Estimating diffusion distances in muscle

Models of fibers and capillaries in cross sections of muscle were used to quantify the relationships between diffusion distances and tissue capillarity. The fibers were constructed as square and hexagonal arrays, and the placement of capillaries around the perimeters of the fibers ordered them in similar arrays. Diffusion distances were measured as the percent cumulative frequency of fiber area within a given distance of a capillary when capillary-to-fiber ratio was increased from 0.5 to 4.0. Equations fitted to the data make it possible to estimate diffusion distances in muscle and to correlate changes in diffusion distances with fiber growth, capillary growth, and the geometrical arrangement of capillaries in the muscle bed.     

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     

Lower capillary density but no difference in VEGF expression in obese vs. lean young skeletal muscle in humans.

Obesity is associated with lower skeletal muscle capillarization and lower insulin sensitivity. Vascular endothelial growth factor (VEGF) is important for the maintenance of the skeletal muscle capillaries. To investigate whether VEGF and VEGF receptor [kinase insert domain-containing receptor (KDR) and Flt-1] expression are lower with obesity, vastus lateralis muscle biopsies were obtained from eight obese and eight lean young sedentary men before and 2 h after a 1-h submaximal aerobic exercise bout for the measurement of VEGF, KDR, Flt-1, and skeletal muscle fiber and capillary characteristics. There were no differences in VEGF or VEGF receptor mRNA at rest between lean and obese muscle. Exercise increased VEGF (10-fold), KDR (3-fold), and Flt-1 (5-fold) mRNA independent of group. There were no differences in VEGF, KDR, or Flt-1 protein between groups. Compared with lean skeletal muscle, the number of capillary contacts per fiber was the same, but lower capillary density (CD), greater muscle cross sectional area, and lower capillary-to-fiber area ratio were observed in both type I and II fibers in obese muscle. Multiple linear regression revealed that 49% of the variance in insulin sensitivity (homeostasis model assessment) could be explained by percentage of body fat (35%) and maximal oxygen uptake per kilogram of fat-free mass (14%). Linear regression revealed significant relationships between maximal oxygen uptake and both CD and capillary-to-fiber perimeter exchange. Although differences may exist in CD and capillary-to-fiber area ratio between lean and obese skeletal muscle, the present results provide evidence that VEGF and VEGF receptor expression are not different between lean and obese muscle.     

Fiber composition and capillarity in growing guinea pigs acclimated to cold and cold plus hypoxia

The central portion of the medial head of the gastrocnemius of control (normoxic and normothermic), hypoxia-, cold-, and cold plus hypoxia-acclimated guinea pigs was analyzed for capillary supply and fiber composition to elucidate changes in capillarity induced by environmental stresses. The muscle was cut at midbelly, frozen, sectioned, and stained for myosin ATPase. Fiber cross-sectional areas; percentages of slow-twitch oxidative (SO), fast-twitch oxidative-glycolytic (FOG), and fast-twitch glycolytic (FG) fibers; and numbers of capillaries around each fiber type were measured. Growth rates of all four guinea pig groups were similar. Capillarity was not affected by acclimation to hypoxia. Cold and cold plus hypoxia acclimation led to increased numbers of capillaries around the fiber in all three fiber types. In addition, significant increases in the percentage of FOG fibers and concomitant decreases in the percentage of FG fibers compared to controls were found in cold and in cold plus hypoxia indicating that a transformation of fiber type from FG to FOG had occurred. The increase in FOGs at the expense of the FGs did not occur in the guinea pigs grown in a hypoxic environment. The increased total capillarity in those muscles studied was the result of more capillaries around all fiber types and was not due to simple transformation of fibers.     

Effect of growth on muscle capillarity and fiber type composition in rat diaphragm

The effect of growth on the capillarity and fiber type composition of the diaphragm, soleus and extensor digitorum longus (EDL) muscles of rats weighing between 55 and 330 g have been studied. Muscle samples obtained from the anesthetized rat were rapidly frozen and sliced transversely in a cryostat. The sections were stained histochemically by the SDH method and the myosin ATPase method after preincubation at pH 4.3 to typify fibers (FG, FOG and SO fibers). To visualize capillaries, the myosin ATPase method after preincubation at pH 4.0 was used. The percentage of FOG fibers decreased in all muscles with growth. While the FG and SO fibers increased in the diaphragm, SO fibers increased in the soleus, and FG fibers increased in the EDL. The capillary density showed a hyperbolic decrease with growth in all muscles, while the number of capillaries around each fiber increased in all muscles with growth. It is concluded that growth causes the changing properties of the motoneurons and the new capillary formation in the diaphragm muscle, as well as the soleus and EDL muscles.     

Effect of decreased O2 supply on skeletal muscle oxygenation and O2 consumption during sepsis: role of heterogeneous capillary spacing and blood flow

One of the main aspects of the initial phase of the septic inflammatory response to a bacterial infection is abnormal microvascular perfusion, including decreased functional capillary density (FCD) and increased blood flow heterogeneity. On the other hand, one of the most important phenomena observed in the later stages of sepsis is an increased dependence of tissue O(2) utilization on the convective O(2) supply. This "pathological supply dependency" is associated with organ failure and poor clinical outcomes. Here, a detailed theoretical model of capillary-to-tissue O(2) transport during sepsis is used to examine the origins of abnormal supply dependency. With use of three-dimensional arrays of capillaries with heterogeneous spacing and blood flow, steady-state O(2) transport is simulated numerically during reductions in the O(2) supply. Increased supply dependency is shown to occur in sepsis for hypoxic (decreased hemoglobin O(2) saturation) and stagnant (decreased blood flow) hypoxia. For stagnant hypoxia, a reduction in FCD with decreasing blood flow is necessary to obtain the observed increase in supply dependency. Our results imply that supply dependency observed under normal conditions does not have its origin at the level of individual capillaries. In sepsis, however, diffusion limitation and shunting of O(2) by individual capillaries occur to a degree that is dependent on the heterogeneity of septic injury and the arrangement of capillary networks. Thus heterogeneous stoppage of individual capillaries is a likely factor in pathological supply dependency.     

Expression of muscle capillary alkaline phosphatase is affected by hypoxia.

Hypoxia stimulates angiogenesis in some microvascular beds, but no clear angiogenic effect of hypoxia has yet been demonstrated in adult skeletal muscle. In this study the distribution of alkaline phosphatase (APase) was compared with a novel microvascular marker, Griffonia simplicifolia I (GSI), to determine whether the respective markers were expressed by muscle capillaries during hypoxic conditions and to probe for the presence or absence of angiogenesis in response to short-term hypoxia. Mice were exposed to normobaric 8% oxygen for 7 or 21 days. Capillary density in the red and white areas of the gastrocnemius muscle was determined with the use of a double-labeling procedure for both APase and fluorescently tagged GSI. Little change in capillary density was found. Focal reductions in APase activity were observed within 1 wk of hypoxia, but no changes were observed in GSI binding. In controls, 74 and 92% of red and white muscle capillaries, respectively, were APase positive. This percentage declined to 60% in red and 43% in white muscle after 21 days of hypoxia. The results indicate that APase expression is labile under certain conditions and warrant a cautious approach to using the enzyme as a marker. Binding of the GSI lectin to muscle capillaries appeared to be unchanged by the exposure to hypoxia, indicating stability of this marker system. No significant change in the number of capillaries around individual muscle fibers was evident at 21 days when GSI was used to detect capillaries. These results confirm the absence of hypoxia-induced angiogenesis in muscle capillaries during the time period studied.     

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.     

Effect of peripheral and central alpha-adrenoceptor blockade on cerebral microvascular and blood flow responses to hypoxia.

The purpose of this study was to evaluate the effects of vascular and central alpha-adrenoceptor blockade on cerebral blood flow (CBF) and utilization of brain arteriolar and capillary reserve in conscious rats during normoxia and hypoxia (8% O2 in N2). Animals were divided into three groups and administered either saline, N-methyl chlorpromazine (does not cross the blood-brain barrier), or phenoxybenzamine (crosses the blood-brain barrier) in equipotent doses. Neither agent affected regional CBF and the utilization of brain microvascular reserve during normoxia. CBF increased from 70.9 +/- 2.9 (SEM) ml/min/100 g in the control normoxic group to 123.8 +/- 4.2 ml/min/100 g in control hypoxic animals. In control, hypoxic flow to pons and medulla of the brain was higher than to cortex, hypothalamus or thalamus. The percent of arterioles/mm2 perfused increased from 49.6 +/- 2.0% during control normoxia to 65.6 +/- 3.0% during control hypoxia. The percentage of capillaries/mm2 perfused changed similarly. Hypoxic CBF was increased similarly after administration of N-methyl chlorpromazine or phenoxybenzamine. Administration of N-methyl chlorpromazine or phenoxybenzamine eliminated regional differences in hypoxic CBF and the utilization of arterioles, and did not affect capillary response. There was no difference between the effect of N-methyl chlorpromazine and phenoxybenzamine on cerebral microvascular and blood flow responses to hypoxia. It was concluded that peripheral alpha-adrenoceptors affect the distribution of regional microvascular and blood flow responses to hypoxia, and central alpha-adrenoceptors probably do not participate in this effect.     

Capillarity and diffusion distances in skeletal muscles in birds

Summary Tissue capillarity and diffusion distances were determined for red and white skeletal muscles of adult birds ranging in mass from 10.8 to 6200 g. In addition, literature values for capillarity and diffusion distances in skeletal muscles of mammals were incorporated into the data set. Muscle mass was closely coupled to body mass. However, no significant allometric relations were found for any of the other variables measured. Number of capillaries per fiber was not correlated with cross sectional area of individual muscle fibers. Thus, capillary density decreased in a hyperbolic manner against fiber area and diffusion distance decreased in a hyperbolic manner against the number of capillaries per muscle fiber. Red muscles had significantly higher numbers of capillaries per fiber and significantly shorter diffusion distances than did white muscles. The patterns for tissue capillarity and diffusion distances in avian muscle reported here are similar to values reported previously for mammalian muscles. In both taxanomic groups capillarity and diffusion distances are independent of body mass. In addition, diffusion distances are characteristic of capillaries distributed in random arrays through the muscle cross section.Abbreviations ALD muscle anterior latissimus dorsi - CD numerical density of capillaries in muscle cross section - C/F number of capillaries per individual muscle fiber - FCSA fiber cross sectional area - GST muscle gastrocnemius - LGST lateral head of muscle gastrocnemius - MGST medial head of muscle gastrocnemius - MM muscle mass - PLD muscle posterior latissimus dorsi     

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.     

Adaptations of the diaphragm in emphysema.

In adult male hamsters the influence of emphysema (EMP) on the in vitro contractile and fatigue properties and the histochemical, morphometric, and metabolic properties of muscle fibers in the costal diaphragm was determined 6 mo after the administration of either elastase or saline (controls, CTL). Isometric contractile properties were determined in vitro using supramaximal direct muscle stimulation. Optimal fiber length for force generation was significantly shorter in the EMP than in the CTL diaphragm. Maximum specific force (i.e., force per unit area) was 25% lower than CTL. Fatigue resistance was significantly improved in the EMP diaphragm compared with CTL. Diaphragm muscle fibers were classified as type I or II on the basis of histochemical staining for myofibrillar adenosinetriphosphatase after alkaline preincubation. The proportions of type I and II fibers were similar between the two groups. Cross-sectional areas of type II fibers were 30% larger in EMP than in CTL diaphragms. Succinate dehydrogenase activities of both type I and II fibers were higher in EMP than in CTL diaphragms. The number of capillaries surrounding both type I and II fibers increased with EMP, but in proportion to the hypertrophy of these fibers. Thus, capillary density (number of capillaries per fiber cross-sectional area) remained unchanged. We postulate that these contractile, morphometric, and metabolic adaptations reflect an increased activation of the diaphragm in response to the loads imposed by EMP.     

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.     

Metabolic and morphometric profile of muscle fibers in chronic hemodialysis patients

Muscle weakness and effort intolerance are common in maintenance hemodialysis (MHD) patients. This study characterized morphometric, histochemical, and biochemical properties of limb muscle in MHD patients compared with controls (CTL) with similar age, gender, and ethnicity. Vastus lateralis muscle biopsies were obtained from 60 MHD patients, 1 day after dialysis, and from 21 CTL. Muscle fiber types and capillaries were identified immunohistochemically. Individual muscle fiber cross-sectional areas (CSA) were quantified. Individual fiber oxidative capacities were determined (microdensitometric assay) to measure succinate dehydrogenase (SDH) activity. Mean CSAs of type I, IIA, and IIX fibers were 33, 26, and 28% larger in MHD patients compared with CTL. SDH activities for type I, IIA, and IIX fibers were reduced by 29, 40, and 47%, respectively, in MHD. Capillary to fiber ratio was increased by 11% in MHD. The number of capillaries surrounding individual fiber types were also increased (type I: 9%; IIA: 10%; IIX: 23%) in MHD patients. However, capillary density (capillaries per unit muscle fiber area) was reduced by 34% in MHD patients, compared with CTL. Ultrastuctural analysis revealed swollen mitochondria with dense matrix in MHD patients. These results highlight impaired oxidative capacity and capillarity in MHD patients. This would be expected to impair energy production as well as substrate and oxygen delivery and exchange and contribute to exercise intolerance. The enlarged CSA of muscle fibers may, in part, be accounted for by edema. We speculate that these changes contribute to reduce limb strength in MHD patients by reducing specific force.     

Capillary supply and mitochondrial content of different skeletal muscle fiber types in untrained and endurance-trained men. A histochemical and ultrastructural study.

The number of capillaries per fiber, per mm2, around each fiber type and relative to fiber area was determined in six untrained subjects (UT) and six elite cross-country skiers (ET). Average values for maximal oxygen uptake were 49.8 ml . kg-1 . min-1 (UT) and 77.9 ml . kg-1 . min-1 (ET). Type I fibers constituted 39.2% (UT) and 68.6% (ET), type II A fibers 39.6% (UT) and 19.2% (ET), while 12.8% (UT) and 6.6% (ET) of the fibers were type II B. The mean fiber area for the type II A fibers was significantly greater (p less than 0.01) than the areas for type I and II B in the untrained group. The average numbers of capillaries around each fiber type (CA) were 4.76-4.84-2.94 (UT) and 7.79-6.63-4.5 (ET) for type I, II A, and II B, respectively. There was a significant difference (p less than 0.01) in the CA values relative to fiber area for all fiber types in both groups, being highest for type I and lowest for type II B. The CA increased linearly with increasing size of the fibers for all fiber types in both groups. The mitochondrial content was determined semiquantitatively for each fiber type. The differences in capillary supply between the fiber types are accompanied by similar differences in mitochondrial content. The results indicate that endurance training increases the capillary supply of all fiber types in the human quadriceps muscle. The fact that light microscopical studies have given lower capillarization values than those obtained with the electron microscope is discussed.     

Effect of hypoxia on percent of arteriolar and capillary beds perfused in the rat brain

The effects of moderate and severe hypoxia on quantitative regional morphometric indexes of the total and perfused arteriolar and capillary network were studied in the rat brain to determine whether diffusion distances were reduced in hypoxia. Fluorescein isothiocyanate (FITC)-labeled dextran was injected into the femoral vein of conscious control and hypoxic rats. After 20 s, the animal was decapitated and the head was frozen in liquid N2. Sections from eight brain regions were photographed to detect the perfused microvessels and then stained for alkaline phosphatase to visualize the total vascular network. There were significant increases in percent perfused arteriolar and capillary morphology between the two groups of hypoxic animals and control animals. In control rats, the percent of capillaries perfused averaged 45.6 +/- 0.6% (mean +/- SE). In moderate hypoxia 63.4 +/- 1.8% of the vessels were perfused and in severe hypoxia 89.4 +/- 0.1% were perfused. The percentage of arterioles perfused changed similarly. There were no significant differences in any index of total or percent perfused arteriolar or capillary morphometry among the regions within any group. During severe hypoxia, a greater percentage of the capillary reserves was utilized. These results demonstrate a uniform response to hypoxia in the capillary and arteriolar network of the conscious rat brain.     

Oxidative capacity and capillary density of diaphragm motor units

Motor units in the cat diaphragm (DIA) were isolated in situ by microdissection and stimulation of C5 ventral root filaments. Motor units were classified based on their isometric contractile force responses and fatigue indexes (FI). The muscle fibers belonging to individual units (i.e., the muscle unit) were identified using the glycogen-depletion method. Fibers were classified as type I or II based on histochemical staining for myofibrillar adenosine triphosphatase (ATPase) after alkaline preincubation. The rate of succinate dehydrogenase (SDH) activity of each fiber was determined using a microphotometric procedure. The location of capillaries was determined from muscle cross sections stained for ATPase after acid (pH = 4.2) preincubation. The capillarity of muscle unit fibers was determined by counting the number of capillaries surrounding fibers and by calculating the number of capillaries per fiber area. A significant correlation was found between the fatigue resistance of DIA units and the mean SDH activity of muscle unit fibers. A significant correlation was also observed between DIA unit fatigue resistance and both indexes of muscle unit fiber capillarity. The mean SDH activity and mean capillary density of muscle unit fibers were also correlated. We conclude that DIA motor unit fatigue resistance depends, at least in part, on the oxidative capacity and capillary density of muscle unit fibers.     

Capillary growth and diffusion distances in muscle

1. Tissue capillarity in muscle was modelled as square-ordered arrays with capillary-to-fiber ratios (C/F) from 0.5 to 'infinity'. 2. C/F up to two had marked effects on diffusion distances, but C/F above had only slight effects on average distances and almost no effect on maximal distances. 3. Capillary growth during normal maturation results in C/F around two. Thus, capillary growth in adult muscle may not be an adaptive mechanism for reducing diffusion distances.     

Effect of sepsis on skeletal muscle oxygen consumption and tissue oxygenation: interpreting capillary oxygen transport data using a mathematical model

Inherent in the inflammatory response to sepsis is abnormal microvascular perfusion. Maldistribution of capillary red blood cell (RBC) flow in rat skeletal muscle has been characterized by increased 1) stopped-flow capillaries, 2) capillary oxygen extraction, and 3) ratio of fast-flow to normal-flow capillaries. On the basis of experimental data for functional capillary density (FCD), RBC velocity, and hemoglobin O2 saturation during sepsis, a mathematical model was used to calculate tissue O2 consumption (Vo2), tissue Po2 (Pt) profiles, and O2 delivery by fast-flow capillaries, which could not be measured experimentally. The model describes coupled capillary and tissue O2 transport using realistic blood and tissue biophysics and three-dimensional arrays of heterogeneously spaced capillaries and was solved numerically using a previously validated scheme. While total blood flow was maintained, capillary flow distribution was varied from 60/30/10% (normal/fast/stopped) in control to 33/33/33% (normal/fast/stopped) in average sepsis (AS) and 25/25/50% (normal/fast/stopped) in extreme sepsis (ES). Simulations found approximately two- and fourfold increases in tissue Vo2 in AS and ES, respectively. Average (minimum) Pt decreased from 43 (40) mmHg in control to 34 (27) and 26 (15) mmHg in AS and ES, respectively, and clustering fast-flow capillaries (increased flow heterogeneity) reduced minimum Pt to 14.5 mmHg. Thus, although fast capillaries prevented tissue dysoxia, they did not prevent increased hypoxia as the degree of microvascular injury increased. The model predicts that decreased FCD, increased fast flow, and increased Vo2 in sepsis expose skeletal muscle to significant regions of hypoxia, which could affect local cellular and organ function.     

3D Visualization and Measurement of Capillaries Supplying Metabolically Different Fiber Types in the Rat Extensor Digitorum Longus Muscle During Denervation and Reinnervation

The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential. (J Histochem Cytochem 57:437–447, 2009)