Histochemical method for simultaneous fiber typing and demonstration of capillaries in skeletal muscle

Summary Since capillary density of skeletal muscle depends on fiber type distribution, fiber typing should accompany measurement of capillary density. To obviate the need for multiple sectioning, we suggest NADH tetrazolium reductase enzyme histochemistry for fiber typing followed (on the same slide) by lectin histochemistry to demonstrate the binding of Ulex europaeus agglutinin, a sensitive marker for endothelium. The method is quick, highly reproducible and gives density estimates comparable to earlier, more tedious methods.     

Effects of Long Term Supplementation of Anabolic Androgen Steroids on Human Skeletal Muscle

The effects of long-term (over several years) anabolic androgen steroids (AAS) administration on human skeletal muscle are still unclear. In this study, seventeen strength training athletes were recruited and individually interviewed regarding self-administration of banned substances. Ten subjects admitted having taken AAS or AAS derivatives for the past 5 to 15 years (Doped) and the dosage and type of banned substances were recorded. The remaining seven subjects testified to having never used any banned substances (Clean). For all subjects, maximal muscle strength and body composition were tested, and biopsies from the vastus lateralis muscle were obtained. Using histochemistry and immunohistochemistry (IHC), muscle biopsies were evaluated for morphology including fiber type composition, fiber size, capillary variables and myonuclei. Compared with the Clean athletes, the Doped athletes had significantly higher lean leg mass, capillary per fibre and myonuclei per fiber. In contrast, the Doped athletes had significantly lower absolute value in maximal squat force and relative values in maximal squat force (relative to lean body mass, to lean leg mass and to muscle fiber area). Using multivariate statistics, an orthogonal projection of latent structure discriminant analysis (OPLS-DA) model was established, in which the maximal squat force relative to muscle mass and the maximal squat force relative to fiber area, together with capillary density and nuclei density were the most important variables for separating Doped from the Clean athletes (regression  =  0.93 and prediction  =  0.92, p<0.0001). In Doped athletes, AAS dose-dependent increases were observed in lean body mass, muscle fiber area, capillary density and myonuclei density. In conclusion, long term AAS supplementation led to increases in lean leg mass, muscle fiber size and a parallel improvement in muscle strength, and all were dose-dependent. Administration of AAS may induce sustained morphological changes in human skeletal muscle, leading to physical performance enhancement.     

Myosin types in human skeletal muscle fibers

By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Myosin types in human skeletal muscle fibers

Summary By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Mathematical modeling of capillary density

Capillary density is important as a determinant for total oxygen transport to tissue. Because both capillary morphology and fiber composition vary considerably from muscle to muscle, measurement of capillary morphology and fiber composition vary considerably from muscle to muscle, measurement of capillary density alone cannot provide the detailed information necessary for analyzing physical phenomena. In this report we consider the capillary:fiber ratio, fiber types, fiber diameters, and fiber composition as components of a unit to express capillary density. We have applied the hexagonal fiber array model to calculate capillary density in cat and dog striated muscle and compared this with experimental data in the literature. The results indicate that this model may be useful for predicting capillary densities from simple biopsy procedures.     

Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies

Muscle fiber typing is conventionally performed using mATPase enzyme histochemistry on cryostat sections. After pre-incubation of sections at pH 4.3, 4.6 and 10.3, based on the pattern of enzyme reactivity, the fibers can be classified in types I, II (subtypes A, AB and B) and the intermediate C (I and II) fibers. We have attempted to perform fiber typing of human psoas muscle by immunohistochemistry, using monoclonal antibodies R11D10 (specific for cardiac and type I skeletal myosin) and MY-32 (specific for fast muscle fibers) on cryostat as well as on paraffin sections. Staining of consecutive cryostat sections showed that type I fibers are R11D10 reactive whereas type II fibers are MY-32 reactive. Subtyping of type II fibers could not be performed by immunohistochemistry. Quantitative analysis of type I and II fibers showed that enzyme histochemical and immunohistochemical analysis are in close agreement.     

Quantitative study of the microvascular pattern in tourniquet-induced muscle ischemia

Experiments were carried out on 40 adult Wistar rats to determine the effect of the application of a pneumatic tourniquet on the capillary network of the tibialis anterior muscles. The tourniquet was applied for four hours to the right hind limb above the knee. During the degeneration processes (6 to 24 hours postischemia), the capillary density and capillary-to-fiber ratio decreased significantly with respect to a control group of untreated animals. In the subsequent regenerative processes, there was a greater increase of fiber density than of capillary density, resulting in a capillary-to-fiber ratio lower than that of the control group. In the last period (30 to 90 days postischemia), both capillary density and fiber density returned to normal values.     

Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies

A novel type of myosin heavy chain (MHC), called 2X, has been recently identified in type 2 fibers of rat skeletal muscles using an immunochemical approach. In the present study, the same panel of anti-MHC monoclonal antibodies was used in immunohistochemistry combined with enzyme histochemistry to identify and compare type 2X fibers in hindlimb skeletal muscles of rat, mouse, and guinea pig. Immunohistochemistry shows that 2X MHC is localized in a large subset of type 2 fibers and is co-expressed with 2A or 2B MHC in a small number of fibers. Enzyme histochemistry shows that type 2X fibers display low myosin ATPase activity after pre-incubation at pH 4.3 and high activity after paraformaldehyde pre-incubation at pH 10.4. After pre-incubation at pH 4.6, myosin ATPase shows intermediate and high activity in rat and mouse 2X fibers, respectively, whereas it is low in guinea pig 2X fibers. Succinate dehydrogenase displays moderate to high activity in 2X fibers of all species. Taken together, these staining patterns allow this novel fiber population to be distinguished from the other type 2 fibers using only enzyme histochemistry. Nevertheless, the combined use of immuno- and enzyme histochemistry prevents incorrect fiber typing due to the interspecies variability of myosin ATPase activity among the correspondent fiber types, and completely modifies the presently used classification of mouse type 2 fibers.     

Morphometric analysis of capillary geometry in pigeon pectoralis muscle

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 livia). 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-sectional area (range, 1,491-5,680 mm-2) depended on fiber size (aerobic fibers, 304-782 microns 2; glycolytic, 1,785-2,444 microns 2), as well as sarcomere length (1.69-2.20 microns), and the relative sectional area of aerobic and glycolytic fibers (aerobic, 42-84% 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 large 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-fold range of capillary density (range, 450-4,670 mm-2), 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 greater than 4,000 mm-2), as compared to samples with a capillary density less than 4,000 mm-2.     

Effect of endurance training on muscle fiber type composition and capillary supply in rat diaphragm

Muscle fiber type composition and capillary supply in rat diaphragm were investigated after 14 weeks of endurance training: body weight and muscle fiber area were significantly decreased, the muscle fiber type composition, capillary to fiber ratio and number of capillaries around each fiber type were unchanged, and the capillary density and number of capillaries around each fiber relative to fiber type areas were significantly increased. These small fiber areas and increased capillary supplies in the trained rats would facilitate oxygen transport to all parts of the muscle fiber during exercise. It is concluded that the changes observed in the trained rat diaphragm appear to enhance the capacity for oxidative metabolism.     

Capillary and fiber size interrelationships in regenerating rat soleus muscle after ischemia: a quantitative study.

The present study examines the influence of ischemia on the muscle fibers and capillarization in rats. Muscle ischemia was achieved by a pneumatic tourniquet at a pressure of 300 mm Hg for 2, 4 and 6 h (groups I, II and III, respectively) to the right hindlimb above the knee. Numerous regenerative fibers were seen at 4 and, especially, 8 and 12 days after ischemia in groups II and III. The quantitative data revealed a significant decrease in the size of muscle fibers (regenerative fibers) in ischemic skeletal muscle, with a concomitant increase in fiber density. The capillary to fiber ratio shows a decrease at 4, 8 and 12 days after ischemia in the three experimental groups: in group I because of a decrease in capillary density; in groups II and III because of an increase in fiber density with respect to capillary density.     

Adaptations in skeletal muscle capillarity following changes in oxygen supply and changes in oxygen demands.

The effects of changes in oxygen supply and oxygen demands on fiber cross-sectional areas, capillary densities and capillary to fiber ratios were determined in three skeletal muscles of rat. The muscles examined were the vastus lateralis, soleus, and diaphragm. Reduced oxygen supply was produced by subjecting rats to ambient hypoxia, and increased oxygen demands were produced by subjecting rats to low ambient temperatures or treatment with thyroxin. Capillaries were visualized by injecting fluorescent dyes into the circulation. Muscles were quick frozen at resting lengths to preserve normal fiber geometry and were subsequently sectioned on a cryostat. All of the muscles sampled from animals in the experimental groups had elevated capillary densities. However, capillary to fiber ratios were not increased significantly in any muscle, for any experimental condition. Thus, all of the observed differences in capillarity were due to changes in the intrinsic rate of muscle fiber growth. Further, the relations of capillary density and capillary to fiber ratio to fiber area were the same as those obtained during normal maturation, suggesting that capillary growth is closely linked to the intrinsic rate of fiber growth.     

Capillary tortuosity in skeletal muscles of mammals depends on muscle contraction

Capillary orientation (anisotropy) was compared in hindlimb muscles of mammals of different size and/or different aerobic capacity (dog, goat, pony, and calf). All muscles were fixed by vascular perfusion at sarcomere lengths ranging from 1.5 to 2.7 micron. The ratios of capillary counts per fiber cross-sectional area on two sets of sections (0 and 90 degrees) to the muscle fiber axis were used to estimate capillary anisotropy and the coefficient c(K,0) relating 1) capillary counts on transverse sections (a commonly used parameter to assess muscle capillarity) and 2) capillary length per volume of fiber (i.e., capillary length density). Capillary orientation parallel to the muscle fiber axis decreased substantially with muscle fiber shortening. In muscles fixed at sarcomere lengths of 2.69 microns (dog vastus intermedius) and 1.52 microns (dog gastrocnemius), capillary tortuosity and branching added 7 and 64%, respectively, to capillary length density. The data obtained in this study are highly consistent with the previously demonstrated relationship between capillary anisotropy and sarcomere length in extended vs. contracted rat muscles, by use of the same method. Capillary anisotropy in mammalian locomotory muscles is curvilinearly related to sarcomere length. No systematic difference was found in capillary tortuosity with either body size, athletic ability, or aerobic capacity. Capillary tortuosity is a consequence of fiber shortening rather than an indicator of the O2 requirements of the tissue.     

Misclassification of hybrid fast fibers in resistance-trained human skeletal muscle using histochemical and immunohistochemical methods

ABSTRACT: Staron, RS, Herman, JR, and Schuenke, MD. Misclassification of hybrid fast fibers in resistance-trained human skeletal muscle using histochemical and immunohistochemical methods. J Strength Cond Res 26(10): 2616-2622, 2012-Sixteen healthy untrained women participated in a 6-week progressive resistance training program to compare 2 common methods of classifying fiber types. The women were a subset from a previous study and were randomly divided into 2 groups: traditional strength training (TS, n = 9) and non-exercising control (C, n = 7). The TS group performed 3 lower limb exercises (leg press, squat, and knee extension) using 6-10 repetitions maximum 2 days per week for the first week and 3 days per week for the remaining 5 weeks (17 total workouts). Pre- and posttraining vastus lateralis muscle biopsies were analyzed for fiber type composition using 2 popular methods: myosin adenosine triphosphatase (mATPase) histochemistry and myosin heavy chain (MHC) immunohistochemistry. Six fiber types (I, IC, IIC, IIA, IIAX, and IIX) were delineated using each method separately and in combination. Because of the subjective nature of each method (visual assessment of staining intensities), IIAX fibers expressing a small amount of MHCIIa were misclassified as type IIX using mATPase histochemistry, whereas those expressing a small amount of MHCIIx were misclassified as type IIA using MHC immunohistochemistry. As such, either method used separately resulted in an underestimation of the type IIAX fiber population. In addition, the use of mATPase histochemistry alone resulted in an overestimation of type IIX, whereas there was an overestimation of type IIA using MHC immunohistochemistry. These fiber typing errors were most evident after 6 weeks of resistance training when fibers were in transition from type IIX to IIA. These data suggest that the best approach to more accurately determine muscle fiber type composition (especially after training) is the combination of mATPase histochemical and MHC immunohistochemical methods.     

Free mobilization and low- to high-intensity exercise in immobilization-induced muscle atrophy

After 3 wk of immobilization, the effects offree cage activity and low- and high-intensity treadmill running (8 wk)on the morphology and histochemistry of the soleus and gastrocnemius muscles in male Sprague-Dawley rats were investigated. In both muscles,immobilization produced a significant(P < 0.001) increase in the meanpercent area of intramuscular connective tissue (soleus: 18.9% inimmobilized left hindlimb vs. 3.6% in nonimmobilized right hindlimb)and in the relative number of muscle fibers with pathologicalalterations (soleus: 66% in immobilized hindlimb vs. 6% in control),with a simultaneous significant (P < 0.001) decrease in the intramuscular capillary density (soleus: mean capillary density in the immobilized hindlimb only 63% of that in thenonimmobilized hindlimb) and muscle fiber size (soleus type I fibers:mean fiber size in the immobilized hindlimb only 69% of that in thenonimmobilized hindlimb). Many of these changes could not be correctedby free remobilization, whereas low- and high-intensity treadmillrunning clearly restored the changes toward control levels, the effectbeing most complete in the high-intensity running group. Collectively,these findings indicate that immobilization-induced pathologicalstructural and histochemical alterations in rat calf muscles are, to agreat extent, reversible phenomena if remobilization is intensified byphysical training. In this respect, high-intensity exercise seems morebeneficial than low-intensity exercise.

     

Training-induced alterations in young and senescent rat diaphragm muscle.

The current study sought to examine the effects of chronic endurance treadmill running on oxidative capacity and capillary density in specific diaphragm muscle fiber types in young (5 mo) and senescent (greater than or equal to 23 mo) female Fischer 344 rats. Both young and senescent animals trained at approximately 75% of maximal O2 consumption for 1 h/day 5 days/wk for 10 wk. Plantaris citrate synthase activity was significantly increased (P less than 0.01) in both young and old trained groups. Densitometric analysis of succinate dehydrogenase (SDH) activity in diaphragm type I, IIa, and IIb muscle fibers was done using a computerized image-processing system. There were no age-related differences in SDH activity between the young and old groups for any of the fiber types. In addition, SDH activity was found to be significantly increased (P less than 0.05) in all three fiber types in both the young and senescent trained animals compared with their sedentary counterparts. Fiber size and capillary density did not differ between young and senescent rats, nor did exercise affect this measure. Each fiber, irrespective of type, had an average of approximately four capillaries in contact with it. However, type IIb fibers had a significantly lower capillary density per unit area than type I or IIa muscle fibers. The results indicate that the senescent costal diaphragm maintains its ability to adapt to an increased metabolic demand brought about by locomotor exercise. Of further interest is the finding that training adaptations occurred in all three fiber types, suggesting that increased work of breathing from moderate exercise leads to recruitment of all three fiber types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recovery from short term intense exercise: its relation to capillary supply and blood lactate concentration

Muscle force recovery from short term intense exercise was examined in 16 physically active men. They performed 50 consecutive maximal voluntary knee extensions. Following a 40-s rest period five additional maximal contractions were executed. The decrease in torque during the 50 contractions and the peak torque during the five contractions relative to initial torque were used as indices for fatigue and recovery, respectively. Venous blood samples were collected repeatedly up to 8 min post exercise for subsequent lactate analyses. Muscle biopsies were obtained from m. vastus lateralis and analysed for fiber type composition, fiber area, and capillary density. Peak torque decreased 67 (range 47-82%) as a result of the repeated contractions. Following recovery, peak torque averaged 70 (47-86%) of the initial value. Lactate concentration after the 50 contractions was 2.9 +/- 1.3 mmol X 1(-1) and the peak post exercise value averaged 8.7 +/- 2.1 mmol X 1(-1). Fatigue and recovery respectively were correlated with capillary density (r = -0.71 and 0.69) but not with fiber type distribution. A relationship was demonstrated between capillary density and post exercise/peak post exercise blood lactate concentration (r = 0.64). Based on the present findings it is suggested that lactate elimination from the exercising muscle is partly dependent upon the capillary supply and subsequently influences the rate of muscle force recovery.     

Capillarity in Rat Skeletal Muscle: Effects of Rapid Freezing versus Perfusion Fixation

We determined the effects of rapid freezing and perfusion fixation on fiber geometry and capillarity in rat skeletal muscle. Fiber areas were significantly decreased, and capillary densities significantly increased, in perfusion-fixed versus quick-frozen muscle. Significant differences in capillary-to-fiber ratios were not observed, suggesting that differences in fiber geometry, not the methods of quantifying capillaries, accounted for the differences in capillary density. We conclude that estimates of fiber geometry, capillarity, and diffusive gas conductances obtained from perfusion-fixed muscles are subject to significant error due to shrinkage.     

Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle

Adult skeletal muscle undergoes adaptation in response to endurance exercise, including fast-to-slow fiber type transformation and enhanced angiogenesis. The purpose of this study was to determine the temporal and spatial changes in fiber type composition and capillary density in a mouse model of endurance training. Long-term voluntary running (4 wk) in C57BL/6 mice resulted in an approximately twofold increase in capillary density and capillary-to-fiber ratio in plantaris muscle as measured by indirect immunofluorescence with an antibody against the endothelial cell marker CD31 (466 ± 16 capillaries/mm² and 0.95 ± 0.04 capillaries/fiber in sedentary control mice vs. 909 ± 55 capillaries/mm² and 1.70 ± 0.04 capillaries/fiber in trained mice, respectively; P < 0.001). A significant increase in capillary-to-fiber ratio was present at day 7 with increased concentration of vascular endothelial growth factor (VEGF) in the muscle, before a significant increase in percentage of type IIa myofibers, suggesting that exercise-induced angiogenesis occurs first, followed by fiber type transformation. Further analysis with simultaneous staining of endothelial cells and isoforms of myosin heavy chains (MHCs) showed that the increase in capillary contact manifested transiently in type IIb + IId/x fibers at the time (day 7) of significant increase in total capillary density. These findings suggest that endurance training induces angiogenesis in a subpopulation of type IIb + IId/x fibers before switching to type IIa fibers. adaptation; capillary density; endothelial cells; fiber type transformation; vascular endothelial growth factor     

Greater capillary-fiber interface per fiber mitochondrial volume in skeletal muscles of old rats.

The objective was to examine whether muscle structural capacity for O2 flux (i.e., capillary-to-fiber surface ratio) relative to fiber mitochondrial volume deteriorates with the muscle atrophy of aging in predominantly slow- (soleus, S) and fast-twitch (extensor digitorum longus, EDL) muscles of old (24 mo) and very old (35 mo) F344BN rats compared with adult (12 mo old). Wet muscle mass decreased 29% (196 +/- 4 to 139 +/- 5 mg) in S and 22% (192 +/- 3 to 150 +/- 3 mg) in EDL between 12 and 35 mo of age, without decline in body mass. Capillary density increased 65% (1,387 +/- 54 to 2,291 +/- 238 mm(-2)) in S and 130% (964 +/- 95 to 2,216 +/- 311 mm(-2)) in EDL, because of the muscle fiber atrophy, whereas capillary per fiber number remained unchanged. Altered capillary geometry, i.e., lesser contribution of tortuosity and branching to capillary length, was found in S at 35 compared with 12 and 24 mo, and not in EDL. Accounting for capillary geometry revealed 55% (1,776 +/- 78 to 2,750 +/- 271 mm(-2)) and 113% (1,194 +/- 112 to 2,540 +/- 343 mm(-2)) increases in capillary length-to-fiber volume ratio between 12 and 35 mo of age in S and EDL, respectively. Fiber mitochondrial volume density was unchanged over the same period, causing mitochondrial volume per micrometer fiber length to decrease in proportion to the fiber atrophy in both muscles. As a result of the smaller fiber mitochondrial volume in the face of the unchanged capillary-to-fiber number ratio, capillary-to-fiber surface ratio relative to fiber mitochondrial volume not only did not deteriorate, but in fact increased twofold in both muscles between 12 and 35 mo of age, independent of their different fiber type.