A theoretical model to study the effects of cellular stiffening on the damage evolution in deep tissue injury

Pressure induced deep tissue injury (DTI) is a severe form of pressure ulcers that is hard to detect in early stages and difficult to prevent and treat. High prevalence figures are partly due to a lack of understanding of pathological pathways involved in DTI. The aim of this study was to investigate, whether changes in material properties of damaged tissue can play a role in DTI aetiology. A numerical model was developed based on muscle microstructure and tissue engineering experiments. A time dependent damage law was proposed and stiffening of dead cells incorporated. The results obtained in the microstructural investigations were used to include the stiffening information in a pre-existing macroscopic model based on animal experiments, which correlated strains to tissue damage measured in the tibialis anterior muscle in rat limbs. With the modelling approach employed in this paper, the damaged area in the rat limb models increased up to 1.65-fold and the rate of damage progression was up to 2.1 times higher in microstructural simulations when stiffening was included.     

Strain-time cell-death threshold for skeletal muscle in a tissue-engineered model system for deep tissue injury

Deep tissue injury (DTI) is a severe pressure ulcer that results from sustained deformation of muscle tissue overlying bony prominences. In order to understand the etiology of DTI, it is essential to determine the tolerance of muscle cells to large mechanical strains. In this study, a new experimental method of determining the time-dependent critical compressive strains for necrotic cell death (E(zz)(c)(t)) in a planar tissue-engineered construct under static loading was developed. A half-spherical indentor is used to induce a non-uniform, concentric distribution of strains in the construct, and E(zz)(c)(t) is calculated from the radius of the damage region in the construct versus time. The method was employed to obtain E(zz)(c)(t) for bio-artificial muscles (BAMs) cultured from C2C12 murine cells, as a model system for DTI. Specifically, propidium iodine was used to fluorescently stain the development of necrosis in BAMs subjected to strains up to 80%. Two groups of BAMs were tested at an extracellular pH of 7.4 (n=10) and pH 6.5 (n=5). The lowest strain levels causing cell death in the BAMs were determined every 15min, during 285-min-long trials, from confocal microscopy fluorescent images of the size of the damage regions. The experimental E(zz)(c)(t) data fitted a decreasing single-step sigmoid of the Boltzmann type. Analysis of the parameters of this sigmoid function indicated a 95% likelihood that cells could tolerate engineering strains below 65% for 1h, whereas the cells could endure strains below 40% over a 285min trial period. The decrease in endurance of the cells to compressive strains occurred between 1-3h post-loading. The method developed in this paper is generic and suitable for studying E(zz)(c)(t) in virtually any planar tissue-engineered construct. The specific E(zz)(c)(t) curve obtained herein is necessary for extrapolating biological damage from muscle-strain data in biomechanical studies of pressure ulcers and DTI.     

Diffusion of water in skeletal muscle tissue is not influenced by compression in a rat model of deep tissue injury

Sustained mechanical loading of skeletal muscle may result in the development of a severe type of pressure ulcer, referred to as deep tissue injury. Recently it was shown that the diffusion of large molecules (10–150 kDa) is impaired during deformation of tissue-engineered skeletal muscle, suggesting a role for impaired diffusion in the aetiology of deep tissue injury. However, the influence of deformation on diffusion of smaller molecules on its aetiology is less clear. This motivated the present study designed to investigate the influence of deformation of skeletal muscle on the diffusion of water, which can be measured with diffusion tensor magnetic resonance imaging (MRI). It could be predicted that this approach will provide valuable information on the diffusion of small molecules. Additionally the relationship between muscle temperature and diffusion was investigated. During deformation of the tibialis anterior a decrease of the apparent diffusion coefficient (ADC) was observed (7.2±3.9%). The use of a finite element model showed that no correlation existed between the maximum shear strain and the decrease of the ADC. The ADC in the uncompressed gastrocnemius muscle decreased with 5.9±3.7%. In an additional experiment a clear correlation was obtained between the decrease of the ADC and the relative temperature change of skeletal muscle tissue as measured by MRI. Taken together, it was concluded that (1) the decreased diffusion of water was not a direct effect of tissue deformation and (2) that it is likely that the observed decreased ADC during deformation was a result of a decreased muscle temperature. The present study therefore provides evidence that diffusion of small molecules, particularly oxygen and carbon dioxide, is not impaired during deformation of skeletal muscle tissue.     

Indices of skeletal muscle damage and connective tissue breakdown following eccentric muscle contractions

 Indirect indices of exercise-induced human skeletal muscle damage and connective tissue breakdown were studied following a single bout of voluntary eccentric muscle contractions. Subjects (six female, two male), mean (SD) age 22 (2) years performed a bout of 50 maximum voluntary eccentric contractions of the knee extensors of a single leg. The eccentric exercise protocol induced muscle soreness (P < 0.05 Wilcoxon test), chronic force loss, and a decline in the 20:100 Hz percutaneous electrical myostimulation force ratio [P < 0.01, repeated measures analysis of variance (ANOVA)]. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities were elevated (P < 0.01, repeated measures ANOVA) following the bout. The mean (SD) CK and LDH levels recorded 3 days post-exercise were 2815 (4144) IU · l^–1 and 375 (198) IU · l^–1, respectively. Serum alkaline phosphatase activity showed no changes throughout the study, and a non-significant increase (P = 0.058, repeated measures ANOVA) in pyridinoline was recorded following the bout. Urinary hydroxyproline (HP) and hydroxylysine (HL) excretion, expressed in terms of creatinine (Cr) concentration, increased after exercise (P < 0.05 and P < 0.01, respectively, repeated measures ANOVA). An increased HP:Cr was recorded 2 days post-exercise and HL:Cr was increased above baseline on days 2, 5, and 9 post-exercise. This indirect evidence of exercise-induced muscle damage suggests that myofibre disruption was caused by the eccentric muscle contractions. Elevated urine concentrations of indirect indices of collagen breakdown following eccentric muscle contractions suggests an increased breakdown of connective tissue, possibly due to a localised inflammatory response. Accepted: 9 October 1996     

Tissue interactions of regenerating muscle tissue with skeletal muscle under the effect of ionizing radiation

The effect of the muscle tissue, ground into a fine chyme, to restoration of the m. gastrocnemius ability to posttraumatic regeneration after x-ray radiation in the doses 20--30 Gy, has been studied in 64 non-inbred white male rats with the body mass about 200 g. The ground muscle is a viscous mass, consisting of finest scraps of muscle fibers, pieces of sarcolemmal tubules with 2--3 nuclei and separate cells. The great destruction of the muscle stroma relieves metabolites to get out and to diffuse. As demonstrate the histological investigations carried out for 1 month, an essential part of the muscle autotransplant, put into the irradiated extremity, regenerates up to formation of differentiated muscle fibers and promotes to restore the regenerative activity in the irradiated m. gastrocnemius.     

Reduction of skeletal muscle injury in composite tissue allotransplantation by heat stress preconditioning

Ischemia-reperfusion injury is a dominant factor limiting tissue survival in any microsurgical tissue transplantation, a fact that also applies to allogeneic hand transplantation. The clinical experience of the 12 human hand transplantations indicates that shorter ischemia times result in reduced tissue damage and, ultimately, in better hand function. Heat stress preconditioning and the accompanying up-regulation of the heat shock protein 72 have been shown to reduce the ischemia-reperfusion injury following ischemia of various organs, including organ transplantation. The aim of this study was to reduce the ischemia-reperfusion injury in a model of composite tissue allotransplantation. Allogeneic hind limb transplantations were performed from Lewis (donor) to Brown-Norway rats. Donor rats in group A (n = 10) received a prior heat shock whereas rats in group B (n = 10) did not receive any prior heat shock. Group C served as a control group without transplantation. The transplantations were performed 24 hours after the heat shock, at which time the heat shock protein 72 was shown to be up-regulated. The outcome was evaluated 24 hours after transplantation by nitroblue tetrazolium staining and wet-to-dry weight ratio of muscle slices (anterior tibial muscle). The nitroblue tetrazolium staining showed a significant reduction of necrotic muscle in group A (prior heat shock) (p = 0.005). The wet-to-dry ratio was significantly reduced in group A (prior heat shock), indicating less muscle edema and less tissue damage (p = 0.05). Heat shock preconditioning 24 hours before an ischemic event leads to an up-regulation of heat shock protein 72 in muscle and to a tissue protection reducing ischemia-reperfusion injury in composite tissue transplantation.     

Serum and urinary markers of skeletal muscle tissue damage after weight lifting exercise

The purpose of this study was to determine whether high intensity weight lifting exercise produces elevations of urinary 3-methylhistidine (3-MH), serum creatine kinase activity (CK), and serum myoglobin concentration (MY), and whether trained weight lifters differed in such responses when compared to a group of untrained subjects. Ten experienced male weight lifters (EWL) and seven untrained male subjects (IWL) performed three sets of six weight lifting exercises at 70%-80% of 1 RM. All subjects consumed a meat-free diet. The 3-MH:creatinine (3-MH:CR) values decreased 24 h and 48 h following exercise (P less than 0.05). The 12-h and 24-h postexercise CK response and the 12-h postexercise MY response increased for both EWL and IWL (P less than 0.05). However, EWL had a lower 24-h postexercise CK response and lower 12-h and 24-h postexercise MY responses compared to IWL (P less than 0.05). Within 48 h following weight lifting exercise, skeletal muscle protein degradation (as assessed by 3-MH:CR values) decreased regardless of prior training experience whereas skeletal muscle tissue damage (as assessed by CK and MY responses) increased. However, prior weight lifting training appeared to diminish the extent of muscle tissue damage.     

Reaction of intact skeletal muscle on the transplantation of minced muscle tissue

The experiments on rats have revealed that grafting of minced muscle tissue to unaffected gastrochemical muscle of the same animal leads to active regeneration in the graft accompanied by the formation of myogenic elements. In the zone of unaffected muscle contact with the graft the plastic state of muscle tissue was observed: sarcoplasm basophily of muscle fibers, groups and chains of round nuclei in them, the presence of satellite cells, ultrastructural changes, indicative of metabolic activity of the muscle fibers. With the resorption of the graft, these phenomena in the unaffected muscle gradually disappeared.     

The effects of estrogen on indices of skeletal muscle tissue damage after eccentric exercise in postmenopausal women

This study examined if estrogen (E) usage (in the form of hormone replacement therapy [HRT]) has a protective effect on skeletal muscle damage in postmenopausal women. Nine postmenopausal women (age 55.2 +/- 9.9 [mean +/- SD]) performed two exercise sessions at 70% of their maximal heart rate on HRT (E-HI) and without HRT (E-LO; following a 28-45 day HRT washout). All subjects followed a condition order of E-HI then E-LO with at least 42 days between exercise sessions. Serum creatine kinase (CK), perceived delayed onset muscle soreness (DOMS), and maximal quadriceps isometric force (MIF) were taken pre-exercise, 24, 48 and 72-hr post exercise. E-HI and E-LO conditions produced a rise in CK (p < 0.001) after exercise; but CK after E-HI was greater than in E-LO (p < 0.001) at 24 hours and at 48 hours. DOMS was significantly elevated at 24, 48, and 72-hr post each exercise session (p < 0.05). The greatest peak DOMS score occurred during the E-HI condition. MIF was similarly reduced after each exercise session (p < 0.05). These results suggest elevated E does not offer a protective effect to skeletal muscle; however, design limitations (i.e., condition order) confound the present data. Interestingly, an association between peak-CK during the E-LO condition and the number of washout days (r = +0.707, p < 0.05) between conditions existed. This suggests a longer washout period may be necessary to elucidate the actual E effects on skeletal muscle. These findings suggest that more work correcting for the present design limitations is warranted on this topic.     

Morphology of connective tissue in skeletal muscle

The arrangement and distribution of connective tissue in six different skeletal muscles and smooth muscle was examined by scanning electron microscopy. The endomysial arrangement of collagen was similar in all types of muscle and consisted of three components: (1) myocyte-myocyte connectives; (2) myocyte-capillary connectives; and (3) a weave network of collagen intimately associated with the basal laminae of the myocytes. The perimysium of the different muscles was qualitatively similar but quantitatively dissimilar. The perimysium consisted of large tendon-like bundles of interwoven collagen which connected with the dense weave collagen that surrounded groups of muscles. The arrangement of the collagen in the perimysium and endomysium would explain differences in the mechanical properties of the different muscle. The contribution of the connective tissue to mechanical properties of muscle is discussed.     

Protective effect of Lycium barbarum polysaccharides on oxidative damage in skeletal muscle of exhaustive exercise rats

The aim of this study was to determine the modulatory effect of Lycium barbarum polysaccharides (LBP) on the oxidative stress induced by an exhaustive exercise. 32 male Wistar rats were taken in the study. The experiment was a 30-day exhaustive exercise program. We determined the lipid peroxidation, glycogen levels, and anti-oxidant enzyme activities in skeletal muscle. The results demonstrated that L. barbarum polysaccharides administration significantly increases glycogen level and anti-oxidant enzyme activities, and decreased malondialdehyde (MDA) level and creatine kinase activities. In conclusion, L. barbarum polysaccharides administration can significantly decrease the oxidative stress induced by the exhaustive exercise.     

Fluostigmine effect on glycogen level in the skeletal muscle

Fluostigmine in a dose not producing evident toxicity reduced the glycogen content in the gastrocnemius muscle in rats, with a consequent decrease of glycogen utilization during contractions of the muscle induced with direct tetanic stimuli. Administration of atropine or atropine with obidoxime failed to change this effect of fluostigmine. The authors suggest that the effect is not due to disturbances of the cholinergic system function.     

Pliometric contraction-induced injury of mouse skeletal muscle: effect of initial length

Hunter, Kam D., and John A. Faulkner. Pliometriccontraction-induced injury of mouse skeletal muscle: effect of initial length. J. Appl. Physiol. 82(1):278-283, 1997.For single pliometric (lengthening) contractionsinitiated from optimal fiber length (L_f), the mostimportant factor determining the subsequent force deficit is the workinput during the stretch. We tested the hypothesis that regardless ofthe initial length, the force deficit is primarily a function of thework input. Extensor digitorum longus muscles of mice were maximallyactivated in situ and lengthened at 2 L_f /s from oneof three initial fiber lengths (90, 100, or 120% of L_f) to one ofthree final fiber lengths (150, 160, or 170% of L_f). Maximalisometric force production was assessed before and after the pliometriccontraction. No single mechanical factor, including thework input(r² = 0.34), was sufficient to explain the differences in force deficits observed among groups. Therefore, the force deficit appears to arisefrom a complex interaction of mechanicalevents. With the data grouped by initial fiber length,the correlation between the average work and the average force deficitwas high(r² = 0.97-0.99). Consequently, differences in force deficits among groups were best explained on the basis of the initial fiber length andthe work input during the stretch.

     

Cellular responses in exertion-induced skeletal muscle injury

Muscle injury is a common result of muscle exertion caused by overload and over-activity. In this presentation, an attempt was made to discuss models of muscle injury which involve exertion but not excessive strain, although most functional activities of the extremities require some eccentric muscle actions. Muscle injury is characterized by cellular and extracellular matrix responses which appear to be common to all types of muscle trauma - even in the absence of bleeding. Using tenotomy and functional over-load of the rat hindlimb muscles as examples, illustrations of several of these responses are presented and discussed.     

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na(v)). The skeletal muscle Na(v) (Na(v)1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na(v)1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na(v)1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na(v)1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.     

Alanine metabolism in skeletal muscle in tissue culture

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L₆) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P < 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P < 0.0005) as well as medium (r = 0.50, P < 0.025) and intracellular (r = 0.85, P < 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P < 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rather to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

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 29:153-163, 2001; Breuls et al. in Ann Biomed Eng 31:1357-1364, 2003; Stekelenburg et al. in J App Physiol 100(6):1946-1954, 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):1946-1954, 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.