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1Otolaryngology and Communication Sciences, and 2Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York
Submitted 8 January 2007 ; accepted in final form 30 July 2007
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ABSTRACT |
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blood supply; larynx; microvascular; blood-tissue exchange
The capillary supply to a muscle fiber is closely correlated to the size and metabolic profile of the fiber, as well as to the metabolic profiles of the surrounding muscle fibers. In fact, increased capillarity precedes exercise-induced fiber-type change (64), suggesting that activity-induced muscle fiber change may be hindered if metabolic support is not present. Capillarity data from studies of aging animals and humans have been variable. Some have reported a decrease (55), while others an increase (45), and still others have reported no change (41) in capillarity. These disparate findings could be due to a number of factors, such as differences in the muscles examined, their fiber-type composition, and how this composition changes during aging, as well as to differences in subject activity levels, sex, and/or differences in sample design.
In most studies of human muscle capillarity, samples were obtained using biopsies and cross sections of the fibers. Under the best of circumstances, a muscle biopsy will sample only a few hundred muscle fibers out of thousands. From these sections, capillary-to-fiber ratio has been commonly estimated based on two-dimensional counts of capillary profiles surrounding muscle fibers in transverse sections. While this type of estimate has produced useful data, it is model-based and assumes that all muscle fibers and capillaries are oriented perpendicular to the plane of section. Although these assumptions may be reasonable for many limb muscles, they are not realistic for the laryngeal muscles, given their complex fiber geometry. The present study has instead used design-based stereological techniques to provide unbiased, three-dimensional quantitative estimates to determine whether there are age-related changes in muscle fiber-type-specific and/or total muscle changes in the capillary length per unit volume (LV cap). Capillary length takes into account length added due to capillary tortuosity and branching, which are not addressed by capillary counts obtained from cross sections. Also, capillary length is an important determinant of both capillary surface area and red cell path length (39).
The O2 flux in muscle is limited by a sharp diffusion gradient localized between the red cell and the sarcolemma (63). Thus it is widely accepted that the maximal rates of O2 diffusion are largely a function of the amount of contact between capillaries and muscle fibers (capillary exchange length), and theoretical modeling supports this view (11, 43). Therefore, the consequences of an age-related decrease in capillarity would be a reduction in the surface area available for O2 diffusion and a diminution in the average red blood cell transit time for O2 transfer. The end result would be a decrease in muscle function.
The laryngeal muscles have evolved to have the specialized functions of airway protection, phonation, and respiration. These functional demands are complex, and laryngeal muscles are adapted to fast and/or variable contraction and fatigue resistance, which is reflected in the fiber-type composition of these muscles. These muscles contain not only type I, II, and IIX myosin heavy chain (MHC) isoforms, but also 
-cardiac MHC. There have also been reports of superfast fibers in laryngeal muscles from rat (57) and rabbit (34), which are similar to those found in extraocular muscles. While in general, the contraction speed of human laryngeal muscle fibers is considerably higher than that found in human limb muscles (56), evidence that superfast fibers are present is not conclusive (58).
The posterior cricoarytenoid muscle (PCA) is the only true laryngeal abductor, and, as such, its role is crucial during inspiration. However, it also participates in the regulation of airflow during expiration by changing airway resistance and is recruited during phonation to stabilize the position of the vocal process. The PCA differs from the other laryngeal muscles, not only in function, but also in the types of MHC isoforms expressed: developmental, -cardiac MHC (3) type I and II, but not IIX MHC (33). It contains a higher proportion of type I fibers (37, 62), and muscle fibers of both types contain a high mitochondrial volume fraction (38), with higher oxidative enzyme profiles (4). These high metabolic demands are supported by blood flow rates that are greater than those of other laryngeal or respiratory muscles during both spontaneous breathing and respiratory recruitment (2). Given the PCA's high-oxidative metabolism, any decrease in blood flow or capillarity could have pronounced effects on its function.
A number of studies have suggested that the PCA may undergo early age-related changes (37, 50, 62). In fact, in one study, the PCA has been reported to display some degree of muscle fiber atrophy as early as age 13 (12). Recently, studies in the rat report that there is an age-related decrease in the ability to abduct the vocal folds during quiet respiration (61), as well as a 42% age-related blood flow decrease to the PCA (35). These later data would suggest that insufficient blood flow may be an underlying cause for age-related dysfunction of the PCA, something that was proposed as early as 1941 by Bach et al. (1).
Since data for age-related changes in head and neck muscles vary greatly between muscles and frequently differ from that typical of limb muscles (36, 46), it is reasonable to assume that the PCA will also differ. Given this assumption, and in conjunction with the animal data presented above, it was hypothesized that there would be age-related changes in muscle fiber-type-specific and/or total capillary exchange lengths within the human PCA.
However, data show no age-related change in LV cap, and, therefore, our original hypothesis is not supported. Instead, data indicate that there are age-related changes in capillary-muscle fiber relationships and capillarity differences between type I and type II muscle fibers within the human PCA.
Stereological Glossary
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METHODS |
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The orientator technique (42) was used to sample the muscles. The first step in this technique is to choose an arbitrary but identifiable vertical direction. The muscle is then oriented with the vertical perpendicular to the table and placed on a clock uniformly divided from 0 to 9, both above and below the horizontal (0–0 direction). The sample is then cut along the vertical axis at a randomly chosen angle, and one of the resulting blocks is randomly chosen and reoriented so that its vertical is running parallel to the table surface. The chosen block is next placed on a cosine-weighted clock that has been numbered 0–9, both above and below the 0–0 horizontal, such that the freshly cut surface is down and the original vertical axis is running in the direction of the 0–0 line of the clock. The block is then cut at a second randomly chosen angle. One of these pieces is placed into a mold containing mounting media with its freshly cut surface either up or down. The other piece is reoriented, allowing free random rotation around the vertical axis, so that the vertical is once again perpendicular to the table and is placed back onto the first clock. This piece is then cut in the 5–5 direction, and one of the remaining pieces is cut in the 0–0 direction. A piece from each of these cuts is placed into the mold with its freshly cut surface either up or down. This procedure results in a block containing three pieces of the muscle having randomly selected, yet perpendicular planar relationships to the vertical. This technique is design based, efficient, and samples the entire muscle volume at all possible orientations without bias. These blocks were frozen in N-methyl butane cooled to near its freezing point with liquid nitrogen. Cryostat sections were cut at 20 µm, collected onto glass slides, and stored at –80°C until processed. Sections were immunolabeled to distinguish type I from type II muscle fibers, as well as for the identification of capillaries. Antigen retrieval was performed using proteinase K, 20 µg/ml (Sigma Chemical, St. Louis, MO). Type II fibers were identified by incubation with a primary antibody, mouse monoclonal (MY-32, Sigma Chemical) to fast MHC, followed by a secondary antibody, horse anti-mouse IgG conjugated with FITC (Vector Laboratories, Burlingame, CA). For identification of the capillaries, the sections were incubated with polyclonal anti-human von Willebrand factor (Dako, Carpinteria, CA), a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories), and then labeled with extravidin conjugated to indocarbocyanine (Cy3; Sigma Chemical). This was followed by mouse anti-human CD-31 (a second endothelial cell marker; Dako), biotinylated goat anti-mouse IgG (Vector Laboratories), and then extravidin Cy3 (Sigma Chemicals). Cell surfaces were imaged using concanavalin A-FITC (Molecular Probes). Appropriate washes and blocking steps were used before, as well as between, all incubation steps.
Stereological data were collected in real time on a Noran Odyssey confocal laser scanning microscope (CLSM, Middleton, WI), which provides high z-axis (<1 µm) resolution, using a x40 oil objective. This system incorporates a multiline laser (457, 488, and 514 nm) and is equipped with computer interfaced x, y, and z stepping motors and custom software that produces an isotropic uniformly random field sampling protocol. The sample fields were distributed in a regular lattice pattern, randomly positioned with respect to the tissue. An unbiased counting frame (13) was used to obtain counts of the variables. Data were collected from 8,395 sample fields, with a mean of 165 from each muscle. From these sample fields, a mean of 543 intersections with type I fibers, 320 intersections with type II fibers, 192 capillaries contacting type I fibers, 90 capillaries contacting type II fibers, 1,375 type I fiber reference points, and 751 type II fiber reference points were counted from each muscle. The stereological abbreviations used in the present study are according to the commonly used nomenclature described by Weibel (65).
Estimates of the type I and type II muscle fiber volume fractions (
V Y, ref) were made using point-counting techniques (65) and the following relationship:
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Surface densities of type I and type II fibers (
V Y, ref) were estimated using counts of line intersections (65) with the following equation:
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Estimates of capillary length densities (
V Y, ref) were made using counts of capillary profiles and the following relationship:
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Using data collected in this manner, it was possible to calculate the length density of capillaries contacting fibers but independent of fiber type (LV cap f, fibers), the fiber-type-specific length densities (LV cap ft, ft), the capillary length density within the interstitial tissue (LV cap it, mus), the fiber-type-specific surface density (SV ft, mus), the total muscle capillary length density (LV cap m, mus), the ratio of the fiber-type-specific capillary length to the corresponding fiber-type-specific surface (LS cap ft, ft), and the ratio of the length of capillaries contacting fibers but independent of type to the surface of muscle fibers (LS cap f, fibers).
Data for the stereological variables were analyzed using SPSS (version 11.5, SPSS, Chicago, IL) general linear model, with sex as a categorical independent variable and age as a continuous independent variable. For comparisons of the variables between type I and type II fibers, the first independent variable was "age group". The "young group" was defined as ages 18–59 yr (15 men, 7 women), and the "old group" as ages 60–98 yr (13 men, 16 women). The second independent variable was sex. These cut points were chosen based on studies demonstrating that muscle strength is relatively constant until the sixth decade, at which time a steep decline in maximal strength begins (16, 31). A probability of P < 0.05 was set for rejection of the null hypothesis.
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RESULTS |
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DISCUSSION |
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Methodological considerations. As previously stated, studies of other muscles have reported an increase, decrease, or no change in vascular density. These disparate results could be due to differences in the muscles examined, the muscle activity levels, the species, and/or differences in sampling. For example, Mitchell et al. (45) reported that, in aging rats, the capillary-to-fiber ratios were different, depending on whether the deep region or superficial portion of the extensor digitorum longus muscle was sampled. Most studies of human muscle have utilized needle biopsies for sampling. While this type of sampling has provided useful data, and in some cases is the only means to obtain samples for biochemical analysis, it is well established that this type of sampling can contain error. This was demonstrated in early studies by Lexell et al. (32). More recently, Hepple and Mathieu-Costello (18) reported that estimates of the capillary-to-fiber interface obtained from needle biopsy samples were significantly smaller compared with those obtained from perfusion-fixed material, even after accounting for differences in sarcomere length. These dissimilarities were similar to those reported by Ogilvie et al. (49) and were ascribed to muscle-fiber swelling in unfixed frozen sections and to fiber shrinkage in perfusion-fixed material. In addition, many of the variables measured are, by necessity, model based. Routinely, these types of studies take data from a selected plane of section, one that is perpendicular to the long axis of the muscle fibers (cross sections). From this biased sampling, variables such as capillary-to-fiber ratio, capillary-to-fiber perimeter exchange index, and share index have been determined. However, these methods do not take into account fibers cut in tangential or longitudinal profile or capillary geometry. Because of this, these models become invalid whenever the basic assumptions are violated. One extremely useful model-based stereological methodology that has taken into account these factors is that of estimating capillary-to-fiber perimeter ratio (40), which is a measure of capillary surface per fiber surface area and could be considered comparable to our capillary length-to-muscle fiber surface (LS cap) calculations. As previously stated, the assumptions made when using these model-based techniques may be reasonable for limb muscles, but they are not realistic for laryngeal muscles, given their complex fiber geometry. It is not uncommon to find that fibers contained in one muscle fascicle are in cross section, while those in an adjacent fascicle are clearly tangential, a situation that the above-mentioned model-based methods cannot adequately address.
The present study has utilized design-based stereological techniques to provide an unbiased quantitative three-dimensional analysis of the capillary length density contacting specific muscle fiber types, as well as the vasculature of the entire PCA. This variable was chosen, as volume length is an important indicator of capillary surface area and red cell path length (39). In addition, it takes into account capillary branching and tortuosity, parameters that capillary counts obtained from cross sections cannot adequately address. This design-based methodology is particularly useful in the larynx, since, to date, laryngeal scaling methods are not useful, including the use of body size. While in general male larynges tend to be larger than female larynges, the variance is so great that significant differences are difficult to determine (36).
Muscle capillarization and aging. Results from the present study indicate that there is no age-related change in the PCA capillary length density as a whole (LV cap m, mus) or with that contacting specific fiber types (LV cap ft, ft). This is similar to results that have been reported for other muscles. Recently, Hepple and Vogell (20) reported no change in the capillarity around fibers in the soleus or gastrocnemius in 28- to 30-mo-old rats. Similarly, Mathieu-Costello et al. (41) found no capillary loss in the aged rat soleus or the extensor digitorum longus, even though they did report muscle fiber atrophy. However, these results do differ from those of Croley et al. (7), who found that, in the vastus lateralis muscle of older women, capillary contacts were lower, specifically for type II fibers. The data of Ryan et al. (55) also show an age-related decreased capillarity in the same muscle from males, regardless of fiber type. Together, these data suggest that age-related change or lack of change may be muscle specific.
The lack of capillarity change in the PCA could be related to its activity level. Compared with that of the typical skeletal muscle, the activity pattern of the PCA is much higher, since portions of it are recruited even during quiet respiration (21) and to a lesser extent during expiration. It is also active during swallowing, coughing, and during different types of phonation. In essence, some portion of the PCA is nearly always active. This activity level was postulated to be the reason that there is a lack of age-related change in neuromuscular junction lengths or in the terminal axonal branching patterns at these junctions within the human PCA (10).
We did find an age-related increase for LV cap it, mus. Since this variable represents both extra- and intrafascicular tissue, it is an indication that there has likely been muscle fiber loss and/or atrophy without a related loss of capillaries. A similar conclusion can also be drawn from the data, indicating an increase in LS cap ft, ft. This could occur if the vasculature remains relatively stable and there is fiber atrophy or loss, which may be occurring, since there is an age-related decrease in SV ft, mus for both fiber types. It could also occur if the fiber sizes were stable and there was an increase in associated capillary length density. However, data from the present study do not support this conclusion, since there was no change in LV cap. In addition, data from another laryngeal muscle, the human thyroarytenoid, show that there is an age-related loss of type I fibers, as well as an increase in diameter of the remaining type I and type II fibers. These data, in conjunction with those from the present study, suggest that there is a combination of fiber loss/atrophy, as well as hypertrophy of the remaining fibers, with a subsequent reorganization of the vasculature. This conclusion is supported by data that there is, in fact, an age-related increase in diameter for both type I and type II muscle fibers in the human PCA (unpublished observations, L. T. Malmgren, C. E. Jones, and L. M. Steer). The hypertrophy likely reflects an increase in metabolic activity of these fibers and suggests that these fibers are recruited more heavily than in younger cases. The vascular remodeling indicated by our data would be important for support of these highly metabolic fibers. In addition, as fiber size increases, the surface-to-volume ratio would decrease. If the mitochondrial volume fraction and the rate of mitochondrial O2 utilization remain constant, a higher O2 flux per unit surface area would be required to maintain maximal O2 utilization, and changes in LS cap would reflect this increased demand. However, it has been demonstrated in rats that declining aerobic performance of aged skeletal muscle is due in part to mitochondrial dysfunction (15).
Muscle oxygen extraction depends on the interaction between the capacity for oxygen flux, which is determined by the relationship of LS cap, and the functional properties of blood oxygen transport capacity of the red cells and capillary bed transit times. It has been shown that maximal O2 flux is probably limited more by the capillary/fiber relationship than by intrafiber diffusion distances (for a review, see Ref. 51). Even though there is a significant age-related increase in the muscle fiber diameter and thus intrafiber diffusion distances, there are compensating vascular changes. Therefore, there is likely little compromise in the metabolic function of these fibers due to lack of anatomic capillarity. This is not to say that O2 delivery is not impaired in aging. Using intravital microscopy, Russell et al. (54) examined the spinotrapezius muscle from rats at rest. They reported that, while there appeared to be no anatomic differences in the percentage of perfused capillaries or in the degree of tortuosity and branching, red cell transit time is significantly reduced. This would most likely result in an impaired diffusive transport of O2. While these data suggest that there is an age-related decrease in functional capillarity, there is also data showing that there is a decrease in O2 extraction at the muscle level. Data from a recent longitudinal study of humans showed that, during maximal exercise, there is a decreased arterial-venous O2 difference, even though cardiac output was similar, indicating that O2 extraction is reduced with aging (44). Support for this conclusion comes from a recent study by Hepple et al. (17), which demonstrated that, in rats with similar levels of convective O2 delivery (arterial O2 x blood flow), the O2 extraction was reduced with aging. This decrease in O2 extraction is likely a result of a reduced oxidative capacity, due at least in part to mitochondrial disfunction (15, 17). It is possible that the increase in LS cap found in the present study is compensating for the age-related decrease in O2 extraction and would have the effect of keeping the diffusion gradient elevated.
Sex differences. Our data indicate that the female type II fibers have a significantly higher LV cap than the male and that this does not change with age. Croley et al. (7) also reported higher capillarity for type II fibers in the vastus lateralis of women, but there was an age-related capillary decrease, which accounted for the majority of the age-related capillary loss reported for this muscle in women. Recently in men, this same muscle was shown to have a similar age-related decrease in capillarity, but type I and II fibers were equally affected (55). This differs from our findings of no change in volume length for either sex and those of Coggan et al. (5), who reported that, in the gastrocnemius muscle of males, there is a decreased capillarity surrounding type II fibers but not type I. Animal studies have also reported sex differences. For example, in the rat plantar flexors, blood flow and fatigue resistance remain unaffected by age in females but are decreased in males (27, 28). Several studies have shown that, at submaximal contraction, females are more resistant to muscle fatigue than males (9, 25). This fatigue may be related to differences in the metabolic capacity of the muscles. It could also be that females recruit type II fibers more frequently than males, necessitating a higher O2 supply and thus higher capillarity. It should be noted that some of this difference in fatigability appears to be negated due to aging (23) or when the subjects are matched for strength or the type of task that is used (24). Recently, however, it has been demonstrated that, even in strength-matched subjects, there is a sex difference in active hyperemia and vascular conductance, in that women have less when the isometric contractions are maintained for the same time (26). The authors concluded that this was the result of sex differences in physiology, which are independent of strength. Support for these conclusions comes from studies of whole body exercise and of isolated whole muscle metabolism (30) that show men may rely more on glycolytic pathways, while women have a greater capacity for utilizing oxidative metabolism. If this is the case, then a major function of the vasculature would be the removal of waste rather than that of supplying O2. A final possibility is that the difference in capillary length density may be related to a smaller type II fiber in women with a similar capillary density (number/mm2). This conclusion is supported by the data of Malmgren et al. (36), who reported that type II fibers in the human thyroarytenoid muscle have a smaller diameter in women, from unpublished data demonstrating that type II fibers in the human PCA have a smaller diameter in women (L. T. Malmgren, C. E. Jones, and L. M. Steer, unpublished observations) and from studies of the human tibialis anterior (53).
Type I vs. type II. Data for the present study indicate that capillarization is higher for type I than type II fibers and that these differences are not dependent on age or sex. This is not surprising, since the oxidative capacity of type I fibers is usually higher than that of type II fibers, which is the case for the human PCA. However, Malmgren and coworkers (37, 38), using a combination of histochemical techniques and electron microscopy, demonstrated very high mitochondrial volume fractions in both fiber types in the human PCA. Recent studies by Poole and Mathieu-Costello (52) have shown that there is a relationship between fiber capillarization and mitochondrial volume density, such that capillary to-muscle-fiber surface appears to be regulated in direct proportion to muscle-fiber mitochondrial volume density. However, it is possible that this relationship is muscle specific, since Hudlicka et al. (22) reported that the normal gracilis muscle in cats did not show any relationship between the mitochondrial volume density and the size of the capillary bed. In addition, this difference between fiber types may not always be present, and the degree of variation may differ from muscle to muscle and even within the same muscle. Stal et al. (60) reported that, in the masseter muscle, there is a 2:1 ratio of capillaries to type I-type II for the anterior portion and a 1.6:1 for the posterior portion. While, as previously stated, it is difficult to do direct comparisons due to methodological differences as well as sampling design (i.e., postmortem vs. needle biopsy, etc.), when multiple muscles have been included in a study, this difference between fiber types appears to be consistent. Sjogaard et al. (59) examined the capillarity of the triceps brachii, vastus lateralis, and soleus and reported that each of these muscles demonstrated a similar difference between type I and type II fibers. This study also reflected the differences in capillarity to support the varying oxidative capacity of the same muscle fiber type within various muscles. That is, type I fibers in the triceps brachii muscle of men have, on average, 4.85 capillaries around each fiber, while in the more oxidative soleus muscle this number is 6.89. Similar results have been reported in animal studies that have compared the soleus muscle to the gastrocnemius (14).
The findings from this study indicate that, while there is no age-related alteration in the overall capillary length density within the human PCA, there are changes indicating that vascular remodeling does occur. This remodeling is likely a response to age-related muscle fiber changes, since data indicate that age-related fiber atrophy as well as possible hypertrophy are taking place in a manner similar to that which has been reported to occur in the rat soleus muscle (19) and the human thyroarytenoid (36). While it does not appear that an alteration in capillarity per se has a role in age-related changes in this muscle, it is unknown whether the vasculature is functionally normal. It is generally accepted that there are a number of age-related changes occurring in the vasculature that lead to decreased blood flow. For example, endothelium-dependent myogenic responses to changes in intraluminal pressure (47) or to flow that is independent of pressure (shear stress; Ref. 48) are significantly diminished with age. Vasodilation induced by acetylcholine also differs in arterioles from different vascular beds (48). Due to these as well as other disparate findings, it will be necessary to perform similar studies on the vasculature of this important laryngeal/respiratory muscle to determine to what extent aging has altered these physiological characteristics.
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FOOTNOTES |
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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.
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