Vita Čebašek

Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

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)

The estimation error of skeletal muscle capillary supply is significantly reduced by 3D method.

Capillary supply of individual skeletal muscle fibers is usually evaluated from two-dimensional (2D) images of thin transverse sections by the number of capillary profiles around a fiber (CAF). This method is inherently inaccurate and the resulting capillary length measurement errors can be avoided by using an alternative three-dimensional (3D) approach where the mean length of capillaries around individual muscle fibers per fiber length (Lcap/Lfib) is measured from 3D images acquired by confocal microscopy. We quantified the error of the 2D method and its reduction by using a 3D approach in realistic geometrical models of muscle fiber capillary bed and in true muscle samples. In models we showed that Lcap/Lfib was sensitive to different arrangements of capillaries, while CAF underestimated capillarization since it could not detect the increased length of capillary bed. In true muscle samples, we detected statistically significant differences in the capillary supply of control and denervated rat soleus muscles by both 2D and 3D methods. Lcap/Lfib was larger than CAF in control muscles reflecting their more complicated capillary bed. Thus, 3D approach is more sensitive in agreement with the analysis of geometrical models. We conclude that the 3D method, though technically more demanding than 2D method, represents a more precise approach to evaluation of muscle capillarization. Moreover, the 3D method could be applied to other organs and we suggest potential medical applications.

Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.

Simultaneous visualization of myosin heavy chain isoforms in single muscle sections.

We developed a staining protocol that enables simultaneous visualization of myosin heavy chain (MHC) pure and hybrid muscle fiber types in rat skeletal muscle. Up to eight different muscle fiber types can be visualized in a single section of the rat extensor digitorum longus muscle, which contains all four adult MHC isoforms and shows plasticity during the denervation-reinnervation process. Triple immunofluorescent staining of MHC-1, MHC-2a and MHC-2b with primary antibodies BA-D5 (isotype IgG2b), SC-71 (isotype IgG1) and BF-F3 (isotype IgM) and with three fluorophore-labeled isotype-specific secondary antibodies displays different muscle fiber types in a merged image of red, green and blue channels, each in its own color. Immunoperoxidase staining with primary antibody 6H1 directed against MHC-2x can be additionally applied on the same tissue section to facilitate the identification of muscle fibers containing MHC-2x. Triple staining can also be used in combination with other staining procedures to derive more information about the number of capillaries or the oxidative potential of muscle fiber types. Simultaneous visualization of multiple fiber types in a single merged image enables economical use of muscle samples and provides simple and rapid identification of all fiber types that are present in rat limb muscles.

Energy metabolism of fibre types within fascicles of human muscles

In human latissimus dorsi muscle a preponderance of type 2b fibres in the first fascicle layer and of type 1 fibres in the second layer was found. NADH-dehydrogenase (NADH) andα-glycero-phosphate dehydrogenase (GPDH) which were measured histophotometrically in type 1, 2a, and 2b fibres showed either extreme or only partial overlapping regarding the activity of metabolic enzymes. In different fascicle layers the average activity of both enzymes did not differ significantly among the fibres of the same type, neither did the NADH and GPDH activity of type 2a and 2b.

Changes in the Capillarity of the Rat Extensor Digitorum Longus Muscle 4 Weeks after Nerve Injury Studied by 2D Measurement Methods

We have previously shown by 3D study that 2 weeks after nerve injury there was no change in the length of capillaries per muscle fibre length in rat extensor digitorum longus muscle (EDL). The primary goal of the present 2D study was to determine the capillarity of rat EDL 4 weeks after various modes of nerve injury. Additionally, we wished to calculate the same capillary/fibre parameters that were used in our 3D stereological study. EDL muscles derived from denervated (4 weeks after nerve injury), re-innervated (4 weeks after two successive nerve crushes) and age-matched controls from the beginning (CON-1) and the end (CON-2) of the experiment were analysed in two ways. Global indices of capillarity, such as capillary density (CD) and capillary/fibre (C/F) ratio, were determined by automatic analysis, local indices as the number (CAF) and the length of capillaries around individual muscle fibres (Lcap) in relation to muscle fibre size were estimated manually by tracing the muscle fibre outlines and the transversally and longitudinally cut segments of capillaries seen in 5-µm-thin muscle cross sections. Four weeks after both types of nerve injury, CD increased in comparison to the CON-2 group (p < 0.001) due to atrophied muscle fibres in denervated muscles and probably proliferation of capillaries in re-innervated ones. Higher C/F, CAF (both p < 0.001) and Lcap (p < 0.01) in re-innervated than denervated EDL confirmed this assumption. Calculated capillary/fibre parameters were comparable to our previous 3D study, which strengthens the practical value to the adapted 2D method used in this study.