Oxidative stress and DNA damage signalling in skeletal muscle in pressure-induced deep tissue injury.

The molecular mechanisms that contribute to the pathogenesis of pressure-induced deep tissue injury are largely unknown. This study tested the hypothesis that oxidative stress and DNA damage signalling mechanism in skeletal muscle are involved in deep tissue injury. Adult Sprague Dawley rats were subject to an experimental protocol to induce deep tissue injury. Two compression cycles with a static pressure of 100 mmHg was applied to an area of 1.5 cm(2) over the mid-tibialis region of right limb of the rats. The left uncompressed limb served as intra-animal control. Muscle tissues underneath compression region were collected for examination. Our analyses indicated that pathohistological characteristics including rounding contour of myofibres and extensive nuclei accumulation were apparently shown in compressed muscles. The elevation of 8OHdG immunopositively stained nuclei indicated the presence of oxidative DNA damage. Increase in oxidative stress was revealed by showing significant elevation of 4HNE and decreases in mRNA abundance of SOD1, catalase and GPx, and protein content of SOD2 in compressed muscles relative to control muscles. Increase in nitrosative stress was demonstrated by significant elevation of nitrotyrosine and NOS2 mRNA content. The activation of tumor suppressor p53 signalling was indicated by the remarkable increases in protein contents of total p53 and serine-15 phosphorylated p53. The transcript expression of the DNA-repairing enzyme, Rad23A, was significantly suppressed in compressed muscles. Our time-course study indicated that increased oxidative/nitrosative stress and proapoptotic signalling were maintained in muscles receiving increasing amount of compression cycles and post-compression time. Furthermore, resveratrol was found to attenuate the histological damage, oxidative/nitrosative stress and proapoptotic signalling in response to prolonged moderate compression. In conclusion, our findings are consistent with the hypothesis that oxidative stress and DNA damage signalling in skeletal muscle are involved in the underlying mechanisms responsible for the pathogenesis of pressure-induced deep tissue injury.

Habitual exercise increases resistance of lymphocytes to oxidant-induced DNA damage by upregulating expression of antioxidant and DNA repairing enzymes.

The underlying mechanisms of adaptation from staying physically active are not completely revealed. This study examined the effects of 8 and 20 weeks of habitual voluntary exercise on the susceptibility of lymphocytes to oxidant-induced DNA damage, antioxidant enzyme activities in cardiac and skeletal muscles, and circulatory antioxidant profile. Forty young adult rats were randomly assigned to sedentary control and exercise groups for an experimental period of 8 or 20 weeks. Animals assigned to exercise groups were subjected to 24 h daily free access to an in-cage running wheel with circumference of 1.19 m. A magnetic digital counter was attached to the running wheel to record daily exercise distance run by the animals. Control rats were housed in cages without a running wheel, located next to the exercised animals. Body weight and food intake were recorded weekly. After the experimental periods of 8 and 20 weeks, blood, left ventricle, soleus and plantaris muscles were collected for analysis. No significant difference was found in plasma total antioxidant capacity between exercised and control animals in the 8 and 20 week groups according to our ferric reducing/antioxidant power (FRAP) analysis. However, modified FRAP for ascorbic acid (FRASC) analysis indicated that plasma ascorbic acid content was significantly increased by 46 and 34% in 8 and 20 week exercise groups, respectively, when compared with the corresponding control groups. Superoxide dismutase (SOD) activity was significantly elevated by 39% in erythrocytes of animals exercised for 8 weeks relative to control animals. In the 20 week exercise group, Glutathione peroxidase (GPx) activity in ventricle and plantaris was significantly upregulated by 477 and 290%, respectively, relative to control values. As demonstrated by comet assay, the oxidant-induced DNA damage was significantly reduced by 21 and 45% in lymphocytes of animals exercised for 8 and 20 weeks, respectively, when compared with the corresponding control lymphocytes. Our qRT-PCR analysis showed that the transcript expression of SOD2 was significantly elevated by 939% in lymphocytes of animals exercised for 8 weeks relative to control animals. Increased expressions of SOD2 (by 19%), catalase (25%), APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1; 46%), Protein kinase, DNA-activated, catalytic polypeptide (Prkdc; 9%) and O-6-methylguanine-DNA methyltransferase (Mgmt; 26%) were found in lymphocytes of animals exercised for 20 weeks relative to control rats. These results demonstrate that habitual exercise confers increased resistance of lymphocytes to oxidant-induced DNA damage, and this protective effect is possibly attributed to the regular exercise-induced elevated expression of antioxidant and DNA repairing enzymes.

Protective effect of caspase inhibition on compression-induced muscle damage.

There are currently no effective therapies for treating pressure-induced deep tissue injury. This study tested the efficacy of pharmacological inhibition of caspase in preventing muscle damage following sustained moderate compression. Adult Sprague-Dawley rats were subjected to prolonged moderate compression. Static pressure of 100 mm Hg compression was applied to an area of 1.5 cm2 in the tibialis region of the right limb of the rats for 6 h each day for two consecutive days. The left uncompressed limb served as intra-animal control. Rats were randomized to receive either vehicle (DMSO) as control treatment (n =8) or 6 mg kg⁻¹ of caspase inhibitor (z-VAD-fmk; n =8) prior to the 6 h compression on the two consecutive days.Muscle tissues directly underneath the compression region of the compressed limb and the same region of control limb were harvested after the compression procedure.Histological examination and biochemical/molecular measurement of apoptosis and autophagy were performed. Caspase inhibition was effective in alleviating the compression-induced pathohistology of muscle. The increases in caspase-3 protease activity, TUNEL index, apoptotic DNA fragmentation and pro-apoptotic factors (Bax, p53 and EndoG) and the decreases in anti-apoptotic factors (XIAP and HSP70) observed in compressed muscle of DMSO-treated animals were not found in animals treated with caspase inhibitor. The mRNA content of autophagic factors (Beclin-1, Atg5 and Atg12) and the protein content of LC3, FoxO3 and phospho-FoxO3 that were down-regulated in compressed muscle of DMSO-treated animals were all maintained at their basal level in the caspase inhibitor treated animals. Our data provide evidence that caspase inhibition attenuates compression-induced muscle apoptosis and maintains the basal autophagy level. These findings demonstrate that pharmacological inhibition of caspase/apoptosis is effective in alleviating muscle damage as induced by prolonged compression.

Proteasome inhibition alleviates prolonged moderate compression-induced muscle pathology.

BACKGROUND: The molecular mechanism initiating deep pressure ulcer remains to be elucidated. The present study tested the hypothesis that the ubiquitin proteasome system is involved in the signalling mechanism in pressure-induced deep tissue injury. METHODS: Adult Sprague Dawley rats were subjected to an experimental compression model to induce deep tissue injury. The tibialis region of the right hind limb was subjected to 100 mmHg of static pressure for six hours on each of two consecutive days. The compression pressure was continuously monitored by a three-axial force transducer within the compression indentor. The left hind limb served as the intra-animal control. Muscle tissues underneath the compressed region were collected and used for analyses. RESULTS: Our results demonstrated that the activity of 20S proteasome and the protein abundance of ubiquitin and MAFbx/atrogin-1 were elevated in conjunction with pathohistological changes in the compressed muscle, as compared to control muscle. The administration of the proteasome inhibitor MG132 was found to be effective in ameliorating the development of pathological histology in compressed muscle. Furthermore, 20S proteasome activity and protein content of ubiquitin and MAFbx/atrogin-1 showed no apparent increase in the MG132-treated muscle following compression. CONCLUSION: Our data suggest that the ubiquitin proteasome system may play a role in the pathogenesis of pressure-induced deep tissue injury.

Muscle apoptosis is induced in pressure-induced deep tissue injury.

Pressure ulcer is a complex and significant health problem. Although the factors including pressure, shear, and ischemia have been identified in the etiology of pressure ulcer, the cellular and molecular mechanisms that contribute to the development of pressure ulcer are unclear. This study tested the hypothesis that the early-onset molecular regulation of pressure ulcer involves apoptosis in muscle tissue. Adult Sprague-Dawley rats were subjected to an in vivo protocol to mimic pressure-induced deep tissue injury. Static pressure was applied to the tibialis region of the right limb of the rats for 6 h each day on two consecutive days. The compression force was continuously monitored by a three-axial force transducer equipped in the compression indentor. The contralateral uncompressed limb served as intra-animal control. Tissues underneath the compressed region were collected for histological analysis, terminal dUTP nick-end labeling (TUNEL), cell death ELISA, immunocytochemical staining, and real-time RT-PCR gene expression analysis. The compressed muscle tissue generally demonstrated degenerative characteristics. TUNEL/dystrophin labeling showed a significant increase in the apoptotic muscle-related nuclei, and cell death ELISA demonstrated a threefold elevation of apoptotic DNA fragmentation in the compressed muscle tissue relative to control. Positive immunoreactivities of cleaved caspase-3, Bax, and Bcl-2 were evident in compressed muscle. The mRNA contents of Bax, caspase-3, caspase-8, and caspase-9 were found to be higher in the compressed muscle tissue than control. These results demonstrated that apoptosis is activated in muscle tissue following prolonged moderate compression. The data are consistent with the hypothesis that muscle apoptosis is involved in the underlying mechanism of pressure-induced deep tissue injury.