Skeletal Muscle Ischemia-reperfusion Research Articles

Colchicine protects rat skeletal muscle from ischemia/reperfusion injury by suppressing oxidative stress and inflammation.

Neutrophils play an important role in ischemia/reperfusion (IR) induced skeletal muscle injury. Microtubules are required for neutrophil activation in response to various stimuli. This study aimed to investigate the effects of colchicine, a microtubule-disrupting agent, on skeletal muscle IR injury in a rat hindlimb ischemia model. Twenty-one Sprague-Dawley rats were randomly allocated into three groups IR group, colchicine treated-IR (CO) group and sham operation (SM) group. Rats of both the IR and CO groups were subjected to 3 hr of ischemia by clamping the right femoral artery followed by 2 hr of reperfusion. Colchicine (1 mg/kg) was administrated intraperitoneally prior to hindlimb ischemia in the CO group. After 2 hr of reperfusion, we measured superoxide dismutase (SOD) and myeloperoxidase (MPO) activities, and malondialdehyde (MDA), tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels in the muscle samples. Plasma creatinine kinase (CK) and lactate dehydrogenase (LDH) levels were measured. We also evaluated the histological damage score and wet/dry weight (W/D) ratio. The histological damage score, W/D ratio, MPO activity, MDA, TNF-α and IL-1β levels in muscle tissues were significantly increased, SOD activity was decreased, and plasma CK and LDH levels were remarkably elevated in both the IR and CO groups compared to the SM group (P<0.05). Colchicine treatment significantly reduced muscle damage and edema, oxidative stress and levels of the inflammatory parameters in the CO group compared to the IR group (P<0.05). Colchicine attenuates IR-induced skeletal muscle injury in rats.

Chronology of mitochondrial and cellular events during skeletal muscle ischemia-reperfusion.

Peripheral artery disease (PAD) is a common circulatory disorder of the lower limb arteries that reduces functional capacity and quality of life of patients. Despite relatively effective available treatments, PAD is a serious public health issue associated with significant morbidity and mortality. Ischemia-reperfusion (I/R) cycles during PAD are responsible for insufficient oxygen supply, mitochondriopathy, free radical production, and inflammation and lead to events that contribute to myocyte death and remote organ failure. However, the chronology of mitochondrial and cellular events during the ischemic period and at the moment of reperfusion in skeletal muscle fibers has been poorly reviewed. Thus, after a review of the basal myocyte state and normal mitochondrial biology, we discuss the physiopathology of ischemia and reperfusion at the mitochondrial and cellular levels. First we describe the chronology of the deleterious biochemical and mitochondrial mechanisms activated by I/R. Then we discuss skeletal muscle I/R injury in the muscle environment, mitochondrial dynamics, and inflammation. A better understanding of the chronology of the events underlying I/R will allow us to identify key factors in the development of this pathology and point to suitable new therapies. Emerging data on mitochondrial dynamics should help identify new molecular and therapeutic targets and develop protective strategies against PAD.

Skeletal muscle ischemia-reperfusion injury and cyclosporine A in the aging rat.

Old patients exhibit muscle impairments and increased perioperative risk during vascular surgery procedures. Although aging generally impairs protective mechanisms, data are lacking concerning skeletal muscle in elderly. We tested whether cyclosporine A (CsA), which protects skeletal muscle from ischemia-reperfusion (IR) in young rats, might reduce skeletal muscle mitochondrial dysfunction and oxidative stress in aging rats submitted to hindlimb IR. Wistar rats aged 71-73 weeks were randomized to IR (3 h unilateral tourniquet application and 2 h reperfusion) or IR + CsA (10 mg/kg cyclosporine IV before reperfusion). Maximal oxidative capacity (VM ax ), acceptor control ratio (ACR), and relative contribution of the mitochondrial respiratory chain complexes II, III, IV (VS ucc ), and IV (VTMPD /Asc ), together with calcium retention capacity (CRC) a marker of apoptosis, and tissue reactive oxygen species (ROS) production were determined in gastrocnemius muscles from both hindlimbs. Compared to the nonischemic hindlimb, IR significantly reduced mitochondrial coupling, VMax (from 7.34 ± 1.50 to 2.87 ± 1.22 μMO2 /min/g; P < 0.05; -70%), and VS ucc (from 6.14 ± 1.07 to 3.82 ± 0.83 μMO2 /min/g; P < 0.05; -42%) but not VTMPD /Asc . IR also decreased the CRC from 15.58 ± 3.85 to 6.19 ± 0.86 μMCa(2+) /min/g; P < 0.05; -42%). These alterations were not corrected by CsA (-77%, -49%, and -32% after IR for VM ax, VS ucc , and CRC, respectively). Further, CsA significantly increased ROS production in both hindlimbs (P < 0.05; +73%). In old rats, hindlimb IR impairs skeletal muscle mitochondrial function and increases oxidative stress. Cyclosporine A did not show protective effects.

COX-2 inhibition attenuates lung injury induced by skeletal muscle ischemia reperfusion in rats

BackgroundSkeletal muscle ischemia reperfusion accounts for high morbidity and mortality, and cyclooxygenase (COX)-2 is implicated in causing muscle damage. Downregulation of aquaporin-1 (AQP-1) transmembrane protein is implicated in skeletal muscle ischemia reperfusion induced remote lung injury. The expression of COX-2 in lung tissue and the effect of COX-2 inhibition on AQP-1 expression and lung injury during skeletal muscle ischemia reperfusion are not known. We investigated the role of COX-2 in lung injury induced by skeletal muscle ischemia reperfusion in rats and evaluated the effects of NS-398, a specific COX-2 inhibitor. MethodsTwenty-four Sprague Dawley rats were randomized into 4 groups: sham group (SM group), sham+NS-398 group (SN group), ischemia reperfusion group (IR group) and ischemia reperfusion+NS-398 group (IN group). Rats in the IR and IN groups were subjected to 3h of bilateral ischemia followed by 6h of reperfusion in hindlimbs, and intravenous NS-398 8mg/kg was administered in the IN group. In the SM and SN groups, rubber bands were in place without inflation. At the end of reperfusion, myeloperoxidase (MPO) activity, COX-2 and AQP-1 protein expression in lung tissue, PGE2 metabolite (PGEM), tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels in bronchoalveolar lavage (BAL) fluid were assessed. Histological changes in lung and muscle tissues and wet/dry (W/D) ratio were also evaluated. ResultsMPO activity, COX-2 expression, W/D ratio in lung tissue, and PGEM, TNF-α and IL-1β levels in BAL fluid were significantly increased, while AQP-1 protein expression downregulated in the IR group as compared to that in the SM group (P<0.05). These changes were remarkably mitigated in the IN group (P<0.05). NS-398 treatment also alleviated histological signs of lung and skeletal muscle injury. ConclusionCOX-2 protein expression was upregulated in lung tissue in response to skeletal muscle ischemia reperfusion. COX-2 inhibition may modulate pulmonary AQP-1 expression and attenuate lung injury.

HIF-P4H-2 deficiency protects against skeletal muscle ischemia-reperfusion injury.

We show here that mice hypomorphic for hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2) (Hif-p4h-2 (gt/gt)), the main regulator of the stability of the HIFα subunits, have normoxic stabilization of HIF-1α and HIF-2α in their skeletal muscles. The size of the capillaries, but not their number, was increased in the skeletal muscles of the Hif-p4h-2 (gt/gt) mice, whereas the amount of glycogen was reduced. The expression levels of genes for glycolytic enzymes, glycogen branching enzyme 1 and monocarboxylate transporter 4, were increased in the Hif-p4h-2 (gt/gt) skeletal muscles, whereas no significant increases were detected in the levels of any vasculature-influencing factor studied. Serum lactate levels of the Hif-p4h-2 (gt/gt) mice recovered faster than those of the wild type following exercise. The Hif-p4h-2 (gt/gt) mice had elevated hepatic phosphoenolpyruvate carboxykinase activity, which may have contributed to the faster clearance of lactate. The Hif-p4h-2 (gt/gt) mice had smaller infarct size following limb ischemia-reperfusion injury. The increased capillary size correlated with the reduced infarct size. Following ischemia-reperfusion, glycogen content and ATP/ADP and CrP/Cr levels of the skeletal muscle of the Hif-p4h-2 (gt/gt) mice were higher than in the wild type. The higher glycogen content correlated with increased expression of phosphofructokinase messenger RNA (mRNA) and the increased ATP/ADP and CrP/Cr levels with reduced apoptosis, suggesting that HIF-P4H-2 deficiency supported energy metabolism during ischemia-reperfusion and protection against injury. Key messages: HIF-P4H-2 deficiency protects skeletal muscle from ischemia-reperfusion injury. The mechanisms involved are mediated via normoxic HIF-1α and HIF-2α stabilization. HIF-P4H-2 deficiency increases capillary size but not number. HIF-P4H-2 deficiency maintains energy metabolism during ischemia-reperfusion.

Fresh frozen plasma reduces edema in skeletal muscle following combined limb ischemia-reperfusion injury and hemorrhagic shock in rats

Exsanguination from extremity vascular injuries is the most common potentially survivable injury on the battlefield. Advances in treatment have dramatically improved survival, increasing the need to address associated morbidities including ischemia-reperfusion injury and extremity compartment syndrome. Despite advances, hemorrhagic shock (HS) requiring fluid resuscitation is common. Plasma-based resuscitation for the treatment of HS has been shown to reduce edema and injury in tissues other than muscle. The objective of this study was to determine if fresh frozen plasma (FFP) resuscitation offered protection in a rat model of combined HS and skeletal muscle ischemia-reperfusion injury. Anesthetized Sprague-Dawley rats underwent 37.5% arterial hemorrhage, producing HS, followed by 3 hours of tourniquet application. Animals were not resuscitated or resuscitated with either FFP (equal to the shed blood volume) or lactated Ringer's solution (three times shed volume) after 30 minutes of ischemia. They were euthanized 24 hours later, and their muscles were analyzed for edema (wet weight-dry weight). Routine histology was performed on muscle cross-sections stained with hematoxylin and eosin and graded using a semiquantitative grading system. All animals developed HS; the mortality rate was 50% in no resuscitation rats. FFP reduced edema by 13% (p = 0.02) compared with lactated Ringer's solution. Pathology scores were not different between treatment groups. FFP resuscitation reduces edema following muscle injury, decreasing the risk of developing extremity compartment syndrome.

Altered exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury.

BackgroundThe NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury.ResultsTo investigate the potential changes in protein expression mediated by NF-κB in secreted exosomes in the serum following I/R injury, the levels of circulating exosomal proteomes in C57BL/6 and NF-κB−/− mice were compared using two dimensional differential in-gel electrophoresis (2-DE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and proteomic analysis. In C57BL/6 mice, the levels of circulating exosomal proteins, including complement component C3 prepropeptide, PK-120 precursor, alpha-amylase one precursor, beta-enolase isoform 1, and adenylosuccinate synthetase isozyme 1, increased following I/R injury. However, in the NF-κB−/− mice, the expression of the following was upregulated in the exosomes: protease, serine 1; glyceraldehyde-3-phosphate dehydrogenase-like isoform 1; glyceraldehyde-3-phosphate dehydrogenase; and pregnancy zone protein. In contrast, the expression of apolipoprotein B, complement component C3 prepropeptide, and immunoglobulin kappa light chain variable region was downregulated in NF-κB−/− mice. Bioinformatic annotation using the Protein Analysis Through Evolutionary Relationships (PANTHER) database revealed that the expression of the exosomal proteins that participate in metabolic processes and in biological regulation was lower in NF-κB−/− mice than in C57BL/6 mice, whereas the expression of proteins that participate in the response to stimuli, in cellular processes, and in the immune system was higher.ConclusionsThe data presented in this study suggest that NF-κB might regulate exosomal protein expression at a remote site via circulation following I/R injury.

Effects of N-acetylcysteine on liver remote injury after skeletal muscle ischemia reperfusion in rats.

This study evaluated the effects of N-acetylcysteine as a scavenger of radical oxygen species on liver injury as a remote organ after skeletal muscle ischemia reperfusion. Twenty male Wistar rats were allocated randomly into two experimental groups: ischemia reperfusion (I/R) and ischemia reperfusion + N-acetylcysteine (I/R+NAC). All animals were undergone 2h of ischemia by occlusion femoral artery and 24h of reperfusion. Rats that were treated with N-acetylcysteine given intravenously at a dose of 150 mg/kg, immediately before reperfusion. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Livers were harvested for histopathological and biochemical studies. Liver tissue malondialdehyde (MDA), glutathione (GSH) and myeloperoxidase (MPO) activity were assayed. The ALT and AST values were significantly lower in I/R+NAC group. Hepatic MDA level and MPO activity were significantly increased in I/R group. The levels of GSH in liver tissue were significantly depressed by ischemia reperfusion. Liver histopathologic study in I/R group showed enlarged sinusoids, sinusoidal congestion, cytoplasmic vacuolation, cellular degenerative changes and necrosis. Histopathologically, there was a significant difference between two groups. Histopatological and biochemical results have shown that N-acetylcysteine was able to protect liver from skeletal muscle ischemia reperfusion injury.

Melatonin attenuates lung injury in a hind limb ischemia–reperfusion rat model

ObjectiveThis study evaluated the protective antioxidant effect of melatonin on lung injury as a remote organ after skeletal muscle ischemia–reperfusion in rats. MethodsThirty male Wistar rats were allocated randomly into three experimental groups: operated with no ischemia (Sham) group, ischemia–reperfusion group and ischemia–reperfusion+melatonin group. Hind limb ischemia was induced by clamping the femoral artery. After 2h ischemia, the clamp was removed and the animal underwent 24h reperfusion. Rats in the ischemia–reperfusion+melatonin group received melatonin (10mg/kg i.v.), immediately before the clamp was removed. At the end of the trial, animals were euthanized and the lungs were removed for water content determination, histopathological and biochemical studies. ResultsIn the ischemia–reperfusion+melatonin group, tissues showed less intense histological abnormalities such as neutrophilic infiltration, intra-alveolar hemorrhage and edema compared with the ischemia–reperfusion group. Histopathologically, there was a significant difference (P<0.05) between the two groups. The lung water content in the ischemia–reperfusion+melatonin group was significantly lower than the ischemia–reperfusion group (P<0.05). Lung tissue myeloperoxidase (MPO) activity and nitric oxide (NO) level were significantly (P<0.05) increased by ischemia–reperfusion. The increase in these parameters was reduced by melatonin.Comparing the ischemia–reperfusion+melatonin group with the sham group, no significant increase in all analyzed aspects of the research was observed. ConclusionsThese findings suggest that melatonin has preventive effects in lung tissue injury after transient femoral artery occlusion.

Hydrogen-rich saline attenuates ischemia–reperfusion injury in skeletal muscle

BackgroundTo investigate the potential beneficial effect of hydrogen-rich saline (HRS) in ischemia–reperfusion (IR) injury of skeletal muscle. MethodsThree experimental groups were established in male Sprague–Dawley rats: (1) sham group, (2) IR with normal saline group, (3) and IR with HRS group. A rat model of skeletal muscle IR injury was induced by 3-h tourniquet occlusion on its left hind limb and 4-h reperfusion. Normal saline and HRS (1.0 mL/100 g) were administered intraperitoneally at 10 min before reperfusion, respectively. Muscle and serum samples were analyzed for detecting the levels of myeloperoxidase (MPO), superoxide dismutase (SOD), malondialdehyde (MDA), and hydroxyl radical (•OH). Muscle samples were assessed by wet/dry rate, hematoxylin and eosin histologic assessment, Bcl2, Bax, cytochrome C, LC3B, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, and electron microscopy. ResultsThe wet/dry ratio increased significantly in the IR group (P < 0.01 compared with that in the sham group) and decreased significantly in IR with HRS groups (4.12 ± 0.14 versus 4.12 ± 0.14, P < 0.01 compared with that in the IR group). Muscle tissues and serum of the IR group had significantly increased levels of MPO, MDA, •OH content, and decreased SOD activities compared with the sham group (P < 0.01). The activity of SOD in the IR with HRS group was greatly elevated compared with that in the IR group (295.028 ± 9.288 versus 249.190 ± 5.450 in muscle tissues; 91.627 ± 2.604 versus 73.4045 ± 6.487 in serum; P < 0.01), whereas the levels of MPO, MDA, and •OH content were clearly reduced (MPO: 0.5649 ± 0.0724 versus 1.0984 ± 0.0824 in muscle tissues; 0.7257 ± 0.1232 versus 1.3147 ± 0.0531 in serum. MDA: 4.457 ± 0.650 versus 7.107 ± 0.597 in muscle tissues; 2.531 ± 0.434 versus 4.626 ± 0.237 in serum. •OH: 16.451 ± 0.806 versus 19.871 ± 0.594 in muscle tissues; 500.212 ± 7.387 versus 621.352 ± 7.591 in serum, P < 0.01). The integrated optical density of positive amethyst staining increased significantly in the IR group (P < 0.01 compared with that in the sham group) and decreased significantly in IR with HRS group (928.79 ± 234.537 versus 3005.972 ± 83.567, P < 0.01 compared with that in the IR group). Muscle tissues of the IR group had significantly increased levels of Bax, cytochrome C, LC3B content, and decreased Bcl2 activities compared with those in the sham group (P < 0.01). The activity of Bcl2 in the IR with HRS group was greatly elevated compared with that in the IR group (0.2635 ± 0.0704 versus 0.1242 ± 0.0662; P < 0.01), whereas the levels of Bax, cytochrome C, and LC3B content were clearly reduced (Bax: 0.3103 ± 0.0506 versus 0.5122 ± 0.0148; cytochrome C: 0.4194 ± 0.1116 versus 0.8127 ± 0.0166; LC3B: 0.5884 ± 0.0604 versus 1.3758 ± 0.0319; respectively, P < 0.01). ConclusionsHRS seems to be effective in attenuating IR injury in skeletal muscle via its antioxidant, anti-apoptosis, and anti-autophagy effect.

Effect of Otostegia persica extraction on renal injury induced by hindlimb ischemia-reperfusion: A rat model

Introduction: It is known that ischemia-reperfusion causes remote organ injury as well as local injury. In traditional systems of medicine, many plants have been documented to be useful for the treatment of various disorders including oxidative esters. This study was designed to investigate whether Otostegia persica extraction pretreatment has a protective effect against renal injury induced by hindlimb ischemia-reperfusion. Methods: Forty male Wistar rats were allocated into five groups as follows: Control, Sham, Otostegia persica, ischemia-reperfusion and ischemia-reperfusion + Otostegia persica groups. Rats in Otostegia persica and ischemia-reperfusion + Otostegia persica groups received Otostegia persica extraction (300 mg/kg) orally 2 days prior to operation. Hindlimb ischemia was induced by clamping the femoral artery for 2 h. After 24 h of reperfusion, blood and urine samples were obtained for kidney function tests and the kidneys were removed for histological analysis and oxidative stress measurement. Results: The decrease in glomerular filtration rate induced by reperfusion was significantly improved by Otostegia persica extraction administration (P < 0.05), which resulted in the decrease in serum urea and creatinine concentrations. Urinary creatinine significantly decreased in ischemia-reperfusion group compared to the other groups (P < 0.05). Urinary excretion rate, water intake and the ratio of kidney/body weight significantly increased in animals with reperfusion injury as compared with other groups (P < 0.05). On histological examination, rats pretreated with Otostegia persica extraction had nearly normal morphology. Skeletal muscle ischemia-reperfusion produced a significant increase in renal tissue malondialdehyde level, while pretreatment with Otostegia persica extraction was associated with a significantly lower malondialdehyde level (P < 0.05). Renal tissue catalase and superoxide dismutase activity and glutathione level were significantly (P < 0.05) decreased by hindlimb ischemia-reperfusion. The increases in these parameters were decreased by pretreatment with Otostegia persica extraction. Conclusions: The results of this study showed that Otostegia persica extraction pretreatment significantly protected the renal injury from skeletal muscle ischemia-reperfusion.

Effects of light-emitting diode (LED) therapy on skeletal muscle ischemia reperfusion in rats.

Low-level laser therapy has been shown to decrease ischemia-reperfusion injuries in the skeletal muscle by induction of synthesis of antioxidants and other cytoprotective proteins. Recently, the light-emitting diode (LED) has been used instead of laser for the treatment of various diseases because of its low operational cost compared to the use of a laser. The objective of this work was to analyze the effects of LED therapy at 904 nm on skeletal muscle ischemia-reperfusion injury in rats. Thirty healthy male Wistar rats were allocated into three groups of ten rats each as follows: normal (N), ischemia-reperfusion (IR), and ischemia-reperfusion + LED (IR + LED) therapy. Ischemia was induced by right femoral artery clipping for 2 h followed by 2 h of reperfusion. The IR + LED group received LED irradiation on the right gastrocnemius muscle (4 J/cm(2)) immediately and 1 h following blood supply occlusion for 10 min. At the end of trial, the animals were euthanized and the right gastrocnemius muscles were submitted to histological and histochemical analysis. The extent of muscle damage in the IR + LED group was significantly lower than that in the IR group (P < 0.05). In comparison with other groups, tissue malondialdehyde (MDA) levels in the IR group were significantly increased (P < 0.05). The muscle tissue glutathione (GSH), superoxide dismutases (SOD), and catalase (CAT) levels in the IR group were significantly lower than those in the subjects in other groups. From the histological and histochemical perspective, the LED therapy has alleviated the metabolic injuries in the skeletal muscle ischemia reperfusion in this experimental model.

Effects of pentoxifylline on skeletal muscle injury induced by acute hindlimb ischaemia–reperfusion: a histopathological study in a rat model

Skeletal muscle ischaemia–reperfusion injury can occur with diseases, trauma and during orthopaedic surgical procedures. Ischaemia–reperfusion injury is characterized by the production of oxygen free radicals leading to disturbances in vasomotility and microvascular permeability. This study evaluated the effects of pentoxifylline, a methylxanthine derivative, on skeletal muscle injury after acute hindlimb ischaemia–reperfusion. Twenty male Wistar rats were allocated randomly into two groups: ischaemia–reperfusion and ischaemia–reperfusion + pentoxifylline. All animals underwent 2 h of ischaemia by occlusion of the femoral artery followed by 24 h of reperfusion. Rats in ischaemia–reperfusion + pentoxifylline group were given a dose of intraperitoneal pentoxifylline injection 40 mg/kg immediately before reperfusion. After the reperfusion period, animals were euthanized and left gastrocnemius muscle harvested for histopathological analysis under light microscopy. In the ischaemia–reperfusion group, the changes were more intense with fibre disruption, neutrophil infiltration and interstitial oedema. There was a significant difference in terms of pathological parameters between ischaemia–reperfusion + pentoxifylline and ischaemia–reperfusion groups as histopathological scores (P < 0.05). The administration of pentoxifylline significantly decreased skeletal muscle injury induced by acute hindlimb ischaemia–reperfusion according to our histological findings.

O10: Effect size of quantitative muscle imaging in Duchenne muscular dystrophy exceeds the effect size of clinical scores of muscle function

s of Oral Presentations / Clinical Neurophysiology 125, Supplement 1 (2014) S1–S339 S31 confirmed Duchenne muscular dystrophy. Physical assessment was performed using the motor function measurement (MFM) scale. Quantitative magnet resonance imaging (qMRI) of thigh muscles was performed using the two-point Dixon method. One year functional and imaging changes were different according to the age and walking abilities at inclusion (group 1: below the age of seven; group 2: seven years and older and ambulant; group 3: non-ambulant). While patients of the first group still showed improvement of motor abilities, in group 2 the largest effect size (1.2) was found in the D1 subscore (standing and transfer function) of the MFM, and in group 3 the largest effect size was found for the total MFM score (0.66). In contrast, the effect sizes using qMRI was much larger consisting of 1.6 in group 1 (all thigh muscles), 4.1 in group 2 (hamstrings), and 2.3 in group 3 (knee extensors). These data suggest that qMRI has an added value compared to clinical outcome measures when designing therapeutic clinical trials in patients with DMD. Power analysis suggest that there are only a few needed patients to show an effect of a putative novel treatment when using quantitive muscle MRI in DMD. O11 Estimation of the muscle fiber density from the motor unit action potential I. Goker1, T. Artug2, O. Osman2, B. Baslo3 1Istanbul Arel University, Biomedical Engineering, Istanbul, Turkey; 2Istanbul Arel University, Electrical and Electronics Engineering, Istanbul, Turkey; 3Istanbul University, Medical Faculty, Istanbul, Turkey Introduction: The aim of this study is the estimation of the muscle fiber density (FD) from the Motor Unit Action Potentials (MUAP) to form a method for monitoring the progress of neuromuscular diseases. Methods: Data groups representing motor units of neurogenic (n=5), myopathic (n=5) and healthy cases (n=5) were created via EMG Simulator v3.6. MUAPs generated from each data group have been detected through a concentric needle electrode of a scanning EMG system. The spikes of the single fiber action potentials within the MUAPs have been found by computing the second-order derivative of MUAPs to estimate the detected number of fibers. MUAPs from 20 different locations within a Motor Unit Territory (MUT) have been acquired. The fiber density (FD) has been computed by taking the average of these numbers of fibers. True FDs have been computed for each MUT. Mean Square Errors (MSE) have been computed for each data group to determine the sensitivity of the method. Results: The test statistics of the FDs (mean ± SD) have been found as 4.13±1.31 (Min: 2 and Max: 8), 2.37±0.98 (Min: 1 and Max: 5) and, 2.40±0.70 (Min: 1 and Max: 4) for neurogenic, myopathic and healthy cases respectively. Mean true fiber densities have been 5.66, 2.57 and 1.50 and MSE have been 4.02, 0.15 and, 0.86 for neurogenic, myopathic and healthy cases respectively. These MSE values have been suggested that this method is more selective for myopathic cases compared to neurogenic and healthy cases. Conclusion: Computation of FD via the second-order derivative of MUAPs can be considered as a beneficial preliminary research. Nevertheless, this method might be extended by taking into account other variables such as maximum amplitude to increase the sensitivity for neurogenic cases.

Effect of recombinant human MG53 protein on tourniquet‐induced ischemia‐reperfusion injury in rat muscle

Skeletal muscle ischemia-reperfusion injury (I-R) is a complex injury process that includes damage to the sarcolemmal membrane, contributing to necrosis and apoptosis. MG53, a muscle-specific TRIM family protein, has been shown to be essential for regulating membrane repair and has been shown to be protective against cardiac I-R and various forms of skeletal muscle injury. The purpose of this study was to determine if recombinant human MG53 (rhMG53) administration offered protection against I-R. rhMG53 was administered to rats immediately before tourniquet-induced ischemia and again immediately before reperfusion. Two days later muscle damage was assessed histologically. rhMG53 offered no protective effect, as evidenced primarily by similar Evans blue dye inclusion in the muscles of rats administered rhMG53 or saline. Administration of rhMG53 does not offer protection against I-R in rat skeletal muscle. Additional studies are required to determine if the lack of a response is species-specific.

0420: Acute exercise preconditioning induced mitochondrial protection against skeletal muscle ischemia–reperfusion injury

Ischemia-reperfusion (I-R) induces mitochondrial function impairments and oxidative stress in skeletal muscle. In cardiac muscle, one-to-several days of exercise attenuate I-R injury. The objective of the present study was to show that a single exercise (EXO) prior to the I-R period protects the hind limb mitochondrial function. Twenty mice were randomly divided into 2 groups: The control group (CTL I-R, n=10) underwent I-R without exercise and the exercise group (EXO I-R, n=10) ran before I-R. Mice underwent 2 hours of ischemia induced by a rubber band tourniquet applied on the right root of the hind limb (the left leg was used as control leg), followed by a reperfusion period of 2 h. The EXO was performed on a treadmill with 10% incline 1h before I-R. The mice ran at a speed of 30cm until 40cm/sec during 30min. Blood lactate obtained from the tip of the tail at the end of EXO was above 9 mmol/L. In skinned gastrocnemius fibers, I-R significantly decreased the maximal mitochondrial respiration (Vmax) of the ischemic leg in comparison to the control leg in the CTL group (23.6 vs 28.6 pmol/(sec*mg wet weight); p<0.05). EXO I-R mice increased Vmax in comparison to CTL I-R mice (39.5 vs 31.8 pmol/(sec*dw) in the ischemic leg and the control leg respectively; p<0.05)). After EXO, the impairment between ischemic leg and control leg was partially restored (p=0.22). Without preconditioning, I-R increased H202 production of the ischemic leg in comparison to the control leg (42.6 vs 37.8 umol/mg dw; p=0.23). EXO I-R mice significantly decreased the H202 production in comparison to CTL I-R mice (26.6 vs 42.6 umol/mg dw; p<0.01) in the ischemic leg. After EXO, the impairment between ischemic leg and control leg was completely abolished (p=0.8). These results show that a preconditioning exercise protect the maximal mitochondrial respiration in mice and reduce the mitochondrial production of ROS following a period of I-R.

Accelerated functional recovery after skeletal muscle ischemia–reperfusion injury using freshly isolated bone marrow cells

BackgroundRelatively little information exists regarding the usefulness of bone marrow–derived cells for skeletal muscle ischemia–reperfusion injury (I/R), especially when compared with I/R that occurs in other tissues. The objectives of this study were to evaluate the ability of freshly isolated bone marrow cells to home to injured skeletal muscle and to determine their effects on muscle regeneration. Materials and methodsFreshly isolated lineage-depleted bone marrow cells (Lin− BMCs) were injected intravenously 2 d after I/R. Bioluminescent imaging was used to evaluate cell localization for up to 28 d after injury. Muscle function, the percentage of fibers with centrally located nuclei, and the capillary-to-fiber ratio were evaluated 14 d after delivery of either saline (Saline) or saline containing Lin− BMCs (Lin− BMCs). ResultsBioluminescence was higher in the injured leg than the contralateral control leg for up to 7 d after injection (P < 0.05) suggestive of cell homing to the injured skeletal muscle. Fourteen days after injury, there was a significant improvement in maximal tetanic torque (40% versus 22% deficit; P < 0.05), a faster rate of force production (+dP/dt) (123.6 versus 94.5 Nmm/S; P < 0.05), and a reduction in the percentage of fibers containing centrally located nuclei (40 versus 17%; P < 0.05), but no change in the capillary-to-fiber ratio in the Lin− BMC as compared with the Saline group. ConclusionsThe homing of freshly isolated BMCs to injured skeletal muscle after I/R is associated with an increase in functional outcomes.

The washout with UW solution and tanshinone II A to lessen skeletal muscle ischemia-reperfusion injury

Objective To assess the effect of intraarterial washout with UW solution and tanshinone Ⅱ A for skeletal muscle preservation during ischemia and reperfusion.Methods Ischemic rat limbs were perfused with UW solution or UW solution plus tanshinone Ⅱ A (0.05,0.1 and 0.2 mg/ml) for 0.5 h before reperfusion; controls received no perfusion.Serum CPK,LDH,and AST were measured pre-ischemia and after reperfusion (2 h,4 h,and 6 h).Muscle water content,MDA,SOD,ATPase were assessed pre-reperfusion and after 6 h reperfusion.ICAM-1 was detected after 6 h reperfusion.Results Intraarterial washout with UW and UW + T could inhibit the express of ICAM-1 in skeletal muscle.The serum levels of CPK,AST,and LDH in UW group were significant lower than those in control group after 2 h of reperfusion,but no difference was observed between UW group and control group after 4 h and 6 h of reperfusion.After 4 h of ischemia,there were significant differences in water content,MDA,SOD,and ATPase in skeletal muscle between UW group and control group,but no difference was found after 6 h of reperfusion.In contrast,all parameters of laboratory test and biochemical analyzing in UW + T(0.05,0.10,and 0.20 mg/ml) groups were significantly different from those in control group at corresponding reperfusion period.Conclusion Intraarterial washout with UW solution is effective in preserving skeletal muscle integrity against I/R insult.Tanshinone Ⅱ A as a beneficial adjunct to UW solution improves the protective effect of UW solution for ischemic skeletal muscle.To better preserve ischemic skeletal muscle,an appropriate dose of tanshinone Ⅱ A (0.1 or 0.2 mg/ml)added to UW solution is required. Key words: Intraarterial washout; UW solution; Tanshinone Ⅱ A; Skeletal muscle

Intramuscular transplantation and survival of freshly isolated bone marrow cells following skeletal muscle ischemia-reperfusion injury

Delayed treatment cellular therapies offer an attractive means to treat extremity injuries involving acute skeletal muscle ischemia-reperfusion injury (I/R). Bone marrow is a rich source of stem and progenitor cells with the potential to improve skeletal muscle regeneration. The extent to which bone marrow cells (BMCs) may be useful for I/R is not known. The purposes of this study were twofold: (1) to evaluate BMC survival following intramuscular injection 0, 2, 7, and 14 days after injury and (2) to determine whether BMCs improve functional recovery following I/R. Magnetic-activated cell sorting was used to isolate lineage-negative (Lin⁻) BMCs and enrich for stem and progenitor cells. To evaluate in vivo cell survival following I/R, Lin⁻ BMCs were injected intramuscularly 0, 2, 7, and 14 days after I/R, and bioluminescent imaging was performed for up to 28 days after cell injections. To assess their ability to improve muscle regeneration, intramuscular injections were performed 2 days after injury, and in vivo muscle function was assessed 14 days later. Lin⁻ BMCs survived throughout the study period regardless of the timing of delivery. Intramuscular injection of Lin⁻ BMCs did not improve maximal isometric torque (300 Hz); however, both saline-injected and Lin⁻ BMC-injected muscles exhibited an increase in the twitch-tetanus ratio, suggesting that damage incurred with the intramuscular injections may have had deleterious consequences for functional recovery. Although BMCs injected intramuscularly survived cell transplantation, they failed to improve muscle function following I/R. The ability of BMCs to persist in injured muscle following I/R lends to the possibility that with further development, their full potential can be realized.

Effect of tramadol on lung injury induced by skeletal muscle ischemia-reperfusion: an experimental study

OBJECTIVE: To determine whether tramadol has a protective effect against lung injury induced by skeletal muscle ischemia-reperfusion. METHODS: Twenty Wistar male rats were allocated to one of two groups: ischemia-reperfusion (IR) and ischemia-reperfusion + tramadol (IR+T). The animals were anesthetized with intramuscular injections of ketamine and xylazine (50 mg/kg and 10 mg/kg, respectively). All of the animals underwent 2-h ischemia by occlusion of the femoral artery and 24-h reperfusion. Prior to the occlusion of the femoral artery, 250 IU heparin were administered via the jugular vein in order to prevent clotting. The rats in the IR+T group were treated with tramadol (20 mg/kg i.v.) immediately before reperfusion. After the reperfusion period, the animals were euthanized with pentobarbital (300 mg/kg i.p.), the lungs were carefully removed, and specimens were properly prepared for histopathological and biochemical studies. RESULTS: Myeloperoxidase activity and nitric oxide levels were significantly higher in the IR group than in the IR+T group (p = 0.001 for both). Histological abnormalities, such as intra-alveolar edema, intra-alveolar hemorrhage, and neutrophil infiltration, were significantly more common in the IR group than in the IR+T group. CONCLUSIONS: On the basis of our histological and biochemical findings, we conclude that tramadol prevents lung tissue injury after skeletal muscle ischemia-reperfusion.