Recently, we evaluated capillary indices without discrimination by fiber type in rat extensor digitorum longus muscle (EDL) 4 weeks after nerve cut (NC), after double nerve crush (double NCR) and in ...two controls, from the start (CON-1) and the end (CON-2) of the experiment. In the present study, we determined the capillary indices related to specific myosin heavy chain (MyHC) fiber types. Fiber-type composition and local capillarity were assessed from a single, composite, multicolor image, where different MyHC-fiber types and capillaries were shown simultaneously. Applying local capillary indices the number of capillaries around fiber (CAF) and the CAF scaled to fiber perimeter (CAF/FP), to specific MyHC-fiber types, we found changes relevant to neuro-muscular studies. In the NC group, only type-2x fibers had a significantly lower CAF, and in the double NCR group, only type-2a fibers had a higher CAF in comparison with both controls. Both types of nerve injury elicited two responses: a coupled regulation of fiber size and capillarity in the oxidative, type 2a fibers and a capillarity independent regulation of fiber size in the glycolytic type-2b fibers. All subtypes of type-2 fibers had a better capillary supply (higher CAF/FP) in the NC and double NCR than in CON-2. The highest improvement was observed in type-2b fibers; this change was mirrored in an oxidative shift only in the double NCR group. Adopting fiber-type-specific capillary indices improves data analysis of rat EDL muscle samples.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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)
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background: In the last years, an important development in the diagnostic procedures and treatment of venous disease of the lower limbs has been achieved. Due to the lack of anatomic names for some ...of the clinically relevant veins, different clinical eponyms were introduced. Recently, an international consensus on the nomenclature of leg veins was accepted to simplify the communication and to minimize the risk of mistreatment. Authors of this article reviewed new literature on he veins of the lower limbs and compared the new terminology with the old one. Conclusions: Through the systematic review of superficial, deep and perforant veins, authors point out the altered and newly introduced names of the veins. The majority of changes refer to the names of superficial veins and perforators. The use of clinical eponyms is discouraged and descriptive terms designating location of the veins are preferred instead. The nomenclature of the deep veins has not been changed much. The term deep femoral vein, already in use in clinical practice was introduced, whereas the use of clinical term superficial femoral vein was discouraged. The most important changes in vein nomenclature are summarized in the table.
We postulated that, in rat extensor digitorum longus muscle (EDL), the length of capillaries per fibre surface area (Lcap/Sfib) and per fibre volume (Lcap/Vfib) could reflect fibre-type ...transformations accompanied by changes in oxidative metabolic profile and selective fibre-type atrophy. We excised rat EDL muscle 2 weeks after the sciatic nerve was cut (acute denervation; DEDL) and 4 weeks after the nerve was crushed (early reinnervation; REDL) and characterised muscle fibre-type transformation by the expression of myosin heavy-chain isoforms and by succinate dehydrogenase (SDH) and nicotinoamide adenine dinucleotide-tetrazolium reductase (NADH-TR) reactions. The numerical percentage (N/N) and area percentage (A/A) of pure and hybrid fibres and their diameter were determined, as was the A/A of SDH- and NADH-TR-positive fibres. The length of capillaries per fibre length (Lcap/Lfib), Lcap/Sfib and Lcap/Vfib were estimated in REDL and Lcap/Vfib in DEDL. In DEDL, the type 2x and 2b fibres evidently atrophied, with the N/N of type 2x fibres being lower and that of hybrid fibres higher. In REDL, the N/N of hybrid fibres was even higher, consequent to a lower N/N of type 2b fibres; however, fibre diameters approached values of the control EDL. Compared with control EDL, denervated and reinnervated muscles exhibited a higher A/A of oxidative fibres. This is probably the result of fibre-type transformation and selective fibre atrophy. We conclude that capillary length does not change during acute denervation and early reinnervation. The obtained higher values of Lcap/Sfib and Lcap/Vfib are related to changes in muscle fibre cross-sectional area.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The goal of this study was to determine the acute effects of permanent denervation on the length density of the capillary network in rat slow soleus (SOL) and fast extensor digitorum longus (EDL) ...muscles and the effect of short-lasting reinnervation in slow muscle only. Denervation was performed by cutting the sciatic nerve. Both muscles were excised 2 weeks later. Reinnervation was studied 4 weeks after nerve crush in SOL muscle only. Capillaries and muscle fibres were visualised by triple immunofluorescent staining with antibodies against CD31 and laminin and with fluorescein-labelled Griffonia (Bandeira) simplicifolia lectin. A recently developed stereological approach allowing the estimation of the length of capillaries adjacent to each individual fibre (Lcap/Lfib) was employed. Three-dimensional virtual test grids were applied to stacks of optical images captured with a confocal microscope and their intersections with capillaries and muscle fibres were counted. Interrelationships among capillaries and muscle fibres were demonstrated with maximum intensity projection of the acquired stacks of optical images. The course of capillaries in EDL seemed to be parallel to the fibre axes, whereas in SOL, their preferential direction deviated from the fibre axes and formed more cross-connections among neighbouring capillaries. Lcap/Lfib was clearly reduced in denervated SOL but remained unchanged in EDL, although the muscle fibres significantly atrophied in both muscle types. When soleus muscle was reinnervated, capillary length per unit fibre length was completely restored. The physiological background for the different responses of the capillary network in slow and fast muscle is discussed.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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 volume was larger in EDL muscle. There was no difference in the length of capillaries per unit fibre surface area between the two muscles. Oxidative and glycolytic fibres differ in the length of capillaries per unit fibre surface area (Lcap/Sfib). This parameter probably reflects the oxidative capacity of muscle fibres. In conclusion, capillary supply is evidently well adapted to different muscle fibre types; consequently, an average capillary supply of a heterogeneous muscle depends on the muscle composition. The estimated mean values blur some intrinsic differences.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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