Validation of an active shape model-based semi-automated segmentation algorithm for the analysis of thigh muscle and adipose tissue cross-sectional areas.

OBJECTIVE: To validate a semi-automated method for thigh muscle and adipose tissue cross-sectional area (CSA) segmentation from MRI. MATERIALS AND METHODS: An active shape model (ASM) was trained using 113 MRI CSAs from the Osteoarthritis Initiative (OAI) and combined with an active contour model and thresholding-based post-processing steps. This method was applied to 20 other MRIs from the OAI and to baseline and follow-up MRIs from a 12-week lower-limb strengthening or endurance training intervention (n = 35 females). The agreement of semi-automated vs. previous manual segmentation was assessed using the Dice similarity coefficient and Bland-Altman analyses. Longitudinal changes observed in the training intervention were compared between semi-automated and manual segmentations. RESULTS: High agreement was observed between manual and semi-automated segmentations for subcutaneous fat, quadriceps and hamstring CSAs. With strength training, both the semi-automated and manual segmentation method detected a significant reduction in adipose tissue CSA and a significant gain in quadriceps, hamstring and adductor CSAs. With endurance training, a significant reduction in adipose tissue CSAs was observed with both methods. CONCLUSION: The semi-automated approach showed high agreement with manual segmentation of thigh muscle and adipose tissue CSAs and showed longitudinal training effects similar to that observed using manual segmentation.

Extracorporeal Shock Wave Therapy Accelerates Regeneration After Acute Skeletal Muscle Injury.

BACKGROUND: Muscle injuries are among the most common sports-related lesions in athletes; however, optimal treatment remains obscure. Extracorporeal shock wave therapy (ESWT) may be a promising approach in this context, because it has gained increasing importance in tissue regeneration in various medical fields. HYPOTHESIS: ESWT stimulates and accelerates regenerative processes of acute muscle injuries. STUDY DESIGN: Controlled laboratory study. METHODS: Adult Sprague-Dawley rats were divided into 4 experimental groups (2 ESWT+ groups and 2 ESWT- groups) as well as an uninjured control group (n ≥ 6 in each group). An acute cardiotoxin-induced injury was set into the quadriceps femoris muscle of rats in the experimental groups. A single ESWT session was administered to injured muscles of the ESWT+ groups 1 day after injury, whereas ESWT- groups received no further treatment. At 4 and 7 days after injury, 1 each of the ESWT+ and ESWT- groups was euthanized. Regenerating lesions were excised and analyzed by histomorphometry and immunohistochemistry to assess fiber size, myonuclear content, and recruitment of satellite cells. RESULTS: The size and myonuclear content of regenerating fibers in ESWT+ muscle was significantly increased compared with ESWT- muscle fibers at both 4 and 7 days after injury. Similarly, at both time points, ESWT+ muscles exhibited significantly higher contents of pax7-positive satellite cells, mitotically active H3P+ cells, and, of cells expressing the myogenic regulatory factors, myoD and myogenin, indicating enhanced proliferation and differentiation rates of satellite cells after ESWT. Mitotic activity at 4 days after injury was doubled in ESWT+ compared with ESWT- muscles. CONCLUSION: ESWT stimulates regeneration of skeletal muscle tissue and accelerates repair processes. CLINICAL RELEVANCE: We provide evidence for accelerated regeneration of damaged skeletal muscle after ESWT. Although further studies are necessary, our findings support the view that ESWT is an effective method to improve muscle healing, with special relevance to sports injuries.

First application of a protein-based approach for time since death estimation.

Awareness of postmortem degradation processes in a human body is fundamental to develop methods for forensic time since death estimation (TDE). Currently, applied approaches are all more or less limited to certain postmortem phases, or have restrictions on behalf of circumstances of death. Novel techniques, however, rarely exceed basic research phases due to various reasons. We report the first application of a novel method, based on decay of muscle proteins, in a recent case of murder-suicide, where other TDE methods failed to obtain data. We detected considerably different protein degradation profiles in both individuals involved and compared the data to our presently available database. We obtained statistical evidence for un-simultaneous death and therefore received valuable information to trace the progression of events based on protein degradation. Although we could not sensibly convert the data to respective times of death, this case highlights the potential for future application and elucidates the necessary further steps to develop a viable TDE method.

Postmortem muscle protein degradation in humans as a tool for PMI delimitation.

Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans.

Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death.

Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0-36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies.

The single nucleotide polymorphism Gly482Ser in the PGC-1α gene impairs exercise-induced slow-twitch muscle fibre transformation in humans.

PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α) is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP) in the PGC-1α gene affects insulin sensitivity, blood lipid metabolism and binding to myocyte enhancer factor 2 (MEF2). Individuals carrying this SNP were shown to have a reduced cardiorespiratory fitness and a higher risk to develop type 2 diabetes. Here, we investigated the responses of untrained men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 x 60 min/week, heart rate at 70-90% VO2peak). Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a training-induced increase in content of slow contracting oxidative fibres. Capillary supply, mitochondrial density, mitochondrial enzyme activities and intramyocellular lipid content increased similarly in both groups. These results indicate that the impaired binding of MEF2 to PGC-1α in humans with this SNP impedes exercise-induced fast-to-slow muscle fibre transformation.

Temperature-dependent modification of muscle precursor cell behaviour is an underlying reason for lasting effects on muscle cellularity and body growth of teleost fish.

Temperature is an important factor influencing teleost muscle growth, including a lasting ('imprinted') influence of embryonic thermal experience throughout all further life. However, little is known about the cellular processes behind this phenomenon. The study reported here used digital morphometry and immunolabelling for Pax7, myogenin and H3P to quantitatively examine the effects of thermal history on muscle precursor cell (MPC) behaviour and muscle growth in pearlfish (Rutilus meidingeri) until the adult stage. Fish were reared at three different temperatures (8.5, 13 and 16°C) until hatching and subsequently kept under the same (ambient) thermal conditions. Cellularity data were combined with a quantitative analysis of Pax7+ MPCs including those that were mitotically active (Pax7+/H3P+) or had entered differentiation (Pax7+/myogenin+). The results demonstrate that at hatching, body lengths, fast and slow muscle cross-sectional areas and fast fibre numbers are lower in fish reared at 8.5 and 13°C than at 16°C. During the larval period, this situation changes in the 13°C-fish, so that these fish are finally the largest. The observed effects can be related to divergent cellular mechanisms at the MPC level that are initiated in the embryo during the imprinting period. Embryos of 16°C-fish have reduced MPC proliferation but increased differentiation, and thus give rise to larger hatchlings. However, their limited MPC reserves finally lead to smaller adults. By contrast, embryos of 13°C-fish and, to a lesser extent, 8.5°-fish, show enhanced MPC proliferation but reduced differentiation, thus leading to smaller hatchlings but allowing for a larger MPC pool that can be used for enhanced post-hatching growth, finally resulting in larger adults.

Size- and age-dependent changes in adductor muscle swimming physiology of the scallop Aequipecten opercularis.

The decline of cellular and especially mitochondrial functions with age is, among other causes, held responsible for a decrease in physiological fitness and exercise capacity during lifetime. We investigated size- and age-related changes in the physiology of exercising specimens of the short lived swimming scallop Aequipecten opercularis (maximum life span 8 to 10 years) from the Isle of Man, UK. A. opercularis swim mainly to avoid predators, and a decrease in swimming abilities would increase the risk of capture and lower the rates of survival. Bigger (older) individuals were found to have lower mitochondrial volume density and aerobic capacities (citrate synthase activity and adenylates) as well as less anaerobic capacity deduced from the amount of glycogen stored in muscle tissue. Changes in redox potential, tissue pH and the loss of glutathione in the swimming muscle during the exercise were more pronounced in young compared to older individuals. This indicates that older individuals can more effectively stabilize cellular homeostasis during repeated exercise than younger animals but with a possible fitness cost as the change in physiology with age and size might result in a changed escape response behaviour towards predators.

Cellularity changes in developing red and white fish muscle at different temperatures: simulating natural environmental conditions for a temperate freshwater cyprinid.

Muscle cellularity patterns in teleost fish have normally been investigated using animals reared under constant temperature conditions. In the present study, Danube bleak (Chalcalburnus chalcoides mento) were reared under two different rising temperature regimes (cold, 12-16 degrees C; warm, 18-20 degrees C) designed to mimic the natural conditions experienced by the fish in temperate freshwater environments. Samples were taken from both groups of animals at intervals during their development. Transverse sections at the level of the anal vent were examined using light and electron microscopy, histochemistry and immunohistochemistry techniques. Total cross-sectional area of red and white muscle, as well as fibre numbers and fibre cross-sectional areas of one epaxial quadrant per specimen, were measured. Analysis of fibre numbers and sizes indicated that white and red myotomal muscles each develop in a different manner. In white muscle, the initial growth phase is dominated by fibre hypertrophy, while the later larval growth phase also includes significant hyperplasia. Red muscle growth is mainly due to hypertrophy within the studied developmental period. The temperature regimes applied in the present study may modify the mechanisms of muscle growth in different ways. For white muscle, pre-hatching hyperplasia (i.e. proliferation of somitic white fibre precursor cells) is reduced under the cold regime whereas post-hatching hyperplasia is not. The inverse is true for white fibre hypertrophy. A similar situation is seen with red muscle except that post-hatching hyperplasia is low and refractory to temperature. Rates of increase in relative amount of red muscle appear to depend not only upon species and temperature but also upon whether the fish have been reared under changing or constant thermal regimes. These findings are discussed in relation to 'landmark' events of early ontogeny (hatching, onset of swimming, start of exogeneous feeding) and to their implications for future accurate interpretation of temperature effects on teleost developmental biology and functional ecology.