jv(c,f) in t h a t muscle (see Methods). The estimates of capillary volume density, Vv(c,f), mean capillary diameter, d(c), and capillary length density, Jv(c,f), calculated via eq. 2, are given in Table 3. The two estimates of Jv(c,f) obtained in each sample by using two different methods (eqs. 1 and 2) were different from each other in only one sample (muscle 4). The slope of the linear regression between the two estimates of Jv(c,f) in each sample (r= 0.93) was not significantly different from 1 (P = 0.42).
DISCUSSION To our knowledge, this is the first study where sarSarcomere length, pn comere length, i.e., the degree of shortening or extension of the muscle fibers, has been considered when Fig. 5. Plot of cross-sectional areas of glycolytic and aerobic fibers estimating fiber size and capillary density in pigeon M. against sarcomere length in both superficial and deep portions of White King (circles),in superficial (filled triangles) and deep portions pectoralis. Such consideration is important because of (open triangles) of Show Racer, and in superficial (filled diamonds) the direct effect of sarcomere length on fiber size (and and deep portions (open diamonds) of White Carneau pigeon pectora- therefore capillary densitytfiber cross-sectional area) lis muscle. Dotted lines indicate the predicted change in fiber cross- and capillary geometry (reviewed by Mathieu-Costello sectional area with sarcomere length assuming a constant fiber volet al., 198913;Mathieu-Costello, 1990). An additional difume, i.e., calculated as a(f) = constant / I,,; constant = product of 1, and a(f) means in the sample group. For glycolytic fibers, all points ficulty presents itself when studying capillary geomewere considered; for aerobic fibers, WK only. Error bars indicate i 1 try in pigeon pectoralis muscle. As described above, it SE (within samples); where a n error bar is not shown, the SE is within is critical to consider the relative area of aerobic and the symbol size. glycolytic fibers in each section, since the estimate of capillary orientation depends on the ratio between capwhere illary countstfiber sectional area in transverse and longitudinal section. The relative area of aerobic and glyUo(K) = (2KF/{r!(2r+l)}. (7) colytic fibers was estimated in each set of sections, r=O after unambiguous identification of aerobic and glycoThe empirical fit to the data for K vs. I, (Fig. 7) was lytic fibers in all tissues. The identification of red and obtained by using non-linear least squares regression white fibers after toluidine-blue staining has been done analysis with the following equation: previously in Canada geese pectoralis muscle (George K = K,, - KOexp {-a(lo - 1.4)} [81 et al., 1983). The estimates of the fitting parameters were K,, = Capillary Density, Fiber Size, and Distribution 1.35, KO = -0.56, and a = -2.26 km. The predicted As expected, capillary density depended on the relavalues for c(K,O) a t sarcomere lengths of 1.6, 2.1, and 2.3 are 1.20, 1.08, and 1.05, respectively (Fig. 8, solid tive area of aerobic vs. glycolytic fibers in the samples. line). For comparison, the curvilinear relationship be- The linear regressions (Fig. 6) yielded values for tween c(K,O) and 1, in various muscles of mammals is Q A ( O k . 1 of 6,184 2 513 mm-2 (WK) and 4,769 2 253 also shown in Figure 8. At a given sarcomere length, mm- (SR) at 100% aerobic fibers. Interestingly, simthe value of c(K,O) was smaller in pigeon M. pectoralis ilar capillary densities (QA(0)z.I range, 4,342-7,033) than in mammalian muscles (Fig. 81, indicating a were found in perfusion-fixed rat myocardium similarger degree of capillary anisotropy in the bird mus- larly prepared (Poole and Mathieu-Costello, 1990). In cle. A larger capillary anisotropy is reflected by a the heart, however, both fiber size [a(f),,, range, 2081.6
1.8
r
2.0
2.2
81
CAPILLARY GEOMETRY IN PIGEON PECTORALIS
TABLE 2. Morphometric estimates of capillary anisotropy and capillary length density, calculated via eq. 1, in each sample (values are means f S.E.)
2,738 t 112 2,279 t 108 3,502 2 259 2,123 t 81 2,968 t 194 1,762 2 93 4,182 2 188 4,163 t 138 3,257 t 139 5,680 t 264 4,155 2 315 1,491 2 85
1 2 3 4 5 6 7 8 9 10 11 12
80 2 3 69 t 4 83 t 4 73 2 4 63 t 4 44 t 6 76 t 3 81 2 3 45 2 3 74 t 3 59 2 4 52 2 4
938 2 54 849 t 75 1,378 t 110 712 t 67 833 t 62 711 t 99 1,045 t 64 832 t 64 699 t 54 1,090 t 93 950 2 95 568 ? 54
2.3 & 0.2 2.1 t 0.3 1.9 t 0.3 2.4 t 0.3 3.0 t 0.4 1.9 t 0.4 3.5 t 0.4 4.8 2 0.6 4.3 t 0.6 5.2 2 0.8 3.9 t 0.9 2.0 & 0.3
1.19 t 0.03 1.22 2 0.04 1.24 t 0.05 1.18 t 0.03 1.13 t 0.03 1.25 2 0.06 1.10 t 0.02 1.06 t 0.01 1.07 t 0.01 1.06 t 0.01 1.08 2 0.03 1.23 t 0.05
3,258 t 157 2,780 2 160 4,342 t 366 2,505 t 115 3,354 ? 236 2,203 t 157 4,600 t 223 4,413 t 152 3,485 2 152 6,021 t 285 4,487 2 362 1,834 ? 128
'Number of capillaries per mm2 fiber sectional area in transverse sections. 'Number of capillaries per mm2 fiber sectional area in longitudinal sections. 3Relative sectional area of aerobic fibersitotal fiber area in longitudinal sections 4Degree of capillary anisotropy. 5Coeffcient relating Q,(O) to Jv(c,f) (eq. 1). 'Capillary length per fiber volume.
7000 6000
?
t
0
hl
I
5000
E
4000
E
-
0
5.0 Y
7
A
Morphometric Analysis of Capillary Geometry in Pigeon Pectoralis Muscle ODILE MATHIEU-COSTELLO Department of Medicine, M-023A, University of California, San Diego, La Jolla, California 92093-0623
crease in oxygen consumption during flight (5 to 10 times that of resting; Butler et al., 19771, remarkable endurance has been reported in pigeons, far beyond the capacity of the most athletic mammals (Pennycuick, 1968). Such endurance has been related to the presence of a large proportion of highly aerobic fibers, which in the pigeon can represent more than 80% of the area of M. pectoralis (reviewed by Rosser and George, 1986). Although the characterization and the distribution of fiber types in the pectoralis muscle of birds are well known, there have been only a few reports on the capillary supply of such highly aerobic skeletal muscles (Rakusan et al., 1971; Khan, 1979; Lundgren and Kiessling, 1988). To our knowledge, the geometry of the capillary network in bird flight muscle has never been analyzed. The importance of considering capillary geometry when estimating muscle capillary supply from histological sections was recognized earlier (Andersen and Kroese, 1978; Appell and Hammersen, 1978). Ellis e t al. (1983) showed that capillary configuration can substantially affect the predicted efficiency of 0, transfer in muscles. We have previously demonstrated that fiber shortening is a major determinant of capillary tortuosity in skeletal muscles of mammals (Mathieu-Costello, 1987; Mathieu-Costello e t al., 1988, 1989a). In mammalian locomotory muscles with large differences in capillarity (capillary countdfiber crosssection range, 450-2,500 mm-,), we found that capillary tortuosity was related to sarcomere length rather than to muscle aerobic capacity (Mathieu-Costello et al., 1989a). The purpose of this study was to examine the relationship(s) between capillary density and geometry in pigeon M. pectoralis. Using samples with large differences in capillary density, we specifically addressed the question of 1) whether or not capillary tortuosity depends on sarcomere length rather muscle aerobic capacity by using samples with larger 0, demands and capillary densities than the mammalian hindlimb muscles previously examined and 2) whether a systematic difference(s) exists in the configuration of the capillary network in the pigeon flight muscle that may contribute to its considerably greater endurance comINTRODUCTION pared to mammalian locomotory muscles. A prelimiThe avian pectoralis muscle is of particular interest nary report has been published (Mathieu-Costello, for the study of structure-function correlations in 1988). blood-tissue gas exchange because of its considerable energy requirements during sustained flight. Except for hovering birds (hummingbirds), practically all the power for flight is generated by one muscle, the downstroke wing depressor, M. pectoralis. In pigeons, the pectoralis muscle constitutes as much as 20% of body Received August 31, 1990 Accepted December 26, 1990. weight (Hartman, 1961). Besides a substantial in-
ABSTRACT The objective of the study was to examine the relationship(s) between the size and the geometry of the capillary network in the flight muscle of pigeon (Columbia Ziuia). To this end, we used morphometry to analyze the degree of anisotropy (i.e., orientation) of capillaries with respect to the axis of the muscle fibers in perfusion-fixed samples of pigeon pectoralis muscles with large difference in capillary density. Capillary number per fiber cross-sectionalarea (range, 1,4914,680 mm-2) depended on fiber size (aerobic fibers, 304-782 pm2; glycolytic, 1,7852,444 pm2), as well as sarcomere length (1.69-2.20 pm), and the relative sectional area of aerobic and glycolytic fibers (aerobic, 4244%of total fiber area). The degree of tortuosity of capillaries, i.e., their bending or sinuosity relative to the muscle fiber axis, was primarily a function of sarcomere length. In spite of largc differences in capillary density, capillary orientation at a given sarcomere length was remarkably similar among samples. In addition to capillaries running parallel to the muscle fiber axis, a unique arrangement of branches running perpendicular to the muscle fiber axis was found in all samples. This arrangement yielded a large circumferential distribution of capillary surface around the muscle fibers. Compared to mammalian limb muscles examined over a 10-foldrange of capillary density (range, 4504,670 rnmp2), the degree of anisotropy of capillaries was greater in all samples of pigeon M. pectoralis. In the pigeon, there was no increase in the amount of capillary surface area available for exchange per microvessel as a result of a greater degree of capillary tortuosity in samples with larger capillary density (capillary number per fiber cross-sectional area > 4,000 mm-2), as compared to samples with a capillary density < 4,000 mrnp2.
0 1991 WILEY-LISS, INC.
75
CAPILLARY GEOMETRY IN PIGEON PECTORALIS
TABLE 1. Measurements (mean pigeon pectoralis muscle Animal No. Weight (gm) Show Racer 1 490 1 490 2 560 2 560 White Carneau 3 50 1 3 501 White King 4 622 4 622 5 617 5 617 6 554 6 554
* SE) of sarcomere length, fiber size and distribution, and capillary density in
Muscle No. Site
Aerobic Glycolytic pm2 ~ ~ (% 0 )g(fg)z.12 ~
1', Pm
~(fa)z.,zkm2
1 2 3 4
sup deep sup deep
1.80 t 0.03 1.83 2 0.10 1.79 t 0.04 1.79 i 0.02
379 i 31 719 t 59 362 2 17 581 t 71
84 68 82 74
5 6
sup deep
1.93 i 0.04 1.87 t 0.12
7 8 9 10 11 12
sup deep sup deep sup deep
2.12 i 0.01 2.20 t 0.01 1.96 i 0.01 1.97 i 0.01 1.69 t 0.03 1.79 t 0.02
'Sarcomere length. 2Cross-sectional area at I , = 2.1 pm. 3Relative cross-sectional area of aerobic fibersitotal fiber area. 4Capillary number per mm2 of muscle fiber cross-sectional area at 1,
MATERIALS AND METHODS
Twelve perfusion-fixed muscles from a total of six pigeons (Columba liuia; body mass Mb, 490-622 gm; Table 1) were used in the study. Three breeds were used in a n attempt to obtain samples with larger differences in capillary density. Although it was not the main purpose of the study, we were interested in possible genetic difference(s1 in flight muscle capillary density and geometry. Show Racers (SR) and White Carneau (WC), both 9 months old, were derived from animals obtained from the Palmetto pigeon plant. They were kept in a barn with flying coops (approx. 10 x 20 x 8 feet). White Kings (WK; 6 months old) were obtained from a local squab ranch where they had been kept in a small aviary (approx. 7 x 6 x 10 feet). All animals were anesthetized by intravenous injection of sodium pentobarbital (3 mg/100 gm). The vascular perfusion of the muscles in situ was performed as described elsewhere in detail (Mathieu-Costello, 1987). Briefly, the entire vasculature was perfused with saline (11.06 gm NaCl/liter; 350 mOsm; 20,000 USP units heparidliter) via a cannula inserted directly into the left ventricle, while the right atrium was cut open to secure outflow. Perfusion fixation followed with a 6.25% solution of glutaraldehyde in 0.1M sodium cacodylate buffer (total osmolarity of the fixative: 1,100 mOsm; pH 7.4) as described previously (Mathieu-Costello, 1987). All perfusions were performed at a nonpulsatile pressure of 150-170 mm Hg. Muscles samples (approx. 1 cm x 4 mm x 1 mm) were taken in the superficial (ventral) and deep portions (dorsal) of the pectoralis muscle a t one sampling site, approximately halfway along the cranial-caudal and the lateral axis of either right or left pectoralis major muscle in each bird. In that site, the thickness of the pectoralis muscle ranged from 5-15 mm (WK) to 20 mm (SR). All samples were cut into thin longitudinal strips, stored in glutaraldehyde fixative, and processed for electron mi-
=
t3 t2 t5
2
1,785 t 96 1,853i 125 2,051 t 225 2,263 i 248
3,194 i 141 2,615 t 189 4,108 2 317 2,491 t 99
428 i 27 782 5 94
62 i 4 48 2 4
2,444 t 298 2,186 2 244
3,229 i 221 1,979 5 164
382 i 27 367 2 22 334 t 22 304 i 19 348 t 27 441 2 38
73 i 4 76 t 3 42 i 2 74 i 3 61 t 5 54 i 4
2,257 t 277 2,078 t 79 1,909 i 219 2,256 t 177 2,284 ? 129 2.045 i 146
4,143 i 187 3,974 t 133 3,490 t 150 6,055 i 283 5,163 t 402 1.749 t 102
2
2.1 pm
croscopy as previously described (Mathieu-Costello, 1987). Tissue Sectioning
One-micrometer sections were cut on a LKB Ultrotome I11 and stained with 0.1% aqueous toluidine blue solution. From each muscle sample, 4-8 blocks were cut into 4 transverse sections (angle between normal to section and fiber axis, a = O O ) , and 4 longitudinal sections ( a= d 2 ) , following a procedure described elsewhere in detail (Mathieu-Costello, 1987). In one sample, we also evaluated capillary orientation distribution as described previously (Mathieu-Costello, 1987) by using a series of sections taken at 0" to 90" to the fiber axis by steps of 5 to 10"from 4 blocks. Sarcomere length (1,) was measured in each longitudinal section (average of 10 measurements of groups of consecutive sarcomeres, systematically sampled over the entire area of each section examined a t magnification of x 630; Mathieu-Costello, 1987). Morphometry
The 1-pm sections were subsampled by a s many systematic, non-overlapping quadrats (i.e., micrographs) a s technically possible from the section area. The number of micrographs per section averaged 6 to 14, yielding about 240-560 fiber profiles in transverse sections for each sample. An average of 8 to 16 micrographs were obtained from each longitudinal section, yielding about 60-110 portions of fiber profiles for each sample. The micrographs were taken on 35-mm direct positive films (Kodak Direct MP 5360) at magnification x 400 by using a Leitz Ortholux light microscope equipped with a Leica camera. A micrograph of a stage micrometer was recorded for calibration. The 35-mm films were projected on a square grid test A 144 (see Weibel, 1979, Appendix 31, a t a final magnification of x 2,060 by using a microfilm reader (Documator DL 2, Jenoptic,
76
0 . MATHIEU-COSTELLO
Jena). The point counts of numerical and volume densities of capillary profiles were collected, stored, and processed by using a n Apple computer. As in previous studies (see Mathieu-Costello, 1987), capillary density estimates were related to the muscle fibers as a reference space in all samples in order to avoid variations due to the unreliable preservation of the intercellular spaces by the preparation procedures in different samples. As discussed previously, it is important to assess the effect of tissue preparation on morphometric estimates of fiber size and sarcomere length (Mathieu-Costello, 1987). In tissues prepared with the same fixatives and procedures as in this study, we found fiber sizes similar to those reported by others in comparable material processed for histochemistry (Zumstein et al., 1983; Mathieu-Costello, 1987). Because histochemical preparation yields a minimum amount of tissue shrinkage, this indicated no major bias in our estimates of fiber size in our glutaraldehyde-fixed material. In the same study, measurements of filament length revealed less than 10% shrinkage compared to filament length in living muscle and compared with data obtained by others (Mathieu-Costello, 1987). The method used to estimate capillary anisotropy in each sample has been described elsewhere in detail (Mathieu et al., 1983).We first estimated the ratio between capillary counts per sectional area of transverse and longitudinal sections of muscle fibers, QA(0) and QA(d2),respectively. This ratio was then used to calculate, via a table or graph of known coefficients, l)the capillary orientation concentration parameter, I 4,000 mm-' (range, 4,342-6,021 mm-2; muscles 3,7,8,10, and 11; Table 3), com ared to samples with Jv(c,f) values < 4,000 mm-'(range 1,834-3,485 mm-'; other muscles, Table 3). Figure 9 shows the plot of the numerical density of capillaries per fiber cross-sectional area, Q A ( a ) , as a function of the sectioning angle, a,on the basis of the Fisher axial distribution model of capillary segments. In the sample considered (muscle lo), capillary number per fiber sectional area was 5,680 2 264 mm-' in transverse sections and 1,090 2 93 mm-2 in longitudinal sections (Table 2). Experimental data were adequately fitted by the Fisher axial distribution model (Fig. 9). Note that the solid line in Figure 9 is not the regression line but the computed value of Q A ( a ) under the Fisher axial distribution model, based on the value of Jv(c,f) in t h a t muscle (see Methods). The estimates of capillary volume density, Vv(c,f), mean capillary diameter, d(c), and capillary length density, Jv(c,f), calculated via eq. 2, are given in Table 3. The two estimates of Jv(c,f) obtained in each sample by using two different methods (eqs. 1 and 2) were different from each other in only one sample (muscle 4). The slope of the linear regression between the two estimates of Jv(c,f) in each sample (r= 0.93) was not significantly different from 1 (P = 0.42).
DISCUSSION To our knowledge, this is the first study where sarSarcomere length, pn comere length, i.e., the degree of shortening or extension of the muscle fibers, has been considered when Fig. 5. Plot of cross-sectional areas of glycolytic and aerobic fibers estimating fiber size and capillary density in pigeon M. against sarcomere length in both superficial and deep portions of White King (circles),in superficial (filled triangles) and deep portions pectoralis. Such consideration is important because of (open triangles) of Show Racer, and in superficial (filled diamonds) the direct effect of sarcomere length on fiber size (and and deep portions (open diamonds) of White Carneau pigeon pectora- therefore capillary densitytfiber cross-sectional area) lis muscle. Dotted lines indicate the predicted change in fiber cross- and capillary geometry (reviewed by Mathieu-Costello sectional area with sarcomere length assuming a constant fiber volet al., 198913;Mathieu-Costello, 1990). An additional difume, i.e., calculated as a(f) = constant / I,,; constant = product of 1, and a(f) means in the sample group. For glycolytic fibers, all points ficulty presents itself when studying capillary geomewere considered; for aerobic fibers, WK only. Error bars indicate i 1 try in pigeon pectoralis muscle. As described above, it SE (within samples); where a n error bar is not shown, the SE is within is critical to consider the relative area of aerobic and the symbol size. glycolytic fibers in each section, since the estimate of capillary orientation depends on the ratio between capwhere illary countstfiber sectional area in transverse and longitudinal section. The relative area of aerobic and glyUo(K) = (2KF/{r!(2r+l)}. (7) colytic fibers was estimated in each set of sections, r=O after unambiguous identification of aerobic and glycoThe empirical fit to the data for K vs. I, (Fig. 7) was lytic fibers in all tissues. The identification of red and obtained by using non-linear least squares regression white fibers after toluidine-blue staining has been done analysis with the following equation: previously in Canada geese pectoralis muscle (George K = K,, - KOexp {-a(lo - 1.4)} [81 et al., 1983). The estimates of the fitting parameters were K,, = Capillary Density, Fiber Size, and Distribution 1.35, KO = -0.56, and a = -2.26 km. The predicted As expected, capillary density depended on the relavalues for c(K,O) a t sarcomere lengths of 1.6, 2.1, and 2.3 are 1.20, 1.08, and 1.05, respectively (Fig. 8, solid tive area of aerobic vs. glycolytic fibers in the samples. line). For comparison, the curvilinear relationship be- The linear regressions (Fig. 6) yielded values for tween c(K,O) and 1, in various muscles of mammals is Q A ( O k . 1 of 6,184 2 513 mm-2 (WK) and 4,769 2 253 also shown in Figure 8. At a given sarcomere length, mm- (SR) at 100% aerobic fibers. Interestingly, simthe value of c(K,O) was smaller in pigeon M. pectoralis ilar capillary densities (QA(0)z.I range, 4,342-7,033) than in mammalian muscles (Fig. 81, indicating a were found in perfusion-fixed rat myocardium similarger degree of capillary anisotropy in the bird mus- larly prepared (Poole and Mathieu-Costello, 1990). In cle. A larger capillary anisotropy is reflected by a the heart, however, both fiber size [a(f),,, range, 2081.6
1.8
r
2.0
2.2
81
CAPILLARY GEOMETRY IN PIGEON PECTORALIS
TABLE 2. Morphometric estimates of capillary anisotropy and capillary length density, calculated via eq. 1, in each sample (values are means f S.E.)
2,738 t 112 2,279 t 108 3,502 2 259 2,123 t 81 2,968 t 194 1,762 2 93 4,182 2 188 4,163 t 138 3,257 t 139 5,680 t 264 4,155 2 315 1,491 2 85
1 2 3 4 5 6 7 8 9 10 11 12
80 2 3 69 t 4 83 t 4 73 2 4 63 t 4 44 t 6 76 t 3 81 2 3 45 2 3 74 t 3 59 2 4 52 2 4
938 2 54 849 t 75 1,378 t 110 712 t 67 833 t 62 711 t 99 1,045 t 64 832 t 64 699 t 54 1,090 t 93 950 2 95 568 ? 54
2.3 & 0.2 2.1 t 0.3 1.9 t 0.3 2.4 t 0.3 3.0 t 0.4 1.9 t 0.4 3.5 t 0.4 4.8 2 0.6 4.3 t 0.6 5.2 2 0.8 3.9 t 0.9 2.0 & 0.3
1.19 t 0.03 1.22 2 0.04 1.24 t 0.05 1.18 t 0.03 1.13 t 0.03 1.25 2 0.06 1.10 t 0.02 1.06 t 0.01 1.07 t 0.01 1.06 t 0.01 1.08 2 0.03 1.23 t 0.05
3,258 t 157 2,780 2 160 4,342 t 366 2,505 t 115 3,354 ? 236 2,203 t 157 4,600 t 223 4,413 t 152 3,485 2 152 6,021 t 285 4,487 2 362 1,834 ? 128
'Number of capillaries per mm2 fiber sectional area in transverse sections. 'Number of capillaries per mm2 fiber sectional area in longitudinal sections. 3Relative sectional area of aerobic fibersitotal fiber area in longitudinal sections 4Degree of capillary anisotropy. 5Coeffcient relating Q,(O) to Jv(c,f) (eq. 1). 'Capillary length per fiber volume.
7000 6000
?
t
0
hl
I
5000
E
4000
E
-
0
5.0 Y
7
A
