Microstructural analysis of deformation-induced hypoxic damage in skeletal muscle

Deep pressure ulcers are caused by sustained mechanical loading and involve skeletal muscle tissue injury. The exact underlying mechanisms are unclear, and the prevalence is high. Our hypothesis is that the aetiology is dominated by cellular deformation (Bouten et al. in Ann Biomed Eng 29:153–63, 2001; Breuls et al. in Ann Biomed Eng 31:1357–364, 2003; Stekelenburg et al. in J App Physiol 100(6):1946–954, 2006) and deformation-induced ischaemia. The experimental observation that mechanical compression induced a pattern of interspersed healthy and dead cells in skeletal muscle (Stekelenburg et al. in J App Physiol 100(6):1946–954, 2006) strongly suggests to take into account the muscle microstructure in studying damage development. The present paper describes a computational model for deformation-induced hypoxic damage in skeletal muscle tissue. Dead cells stop consuming oxygen and are assumed to decrease in stiffness due to loss of structure. The questions addressed are if these two consequences of cell death influence the development of cell injury in the remaining cells. The results show that weakening of dead cells indeed affects the damage accumulation in other cells. Further, the fact that cells stop consuming oxygen after they have died, delays cell death of other cells

Muscle Fiber Viability, a Novel Method for the Fast Detection of Ischemic Muscle Injury in Rats

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury

Changes in intracellular calcium during compression of C2C12 myotubes

In recent years, damage directly due to tissue deformation has gained interest in deep pressure ulcer aetiology research. It has been shown that deformation causes muscle cell damage, though the pathway is unclear. Mechanically induced skeletal muscle damage has often been associated with an increased intracellular Ca2+ concentration, e.g. in eccentric exercise (Allen et al., J Physiol 567(3):723–735, 2005). Therefore, the hypothesis was that compression leads to membrane disruptions, causing an increased Ca2+-influx, eventually leading to Ca2+ overload and cell death. Monolayers of differentiated C2C12 myocytes, stained with a calcium-sensitive probe (fluo-4), were individually subjected to compression while monitoring the fluo-4 intensity. Approximately 50% of the cells exhibited brief calcium transients in response to compression, while the rest did not react. However, all cells demonstrated a prolonged Ca2+ up-regulation upon necrosis, which induced similar up-regulations in some of the surrounding cells. Population heterogeneity is a possible explanation for the observed differences in response, and it might also become important in tissue damage development. It did not become clear however whether Ca2+-influxes were the initiators of damag