Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy (original) (raw)
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Characterization of the Capillary Network in Skeletal Muscles From 3D Data
Physiological Research, 2011
In this review we present immunohistochemical methods for visualization of capillaries and muscle fibres in thick muscle sections. Special attention is paid to the procedures that preserve good morphology. Applying confocal microscopy and virtual 3D stereological grids, or tracing of capillaries in virtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to a muscle fibre per fibre length, fibre surface area or fibre volume can be evaluated by an unbiased approach. Moreover, 3D models of capillaries and muscle fibres can be produced. Comparison of the developed methods with counting capillary profiles from 2D sections is discussed and the reader is warned that counting capillary profiles from 2D sections can underestimate the capillary length by as much as 75 percent. Application of the described 3D methodology is illustrated by the anatomical remodelling of capillarity during acute denervation and early reinnervation in the rat soleus and exte...
Capillary Network in Slow and Fast Muscles and in Oxidative and Glycolytic Muscle Fibres
Image Analysis & Stereology, 2011
The aim of this study was to compare capillary network in slow and fast muscles and also in oxidative and glycolytic muscle fibres. Soleus (SOL) and extensor digitorum longus (EDL) muscles were excised from five female rats. Capillaries and muscle fibres were demonstrated on thick tissue sections by a triple immunofluorescent method. Stacks of perfectly registered optical images were captured by a confocal microscope and further analysed. Applying stereological methods (POINTGRID, FAKIR and SLICER plugin- modules of the Ellipse programme), we estimated the mean length of capillaries, adjacent to individual muscle fibre, per unit fibre length (Lcap/Lfib), per unit surface area of the fibre (Lcap/Sfib) and per unit fibre volume (Lcap/Vfib) in the slow SOL and in predominantly fast EDL muscle, and separately in oxidative and glycolytic fibres of EDL muscle. The length of capillaries per unit fibre length was larger in SOL than in EDL muscle, however, capillary length per unit fibre vol...
A novel staining method for quantification and 3D visualisation of capillaries and muscle fibres
European Journal of Histochemistry, 2009
The aim of this study was to introduce a combined fluorescent staining that clearly demonstrates capillaries and distinguishes them from the basal lamina of muscle fibres in skeletal muscle tissue. The triple staining with CD31, Griffonia (Bandeira) simplicifolia lectin (GSL I) and laminin efficiently distinguishes vascular endothelium from the basal lamina of skeletal muscle fibres in physiological and pathological conditions. The presented triple staining method has several advantages, which facilitate quantitative analysis of the capillary network, and its relation to individual muscle fibres.
Journal of Histochemistry & Cytochemistry, 2009
S U M M A R Y 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) K E Y W O R D S 3D visualization capillaries denervation muscle fiber types reinnervation Correspondence to: Ida Eržen,
Acta medica Okayama, 2010
The skeletal muscle is classified into 2 types, slow oxidative or fast glycolytic muscle. For further characterization, we investigated the capillary architecture in slow and fast muscles. The rat soleus and extensor digitorum longus (EDL) muscles were used as representatives of slow and fast muscles, respectively. To investigate capillary density, sections of both types of muscle were stained with alkaline phosphatase; the soleus muscle showed more intense reactivity, indicating that it had a denser capillary structure than the EDL muscle. We then injected fluorescent contrast medium into samples of both muscle types for light and confocal-laser microscopic evaluation. The capillary density and capillary-to-fiber ratio were significantly higher, and the course of the capillaries was more tortuous, in the soleus muscle than in the EDL muscle. Capillary coursed more tortuously in the soleus than in the EDL muscle. Succinate dehydrogenase (SDH) activity, an indicator of mitochondrial ...
Microvascular …, 1984
The stereomorphological arrangement of skeletal muscle capillaries depends on many factors. Genetic biochemical composition of muscle fibers, age, and training can interfere with the growth of the capillary network. This study is focused on the age effect of chronic reduction of blood flow, as in arterial stenosis, in skeletal muscle network of young rats when compared with adult rats. It has been observed that there is a statistically significant decrease of the length of short capillaries which cross the muscle fibers. In adult rats the opposite occurs.
Microvascular Research, 1990
It is generally assumed that when a muscle is shortened or extended the total length of capillaries does not change, implying that capillaries are nondistensible, longitudinally. On the basis of stereological estimates of capillary anisotropy versus sarcomere length, we propose that as long as capillaries are in a tortuous configuration muscle extension will merely decrease the tortuosity, leaving vessel length unaltered. Once capillaries have been pulled into a straight configuration, further extension of the muscle will cause the vessels to stretch. By means of intravital videomicroscopy we have demonstrated that stretching of individual capillaries does indeed occur over a sarcomere length range of 2.1 to 2.9 microns in rat extensor digitorum longus muscle. In vivo measurements of the lengths of six capillaries together with the sarcomere lengths of adjacent fibers were made in muscles positioned at various degrees of extension. Normalized data indicated that four capillaries stretched to the same degree as the muscle, one stretched more and another less. This may reflect differences in distensibility or tortuosity of capillaries in series with one another. The elastic stretching of capillaries during muscle activity may have important consequences in terms of shifts in permeability and increases in capillary surface area.
Validation of a New Semi-Automated Technique to Evaluate Muscle Capillarization
Advances in Experimental Medicine and Biology, 2016
The method of capillary domains has often been used to study capillarization of skeletal and heart muscle. However, the conventional data processing method using a digitizing tablet is an arduous and time-consuming task. Here we compare a new semi-automated capillary domain data collection and analysis in muscle tissue with the standard capillary domain method. The capillary density (1481 ± 59 vs. 1447 ± 54 caps mm(-2); R(2):0.99; P < 0.01) and heterogeneity of capillary spacing (0.085 ± 0.002 vs. 0.085 ± 0.002; R(2):0.95; P < 0.01) were similar in both methods. The fiber cross-sectional area correlated well between the methods (R(2):0.84; P < 0.01) and did not differ significantly (~8 % larger in the old than new method at P = 0.08). The latter was likely due to differences in outlining the contours between the two methods. In conclusion, the semi-automated method gives quantitatively and qualitatively similar data as the conventional method and saves a considerable amount of time.
Microcirculation, 2014
Objective-To provide insight into mitochondrial function in vivo, we evaluated the 3D spatial relationship between capillaries, mitochondria, and muscle fibers in live mice. Methods-3D volumes of in vivo murine Tibialis anterior muscles were imaged by multi-photon microscopy (MPM). Muscle fiber type, mitochondrial distribution, number of capillaries, and capillary-to-fiber contact were assessed. The role of myoglobin-facilitated diffusion was examined in myoglobin knockout mice. Distribution of GLUT4 was also evaluated in the context of the capillary and mitochondrial network. Results-MPM revealed that 43.6 ± 3.3% of oxidative fiber capillaries had ≥ 50% of their circumference embedded in a groove in the sarcolemma, in vivo. Embedded capillaries were tightly associated with dense mitochondrial populations lateral to capillary grooves and nearly absent below the groove. Mitochondrial distribution, number of embedded capillaries, and capillary-to-fiber contact were proportional to fiber oxidative capacity and unaffected by myoglobin knockout. GLUT4 did not preferentially localize to embedded capillaries. Conclusions-Embedding capillaries in the sarcolemma may provide a regulatory mechanism to optimize delivery of oxygen to heterogeneous groups of muscle fibers. We hypothesize that mitochondria locate to paravascular regions due to myofibril voids created by embedded capillaries, not to enhance the delivery of oxygen to the mitochondria.