Natalio Banchero

Effects of Hypoxia on Capillary Density and Fiber Composition in Rat Skeletal Muscle

SummaryCapillary density, fiber composition and myoglobin concentration were determined in skeletal muscle of Sprague-Dawley female rats. PIO2 for control (C) rats was 123 mm Hg; for hypoxic (H) rats the O2 concentration in air was lowered to 12.6% (PIO2=73 mm Hg) over 4 days and then kept at this level for 39 days. Body weight (BW) in C rats went from 182 to 258 g in 6 weeks. H rats showed an initial 13 g drop in BW but later gained weight at the same rate. A difference of 20 g was maintained between C and H rats. Five groups of about 10 rats each (3 C and 2 H) were studied by histochemistry using frozen samples from the tibialis anterior, gastrocnemius (medial head) and soleus muscles. 20 μm slices, cut transversely, were treated for alkaline phosphatase and ATPase after preincubation at pHs of 3.8 to 9.4 in order to visualize capillaries and typify fibers. In both C and H rats, capillary density decreased at the same rate with increasing BW, as a consequence of the increase in the size of the fibers resulting from growth. At the same BW capillary densities for a given muscle were not statistically different. No consistent differences in fiber composition were found in the soleus after exposure to hypoxia. In the gastrocnemius and the tibialis anterior, however, the percentage population of white fibers tended to be higher in the H rats than in the C rats, while the population of red fibers decreased. Myoglobin concentration was higher in the soleus, a muscle with a preponderant intermediate fiber population, than in the other two muscles. Hypoxia did not systematically affect myoglobin concentration.

Changes in the capillarity of skeletal muscle in the growing rat.

Capillary density (CD), capillary to fiber ratio (C/F), fiber cross sectional area (FCSA) and fiber composition were measured in the soleus and the gastrocnemius (medial head) muscles of rats weighing between 99 and 666 g. Muscle samples obtained from the anesthetized animal were rapidly frozen (−130°C) sliced transversely at 16–18 μm, and treated histochemically by the ATPase method after preincubation at pHs of 4.0 and 4.4 to visualize capillaries and typify fibers. In both muscles the FCSA was positively related to body weight (BW) and muscle weight. At a given BW, the FCSA of the soleus was greater than that of the gastrocnemius. In both muscles CD decreased hyperbolically with FCSA (soleus: CD=1.0613 ×106/FCSA+298.71; gastrocnemius: CD=1.0349 ×106/FCSA+240.74). At the same time a positive linear correlation between C/F and FCSA was found (soleus: C/F=3.92×10−4 FCSA+0.82; gastrocnemius: C/F=2.90×10−4 FCSA+0.93). At a given FCSA, CD and C/F were greater in the soleus than in the gastrocnemius because of differences in fiber composition between the two muscles. The soleus had only oxidative fibers (STO and FTOG) whereas the gastrocnemius had 54% glycolytic fibers (FTG). The very large variability in CD and C/F values reported in the literature could, in part, be due to the differences in capillarity observed with maturation. A change in fiber composition with BW was observed in the soleus, but no systematic change occurred in the gastrocnemius.

Oxygen transport in the llama (LAma glama)

Abstract Three male llamas 5–14 months old and born at sea level were studied first at 260 m altitude, and again after 5 and 10 weeks at 3420 m. Observations were made with the animals awake, standing and unsedated, at rest breathing air, acute hypoxia, acute hyperoxia and during treadmill exercise. O 2 tension, O 2 content, O 2 saturation and pH were measured on arterial and mixed venous blood. These data were adjusted to pH 7.40 and 37.0 °C to permit the construction of Hb-O 2 dissociation curves. Cardiac output () was measured by dye dilution. Llama Hb has a high affinity for O 2 : P 50 of 22.7 mm Hg at sea level, and 23.7 mm Hg at altitude, indicating no decrease in the affinity of Hb for O 2 at altitude. Exposure to 3420 m resulted in a decrease in Pao-from 87 to 51–53 mm Hg, but SaO 2 remained above 92%. With an increase in Hb concentration from 10.6 to 11.8 g per 100 ml, there was no decrease in CaO 2 . Furthermore, there was no change in , (CaO 2 -CVO 2 ), or systemic O 2 transport ( × CaO 2 ). Under all conditions, PVO 2 was lower than in most other mammals, but always exceeded 20 mm Hg, reflecting the efficient mechanisms of O 2 extraction in the llama.

Visualization of capillaries in skeletal muscle by the ATPase reaction

SummaryA simple and reliable technique for the visualization of capillaries in skeletal muscle of dogs, guinea pigs and rats is described. 10–20 μm frozen sections were incubated in a medium containing Ca2+ and ATP following acid preincubation (pH 3.8–4.2). The capillaries stained in black and were readily seen surrounding the muscle fibers. Serial sections were also treated for alkaline phosphatase. Values for capillary density using both methods were not different.

Lung morphometry in guinea pigs acclimated to hypoxia during growth.

The effect of chronic hypoxic exposure on lung development has been assessed in growing guinea pigs (Cavia porcellus). Weanling males of initial W = 229 g were acclimated to a PO2 of 80 Torr for 2-14 weeks before sacrifice (range of W = 244-965 g). Growth was the same in hypoxic animals as in controls maintained at a PO2 of 133 Torr (range of W in controls = 89-1274 g). Lungs were fixed by tracheal instillation of glutaraldehyde and examined morphometrically with the electron microscope. Within 3 weeks of exposure, lung volume (VL) and alveolar surface area (Sa) were significantly increased by 32% and 27% respectively in the hypoxia acclimated animals compared to controls of similar W. However, these differences were progressively reduced with increasing time of exposure, and mean values of VL and Sa were not different between groups when W greater than 900 g. Chronic hypoxia accelerated lung development towards normal adult dimensions to a degree remarkably similar to that reported in cold acclimated guinea pigs. These findings are compatable with the theory of adaptive lung growth mediated by increased pulmonary blood flow, and suggest anatomical limitations to such growth related to an animals age.

The closest-individual method in the analysis of the distribution of capillaries ☆

Abstract An ecological survey method termed the closest-individual method was adapted for measuring the distribution of capillaries in tissue. Models were constructed in which dots represented transversely sectioned capillaries in known densities, and in ordered, random, or contagious (clustered) array. The distances from randomly selected points on a model to the nearest capillary were measured, and the frequency distributions of these distances were plotted. This distribution was compared to the mathematically predicted distribution of distances for each model. All values derived by the closest-individual method were statistically similar to predicted values, and were within approximately 5% of predictions. In addition, the kurtosis of each set of data was used to evaluate whether the data from the closest-individual method could correctly identify the nature of the array of capillaries in each model. This method is potentially of great use to studies of capillarity since it calculates median diffusion distance more accurately than estimates from capillary density alone; it calculates maximal diffusion distance, which cannot be estimated from capillary density alone; and by identifying the array of capillaries as either random, ordered, or contagious, it assesses the nature of interaction among capillaries. Other similar measurement methods are discussed and compared.

Capillary density of skeletal muscle in dogs exposed to simulated altitude (38556).

A number of anatomical and physiological changes have been reported in man and in animals living in chronic hypoxia. It is generally believed that these changes are adaptative in nature, and they help the animal tolerate a low Po2 environment. Possibly these features or mechanisms are optimally developed in man and in animals native to high altitude. However, the study of the acute situation, when a lowland animal is exposed to a low oxygen environment, offers the opportunity to ascertain how fast these changes occur, and, on occasion, provides insight into the underlying mechanisms. Valdivia (1) has found that high altitude guinea pigs (at 4540 m) have a greater number of open capillaries in skeletal muscle than sea level controls. He regarded this as an important adaptative mechanism to chronic hypoxia even though muscle fiber size was approximately the same in all animals. More recently Cassin et al. (2) showed that the number of capillaries per unit surface area in skeletal muscle of rats exposed to 6,100 m for 5 wk increased significantly. However, the evidence of an increased capillarity in hypoxic conditions and its role in facilitating O2 delivery is not conclusive. Furthermore, there is no experimental evidence that exposure of larger mammals to hypoxia results in structural alterations of peripheral tissues. Materials and Methods. Three adult mongrel dogs (two males and one female) native to Denver (1610 m PB 635 mmHg) were studied before and after a 3-wk exposure to a simulated altitude of about 4880 m (ambient pressure 435 mmHg) during which time they received food and water ad libitum. Because the dogs were trained to run on a treadmill, the sternothyroideous muscle was chosen for study in order to minimize the possible effects of exercise on the skeletal muscle.

Effect of maturation on capillary density, fiber size and composition in rat skeletal muscle.

Summary Capillary density, fiber size and composition, were quantitated by histo-chemical methods in the tibialis anterior, soleus and gastrocnemius (medial head) of rats of different body weights. The capillary density was different in these muscles and it decreased with increasing body weight in a linear fashion due to a concomitant increase in the average cross sectional area of the fibers. This observation suggests that part of the variability in capillary density found in the literature is due to differences in body size. A change in muscle fiber composition with maturation was also observed. These were more marked in the tibialis anterior and in the soleus than in the gastrocnemius. The authors gratefully acknowledge the assistance of Ms. Sheila H. Eby.

Increased capillary supply in skeletal muscle of guinea pigs acclimated to cold

The ATPase technique was used to visualize blood capillaries and to study fiber composition in 10-micrometer transverse sections of guinea pig gastrocnemius and soleus muscles. A control group of newborn, weanling, juvenile and adult male guinea pigs (GP) (BW = 89-1274 g) was studied in a 20-24 degrees C environment (22 degrees C GP) while 2-3 week old animals were exposed continuously to 5 degrees C for 2-18 weeks before sacrifice (5 degrees C GP) (BW = 239-1074 g). Body weight gain was not affected by cold exposure; however, the gastrocnemius and soleus muscles of the 5 degrees C GP grew at a slower rate than did the muscles of the 22 degrees C GP. The equations relating fiber cross sectional area (FCSA) and muscle weight (MW) were not different between the 22 degrees C GP and 5 degrees C GP for the soleus and gastrocnemius. Therefore, in both muscles at the same BW, FCSA was smaller in the 5 degrees C GP than in the 22 degrees C GP. In both of the two muscles of each group, capillary density (CD) decreased hyperbolically with increasing FCSA, while the capillary to fiber ratio (C/F) and the average number of capillaries around each fiber (CAF) increased linearly with increasing FCSA. The regression lines for CD, C/F and CAF versus FCSA for both muscles were parallel between groups, but at any FDSA, the CD, C/F and CAF were greater in the 5 degrees C GP than in the 22 degrees C GP. Percent fiber composition of the gastrocnemii of the 22 degrees C GP and 5 degrees C GP were not different; however, at the same FCSA each fiber type had a greater capillary supply in the 5 degrees C GP. The increased capillarity in the gastrocnemius and soleus muscles of the 5 degrees C GP suggests an improved capacity for oxygenation, a response which would correlate well with the increased oxygen utilization during prolonged cold exposure.

How to quantify skeletal muscle capillarity.

The capillarity in skeletal muscle is not adequately defined by either capillary density or by capillary to fiber ratio. Capillary density gives no clue as to the evenness of the distribution of the capillaries in muscle cross sections but the reciprocal of capillary density provides an estimate of the area of tissue cross section served by the average capillary, a value used by Krogh and other investigators (Tenney, 1974). The capillary to fiber ratio renders no information on the area of tissue served by each capillary. Further, capillary density and C/F give no indication of the maximal diffusion path length, important in assessing the capacity of muscle for oxygenation. For these reasons, we have been interested in finding a better method of obtaining data that could be used to estimate diffusion distances. This paper describes capillarity in skeletal muscle using several methods of quantification that, we believe, provide a better understanding of the transport of oxygen within the muscle.