ABSTRACT
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.
Subject(s)
Capillaries/ultrastructure , Microscopy, Confocal/methods , Muscle Fibers, Skeletal/ultrastructure , Muscle, Skeletal/blood supply , Muscle, Skeletal/cytology , Animals , Capillaries/anatomy & histology , Image Processing, Computer-Assisted , Imaging, Three-Dimensional/methods , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Capillaries/cytology , Fluorescent Dyes/chemistry , Muscle Fibers, Skeletal/ultrastructure , Muscle, Skeletal/cytology , Staining and Labeling/methods , Animals , Capillaries/chemistry , Coloring Agents/chemistry , In Vitro Techniques , Muscle Fibers, Skeletal/chemistry , Muscle, Skeletal/blood supply , Muscle, Skeletal/chemistry , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Muscle Fibers, Skeletal/cytology , Muscle, Skeletal/blood supply , Muscle, Skeletal/cytology , Animals , Capillaries/anatomy & histology , Image Processing, Computer-Assisted , Male , Microscopy, Confocal , Rats , Rats, WistarABSTRACT
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) and alpha-glycerophosphate 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.
