Horizontal deformation of skeletal muscle thick sections visualised by confocal microscopy.

Certain morphological parameters of the skeletal muscle tissue can be better understood via 3D considerations. Fluorescent confocal microscopy of thick tissue sections is a well-established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, thick tissue sections are prone to deformations which may significantly influence some stereological and morphometric results like muscle fibre diameter and capillary length, but not dimensionless parameters like object number and Euler-Poincaré characteristics. To better understand this phenomenon, we studied the horizontal deformation of thick (100 µm) transverse skeletal muscle sections, by comparing the muscle fibre diameters measured on thick sections to muscle fibre diameters measured on thin (10 µm) sections of the same sample. Diameter changes were further correlated with shrinkage in the Z direction (axial shrinkage) and deviation of the muscle fibre preferential axis from the Z-axis. We showed that the thick sections dilated in horizontal and shrunk in Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in the Z direction. The latter was more pronounced in transversely than obliquely cut tissue sections. The results emphasise that even when shrinkage in the Z direction can be corrected using calibration, it is important to optimise histological protocols to minimise the Z-axis collapse that could cause horizontal dilation. LAY DESCRIPTION: In skeletal muscle research, 3D analysis is especially important for studying the microvasculature. Laser scanning confocal microscopy of skeletal muscle thick tissue sections is a well-established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, such sections are prone to deformations which may significantly influence the study results. To better understand this phenomenon, we studied the horizontal deformation of thick transverse skeletal muscle sections. We compared the average muscle fibre diameters measured on thick skeletal muscle sections, thin fixed skeletal muscle sections and immunohistochemically stained thin skeletal muscle sections with the muscle fibre diameters measured on thin native skeletal muscle sections of the same sample, with the latter condition serving as the standard diameters (ie the control condition). We further studied the association among muscle fibre diameter changes, shrinkage of the thick skeletal muscle sections in the Z direction and their sectioning angle. We showed that the thick skeletal muscle sections dilated in the horizontal direction and shrunk in the Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in Z direction. The shrinkage in the Z direction was more pronounced in transversely than obliquely cut tissue sections. These results emphasise that even when shrinkage in the Z direction can be corrected using Z-axis calibration, it is very important to optimise histological protocols to minimise the Z-axis collapse that could cause horizontal dilation in order to enhance the integrity of study results.

3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice.

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

A novel method for evaluation of capillarity in human skeletal muscles from confocal 3D images.

A well developed capillary bed is essential for proper function of skeletal muscles. We present for the first time a triple immunofluorescent method suitable for staining capillaries and muscle fibre outlines in thick sections of human skeletal muscle, applying antibodies against collagen IV (in red) and F8 (in green) as well as Ulex europaeus lectin, visualized in green fluorescence. Further, we present possibilities for quantitative evaluation of the capillary network which implies the length of capillaries per unit volume of muscle tissue (Lcap/Vmuscle) and the length of capillaries supplying individual muscle fibres per unit fibre length (Lcap/Lfib), per surface area (Lcap/Sfib) and per volume (Lcap/Vfib) as well as the course of capillaries in the muscle. The latter can be described by the tortuosity, orientation and mean capillary length. To get reasonable results we met the following requirements: i) high quality thick tissue sections, from which 3D image data were acquired; ii) immunofluorescent methods suitable for confocal microscopy; iii) penetration of the fluorescent dyes throughout the tissue section; iv) proper 3D image analysis methods for performing reliable measurements and v) control over relevant tissue deformations. The developed methodology is illustrated by results obtained from autopsy or biopsy samples of three human muscles, i.e. vastus lateralis, multifidus and masseter muscle that exhibit differences in genetic background, innervation, tasks and functional activity.

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.

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.

Histochemical and immunohistochemical profile of human and rat ocular medial rectus muscles.

PURPOSE: To compare the organization of human and rat ocular medial recti muscles (MR). METHODS: The cryosections of human and rat MR were processed for myofibrillar ATPase (mATPase), succinate dehydrogenase and glycerol-3-phosphate dehydrogenase. To reveal myosin heavy chain (MyHC) isoforms, specific monoclonal antibodies against MyHC-1/beta- slow, alpha-cardiac (-alpha), -2a, -2x, -2b, -extraocular (eom), -embryonic (-emb) and -neonatal (-neo) were applied. The MyHC gene expression was studied by in situ hybridization in human muscle. RESULTS: The muscle fibers were arranged in two distinct layers in both species. In the orbital layer most fibers were highly oxidative and expressed fast MyHC isoforms, whereas slow and oxidative fibers expressed MyHC-1 and -alpha, some of them also MyHC-2a, -2x, -eom, very rarely -emb, and -neo. In the global layer, slow fibers with very low oxidative and glycolytic activity and three types of fast fibers, glycolytic, oxidative and oxidative-glycolytic, could be distinguished. The slow medium-sized fibers with mATPase activity stable at pH 4.4 expressed mostly MyHC-1 and -alpha in rat, while in humans they co-expressed MyHC-1 with -2b, -2x, -eom, and -neo. In both species, the fast fibers showed variable mATPase activity after preincubation at pH 9.4, and co-expressed various combinations of MyHC-2b, -2x, -2a and -eom but not -emb and -neo. MyHC-2b expressing fibers were larger and glycolytic, while MyHC-2a expressing fibers were smaller and highly oxidative in both species. To our knowledge, the present study is the first that demonstrated the expression of MyHC-2b in any of human skeletal muscles. Though the expression of MyHC genes did not correlate with the immunohistochemical profile of fibers in human MR, the expression of MyHC-2b gene was undoubtedly confirmed. CONCLUSIONS: Rat MR represent a good model that can be applied to study human MR in experiment or disease, however certain differences are to be expected due to specific oculomotor demands in humans.

Capillary Network Morphometry of Pig Soleus Muscle Significantly Changes in 24 Hours After Death.

Capillary network characteristics are invaluable for diagnostics of muscle diseases. Biopsy material is limited in size and mostly not accessible for intensive research. Therefore, especially in human tissue, studies are performed on autopsy material. To approach the problem whether it is reliable to deduce hypotheses from autopsy material to explain physiological and pathological processes, we studied capillarity in pig soleus muscle 1 and 24 hr after death. Capillaries and muscle fibers were immunofluorescently marked, and images were acquired with a confocal microscope. Characteristics of the capillary network were estimated by image analysis methods using several plugins of the Ellipse program. Twenty-four hours after death, the measured characteristics of the capillary network differ by up to 50% when compared with samples excised 1 hr after death. Muscle fiber diameter, the measured capillary length, and tortuosity were reduced, and capillary network became more anisotropic. The main postmortem change that affects capillaries is evidently geometric deformation of muscle tissue. In conclusion, when comparing results from biopsy samples with those from autopsy samples, the effect of postmortem changes on the measured parameters must be carefully considered.

Frequency of M-cadherin-stained satellite cells declines in human muscles during aging.

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30- microm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 +/- 2.3 years) and six old (70.8 +/- 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group (p < 0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.

Persistent expression of the alpha1S-dihydropyridine receptor in aged human skeletal muscle: implications for the excitation-contraction uncoupling hypothesis of sarcopenia.

Previous studies on aged animal muscle suggest that excitation-contraction uncoupling and fibre transitions play a central role in sarcopenia, the progressive loss and functional decline of aging skeletal muscle fibres. A drastic reduction in the voltage-sensing alpha1S-subunit of the transverse-tubular dihydropyridine receptor is believed to be the underlying cause for a decreased transmission of the surface depolarization signal into Ca2+-mediated muscle contraction. Extending these studies to human muscle, we asked whether potential changes in the relative expression of the voltage sensor occur in senescent human fibres. For internal standardization and as markers of potential fast-to-slow transitions, the fast isoforms of the Ca2+-binding element calsequestrin and the myosin heavy chain were employed. Besides small inter-individual variations in expression levels, the microsomal immunoblot analysis of vastus lateralis autopsy specimens from male humans aged 18 to 82 years of age showed no major changes in the relative abundance of the alpha1S- and alpha2-dihydropyridine receptor, fast calsequestrin and the slow/fast myosin heavy chains. The oligomeric status of the alpha1S-dihydropyridine receptor was unaltered in aged fibres. Biochemical assays revealed no significant modifications in Ca2+-ATPase activity and a reduced Ca2+-binding capacity in aged human muscle preparations. Although impairments of other Ca2+-regulatory proteins and/or disturbed protein-protein interactions might be involved in the pathophysiological changes of sarcopenia, dihydropyridine receptor and calsequestrin expression seem to be preserved during the aging process of human skeletal muscle fibres. Hence, the supposition that excitation-contraction uncoupling is responsible for sarcopenia can not be transferred from animal models to senescent human muscle without modifications.

Early effects of denervation on Ca(2+)-handling proteins in skeletal muscle.

The adaptive response of skeletal muscle fibres depends on a variety of biological factors including loading conditions and neuromuscular activity. An extreme type of atrophy-inducing change in contractile activity is represented by the physical disconnection between the motor nerve and its respective fibre unit. Since fibre type alterations have a striking effect on the Ca(2+)-regulatory apparatus, we have investigated the fate of a key Ca(2+)-pump and essential Ca(2+)-binding proteins in extensor digitorum longus specimens two weeks after nerve crush or complete denervation. In contrast to increased levels of sarcalumenin, immunoblotting revealed that the expression of the fast SERCA1 Ca(2+)-ATPase isoform is drastically decreased and fast calsequestrin is slightly reduced. Analysis of myosin heavy chain isoforms agreed with this result and showed a fast-to-slow fibre type shifting process following denervation. Hence, changes in muscle activity appear to have a profound effect on the abundance and isoform expression pattern of Ca(2+)-handling elements.

Human masseter muscle fibers from the elderly express less neonatal Myosin than those of young adults.

In contrast to limb muscles where neonatal myosin (MyHC-neo) is present only shortly after birth, adult masseter muscles contain a substantial portion of MyHC-neo, which is coexpressed with mature MyHC isoforms. Changes in the numerical and area proportion of muscle fibers containing MyHC-neo in masseter muscle with aging could be expected, based on previously reported findings that (i) developmental MyHC-containing muscle fibers exhibit lower shortening velocities compared to fibers with exclusively fast MyHC isoforms and (ii) transformation toward faster phenotype occurs in elderly compared to young masseter muscle. In this study, we detected MyHC isoforms in the anterior superficial part of the human masseter muscle in a sufficiently large sample of young, middle-aged, and elderly subjects to reveal age-related changes in the coexpression of MyHC-neo with adult MyHC isoforms. MyHC isoforms were visualized with immunoperoxidase method and the results were presented by (i) the area proportion of fibers containing particular MyHC isoforms and (ii) the numerical proportion of fiber types defined by MyHC-1, -2a, -2x, and -neonatal isoform expression from a successive transverse sections. We found a lower numerical and area proportion of fibers expressing MyHC-neo as well as a lower area proportion of fibers containing MyHC-1 in elderly than in young subjects. We conclude that the diminished expression of MyHC-neo with age could point to a lower regeneration capacity of masseter muscle in the elderly.

Localization and properties of cholinesterases in the common prawn (Palaemon serratus): A kinetic-histochemical study.

Due to the diversity in biochemical properties and tissue distribution of cholinesterase (ChE) enzymes, a characterization should be performed before they are used as biomarkers for monitoring pesticide contamination. In this study we investigate the distribution of ChEs and their kinetic properties in diverse tissues of the common prawn Palaemon serratus. The results concerning the histochemical localization of ChEs suggest that the highest amount of ChE activity occurs in prawn eyes, followed by the brain, gills, and digestive tract. Negligible staining was observed in the muscle. We describe the kinetic properties of ChEs in eyes, gills, and hepatopancreas, investigating their substrate preferences with different thiocholine esters and their sensitivity to inhibition with selective inhibitors. The results suggest that the studied enzymes are ChEs and not nonspecific esterases, due to their apparent affinity for choline esters, with a distinct preference for the substrate acetylthiocholine, and their high sensitivity to inhibition by eserine sulfate. P. serratus eyes can be considered the best tissue for recovery of ChE enzyme: this tissue is easy to isolate, it expresses high levels of ChE, and the enzyme shows properties of a vertebrate AChE.

Adaptation of muscle fibre types and capillary network to acute denervation and shortlasting reinnervation.

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.

Nerve injury affects the capillary supply in rat slow and fast muscles differently.

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.

Age dependence of the human skeletal muscle stem cell in forming muscle tissue.

Human skeletal muscle stem cells from healthy donors aged 2-82 years (n = 13) and from three children suffering from Duchenne Muscular Dystrophy (DMD) were implanted into soleus muscles of immunoincompetent mice and were also expanded in vitro until senescence. Growth of implanted cells was quantified by structural features and by the amount of human DNA present in a muscle. Proliferative capacity in vitro and in vivo was inversely related to age of the donor. In vitro, a decline of about two mean population doublings (MPDs) per 10 years of donor's age was observed. Muscle stem cells from DMD children were prematurely aged. In general, cell preparations with low or decreasing content in desmin-positive cells produced more MPDs than age-matched high-desmin preparations and upon implantation more human DNA and more nonmyogenic than myogenic tissue. Thus, a "Desmin Factor" was derived which predicts "quality" of the human muscle tissue growing in vivo. This factor may serve as a prognostic tool.