Intramuscular beta2-agonist administration enhances early regeneration and functional repair in rat skeletal muscle after myotoxic injury.

Systemic administration of beta(2)-adrenoceptor agonists (beta(2)-agonists) can improve skeletal muscle regeneration after injury. However, therapeutic application of beta(2)-agonists for muscle injury has been limited by detrimental cardiovascular side effects. Intramuscular administration may obviate some of these side effects. To test this hypothesis, the right extensor digitorum longus (EDL) muscle from rats was injected with bupivacaine hydrochloride to cause complete muscle fiber degeneration. Five days after injury, half of the injured muscles received an intramuscular injection of formoterol (100 mug). Muscle function was assessed at 7, 10, and 14 days after injury. A single intramuscular injection of formoterol increased muscle mass and force-producing capacity at day 7 by 17 and 91%, respectively, but this effect was transient because these values were not different from control levels at day 10. A second intramuscular injection of formoterol at day 7 prolonged the increase in muscle mass and force-producing capacity. Importantly, single or multiple intramuscular injections of formoterol did not elicit cardiac hypertrophy. To characterize any potential cardiovascular effects of intramuscular formoterol administration, we instrumented a separate group of rats with indwelling radio telemeters. Following an intramuscular injection of formoterol, heart rate increased by 18%, whereas systolic and diastolic blood pressure decreased by 31 and 44%, respectively. These results indicate that intramuscular injection can enhance functional muscle recovery after injury without causing cardiac hypertrophy. Therefore, if the transient cardiovascular effects associated with intramuscular formoterol administration can be minimized, this form of treatment may have significant therapeutic potential for muscle-wasting conditions.     

Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ～80% from pre-EP levels. Within 3 h, torque recovered to ～50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ～25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ～20% and ～70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.     

Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.     

Imaging of skeletal muscle function using 18FDG PET: force production, activation, and metabolism

The purpose of thisstudy was to determine whether [¹⁸F]fluorodeoxyglucose(FDG) positron emission tomography (PET) can be used to evaluate muscleforce production, create anatomic images of muscle activity, andresolve the distribution of metabolic activity within exercisingskeletal muscle. Seventeen subjects performed either elbow flexion,elbow extension, or ankle plantar flexion after intravenous injectionof FDG. PET imaging was performed subsequently, and FDG uptake wasmeasured in skeletal muscle for each task. A fivefold increase inresistance during elbow flexion increased FDG uptake in the bicepsbrachii by a factor of 4.9. Differences in relative FDG uptake weredemonstrated as exercise tasks and loads were varied, permittingdifferentiation of active muscles. The intramuscular distribution ofFDG within exercising biceps brachii varied along the transverse andlongitudinal axes of the muscle; coefficients of variation along theseaxes were 0.39 and 0.23, respectively. These findings suggest FDG PETis capable of characterizing task-specific muscle activity andmeasuring intramuscular variations of glucose metabolism withinexercising skeletal muscle.

     

Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle

The presence and potential physiological role of the erythropoietin receptor (Epo-R) were examined in human skeletal muscle. In this study we demonstrate that Epo-R is present in the endothelium, smooth muscle cells, and in fractions of the sarcolemma of skeletal muscle fibers. To study the potential effects of Epo in human skeletal muscle, two separate studies were conducted: one to study the acute effects of a single Epo injection on skeletal muscle gene expression and plasma hormones and another to study the effects of long-term (14 wk) Epo treatment on skeletal muscle structure. Subjects (n = 11) received a single Epo injection of 15,000 IU (double blinded, cross over, placebo). A single Epo injection reduced myoglobin and increased transferrin receptor and MRF-4 mRNA content within 10 h after injection. Plasma hormones remained unaltered. Capillarization and fiber hypertrophy was studied in subjects (n = 8) who received long-term Epo administration, and muscle biopsies were obtained before and after. Epo treatment did not alter mean fiber area (0.84 +/- 0.2 vs. 0.72 +/- 0.3 mm(2)), capillaries per fiber (4.3 +/- 0.5 vs. 4.4 +/- 1.3), or number of proliferating endothelial cells. In conclusion, the Epo-R is present in the vasculature and myocytes in human skeletal muscle, suggesting a role in both cell types. In accordance, a single injection of Epo regulates myoglobin, MRF-4, and transferrin receptor mRNA levels. However, in contrast to our hypothesis, prolonged Epo administration had no apparent effect on capillarization or muscle fiber hypertrophy.     

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.     

Distribution of Penicillin G in Serum and Tissue Cage Fluid in Cattle*

The penetration of penicillin into tissue cage fluid (TCF) in calves was studied after intravenous and intramuscular injection. The penicillin concentrations in TCF were lower than in serum and maximum was reached much later. Intravenous injection of benzyl-penicillin gave significantly higher levels in TCF than intramuscular injection. The penetration after procaine penicillin was very slow. The results showed that the serum peak rather than the area under curve determines the penetration of penicillin. Repeated intramuscular injections of benzylpenicillin and procaine penicillin caused an accumulation of penicillin in TCF. Similar levels were however reached by one single intravenous injection. The clinical counterparts to the used tissue cage model are abscesses. It was concluded that if high penicillin concentration are desireable in such foci, the drug must be given in a way that gives as high serum peaks as possible.     

Serum half-life, distribution, hepatic uptake and biliary excretion of alpha-tocopherol in rats

The serum clearance of alpha-[3H]tocopherol has been studied after intravenous injection of intestinal lymph labeled in vivo with radioactive alpha-tocopherol. The half-life of the injected alpha-[3H]tocopherol was approx. 12 min. Fractionation of plasma by ultracentrifugation 10 min after injection of lymph showed that 91% of the radioactive alpha-tocopherol remaining in plasma was located in chylomicrons (d less than 1.006 g/ml) and 7.8% in high-density lipoproteins (HDL, 1.05 less than d less than 1.21 g/ml). 2 h after administration of alpha-tocopherol, about 35% of the radioactivity recovered in plasma was associated with chylomicrons and approx. 51% with HDLs. alpha-[3H]Tocopherol was initially taken up by the liver, which contained more than 50% of the injected radioactivity after 45-60 min. Separation of parenchymal and nonparenchymal cells demonstrated a preferential uptake of alpha-[3H]tocopherol by the parenchymal liver cells. After 24 h about 11% of the injected dose was recovered in the liver. Considering whole organs the liver, adipose tissue and skeletal muscle had the highest content of radioactivity after 24 h. Furthermore, about 14% of the administered dose was recovered in bile during 24 h draining.     

Expression of luciferase in selected organs following delivery of naked and formulated DNA to rainbow trout (Oncorhynchus mykiss) by different routes of administration

In the present work, the expression of luciferase in selected organs following administration of DNA delivered as naked, liposome-formulated or chitosan-formulated by different routes of administration (intramuscular, intraperitoneal and intravenous injection, immersion and anal intubation) was studied in rainbow trout (Oncorhynchus mykiss). The different formulations and routes of administration both influenced in which organs luciferase was expressed and the magnitude of expression. The highest expression levels of luciferase in the head kidney and liver were found after an intraperitoneal injection of lipoplex 2. In the spleen, the highest levels were detected after injection of naked DNA (intraperitonal or intramuscular) and lipoplex 2 (intraperitoneal). Following intravenous injection, naked DNA gave higher expression levels in the organs than the formulated plasmids and immersion and anal intubation were not effective routes of delivery as no expression of luciferase could be detected in any of the organs tested. Additionally, PCR using a primer specific for a 600 bp region of the luciferase gene pcDNA3-luc was used to assess the distribution of the plasmid itself after intramuscular and intraperitoneal injection. Positive amplification was obtained in spleen, head kidney, liver and muscle at the injection site following injection of formulated plasmids, while only muscle tissue from the injection site was positive when naked DNA was used.     

Interaction between troponin and myosin enhances contractile activity of myosin in cardiac muscle

Ca(2+) signaling in striated muscle cells is critically dependent upon thin filament proteins tropomyosin (Tm) and troponin (Tn) to regulate mechanical output. Using in vitro measurements of contractility, we demonstrate that even in the absence of actin and Tm, human cardiac Tn (cTn) enhances heavy meromyosin MgATPase activity by up to 2.5-fold in solution. In addition, cTn without Tm significantly increases, or superactivates sliding speed of filamentous actin (F-actin) in skeletal motility assays by at least 12%, depending upon [cTn]. cTn alone enhances skeletal heavy meromyosin's MgATPase in a concentration-dependent manner and with sub-micromolar affinity. cTn-mediated increases in myosin ATPase may be the cause of superactivation of maximum Ca(2+)-activated regulated thin filament sliding speed in motility assays relative to unregulated skeletal F-actin. To specifically relate this classical superactivation to cardiac muscle, we demonstrate the same response using motility assays where only cardiac proteins were used, where regulated cardiac thin filament sliding speeds with cardiac myosin are >50% faster than unregulated cardiac F-actin. We additionally demonstrate that the COOH-terminal mobile domain of cTnI is not required for this interaction or functional enhancement of myosin activity. Our results provide strong evidence that the interaction between cTn and myosin is responsible for enhancement of cross-bridge kinetics when myosin binds in the vicinity of Tn on thin filaments. These data imply a novel and functionally significant molecular interaction that may provide new insights into Ca(2+) activation in cardiac muscle cells.     

Des-enkephalin-gamma-endorphin: bioavailability in rats following the subcutaneous and intramuscular route of administration

A pharmacokinetic study with [3H]des-enkephalin-gamma-endorphin (3H-DE gamma E) was performed in rats after the intravenous, subcutaneous and intramuscular route of administration. Disappearance of non-metabolized 3H-DE gamma E from blood upon intravenous dosing followed a biphasic decay with half-lives of 0.7 +/- 0.3 (+/- S.D.) min for the initial distribution phase and 6.3 +/- 2.7 min for the terminal elimination phase. The central and peripheral volumes of distribution were strikingly high (0.38 and 0.55 1 X kg-1, respectively). Extensive metabolism occurred already within the first minutes after injection. The blood clearance rate was found to be 0.29 +/- 0.12 1 X min-1 X kg-1, which value points to remarkable extrahepatic elimination of the neuropeptide. As compared to the intravenous route of administration, subcutaneous or intramuscular injection of 3H-DE gamma E resulted in low but longer-lasting peptide levels in blood. These levels reached already peak values at 2 min after both routes of administration and then declined to below the limit of detection at 2-3 h. The absolute bioavailability of DE gamma E after subcutaneous injection amounted to 30.9 +/- 16.3% (range 16.0-46.9%), whereas the bioavailability after intramuscular injection was observed to be 3.5 times lower (8.5 +/- 3.0%; range 4.6-12.0%). These data suggest that subcutaneous dosing of DE gamma E might be more effective in displaying CNS activity than the intramuscular route.     

Myoglobin synthesis in cell cultures of red and white muscle

Myoglobin synthesis was compared in cell cultures of leg (red) and breast (white) muscle of chick embryos. In leg muscle cultures a rapidly increasing amino acid incorporation into myoglobin begins two days after muscle cell fusion; in breast muscle cultures no comparable increase was observed. This qualitative difference in cultures of the two muscle cell types provides possibilities for the further study of the mechanism of myoglobin synthesis.     

注射光合细菌后罗氏沼虾血细胞组成的变化

以光合细菌作为刺激物,对罗氏沼虾进行肌肉注射,在注射后12、24、36、48、60h依次从罗氏沼虾心脏取血,用改良后的瑞氏染色法制成血涂片,光镜观察罗氏沼虾血细胞组成的变化.结果发现罗氏沼虾透明细胞在循环血细胞中所占地比例由正常状态下的66.62%略微下降至60%左右;36h左右所占比例急剧下降到30%左右;48h左右开始逐步回升;60h恢复至55%;接受刺激后的变化遵循缓慢下降、急剧下降、缓慢回升的过程.     

Myogenic potential of muscle side and main population cells after intravenous injection into sub-lethally irradiated mdx mice.

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10(4) muscle SP cells or with 10(6) MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells.     

Two-step mechanism of myofibrillar protein degradation in acute plasmocid-induced muscle necrosis

Acute muscle necrosis was induced in rats by intramuscular injection of plasmocid, a known myotoxic agent. A single injection of 5 mg/ml plasmocid produced massive fiber necrosis with extensive phagocytosis. Plasmocid administration led to a preferential decrease of alpha-actinin with preservation of other structural proteins within 3 h after injection, and large increases (2-7-fold) in the activities of acid hydrolases, cathepsins B and L, cathepsin D and alpha-galactosidase within 48 h after injection. The plasmocid-induced stimulation of alpha-actinin loss seen at 3 h, when no increases of acid hydrolases occurred, could be inhibited by a cysteine protease inhibitor, Ep-475 (E-64-c), and EGTA. On the other hand, increased lysosomal enzyme activity seemed to have a close correlation with the appearance of invading mononuclear cells, probably macrophages, and not muscle lysosomes. These observations suggest that a two step mechanism of protein degradation (nonlysosomal and lysosomal processes) possibly occurs in plasmocid-induced muscle degradation and macrophages can serve as a main endogenous reservoir of proteases in pathological states.     

Transfer of Adoptive Immunity to Plasmodium berghei: A Comparison of Routes of Injection*

SYNOPSIS. The ability to protect rats against Plasmodium berghei by the transfer of spleen cells by various routes of injection was examined. Intraperitoneal and intravenous injections of cells taken from spleens of recovered rats transferred immunity. Intramuscular injection of the same material was unsuccessful. All injections were derived from the same pool of immune spleen cells and contained equal numbers of trypan blue-excluding cells.     

Induction of histiocytic sarcoma in mouse skeletal muscle

Myeloid sarcomas are extramedullary accumulations of immature myeloid cells that may present with or without evidence of pathologic involvement of the bone marrow or peripheral blood, and often coincide with or precede a diagnosis of acute myeloid leukemia (AML). A dearth of experimental models has hampered the study of myeloid sarcomas and led us to establish a new system in which tumor induction can be evaluated in an easily accessible non-hematopoietic tissue compartment. Using ex-vivo transduction of oncogenic Kras(G12V) into p16/p19(-/-) bone marrow cells, we generated transplantable leukemia-initiating cells that rapidly induced tumor formation in the skeletal muscle of immunocompromised NOD.SCID mice. In this model, murine histiocytic sarcomas, equivalent to human myeloid sarcomas, emerged at the injection site 30-50 days after cell implantation and consisted of tightly packed monotypic cells that were CD48+, CD47+ and Mac1+, with low or absent expression of other hematopoietic lineage markers. Tumor cells also infiltrated the bone marrow, spleen and other non-hematopoietic organs of tumor-bearing animals, leading to systemic illness (leukemia) within two weeks of tumor detection. P16/p19(-/-); Kras(G12V) myeloid sarcomas were multi-clonal, with dominant clones selected during secondary transplantation. The systemic leukemic phenotypes exhibited by histiocytic sarcoma-bearing mice were nearly identical to those of animals in which leukemia was introduced by intravenous transplantation of the same donor cells. Moreover, murine histiocytic sarcoma could be similarly induced by intramuscular injection of MLL-AF9 leukemia cells. This study establishes a novel, transplantable model of murine histiocytic/myeloid sarcoma that recapitulates the natural progression of these malignancies to systemic disease and indicates a cell autonomous leukemogenic mechanism.     

Recovery of acetylcholine release and choline acetyltransferase activity after freezing-induced degeneration of rat soleus muscle

In order to study the effect of synaptic contact on the amounts of choline acetyltransferase (ChAT) and acetylcholine (ACh) in the nerve terminals and on their ability to release ACh, a freeze—thaw procedure was developed as a means to induce long lasting degeneration of rat soleus muscle. It was found that 4 days after the freeze—thaw procedure the preparation did not contract upon direct electric stimulation and the level of creatine kinase (CK) was below detection. The preparation contained about 15% of the ChAT activity and 15% of the ACh content of the controls. The ACh release evoked by 50 mM KCl was 25% of controls, but it was, when expressed as a fraction of the ACh content, about twice as high as that in control muscles. At day 12, the preparation still did not contract and the level of CK was less than 5% of controls. The ChAT activity and the ACh content were 40%) and 20% of controls, respectively. However, no release of ACh could be evoked by 50 mM KCl. At days 28 and 58 the preparation contracted upon stimulation of the nerve; the CK activity had recovered to about 20% and the ACh content to 40%, while the ChAT activity did not increase above 40%. The KCl–evoked ACh release had recovered to 20—30% of controls. The results indicate that freezing destroyed muscle cells and most intramuscular nerve branches. Subsequent regeneration of muscle fibres was slow, probably because freezing had killed many satellite cells in the muscle. Because the ChAT activity at day 12 had recovered when CK was almost absent and the preparation failed to contract, we conclude that there was expression of ChAT activity in ‘nerve terminals’ which do not make contact with regenerated muscle cells, although little if any ACh was released from these sites. ©1998 Elsevier Science Ltd. All rights reserved.     

Altered reabsorption of protein by the renal cortex in rats treated with hypertonic saline or mannitol.

The reabsorption of horseradish peroxidase (HRP) by the proximal tubule cells of rat kidneys was investigated by measuring the concentration of HRP in total particulate fractions of the cortex 1/4 and 1 hr after intravenous injection, and by correlated cytochemical observations. When compared to the corresponding values of the control animals, the concentration of HRP 1 hr after injection was decreased approximately 10-fold in the renal cortex of rats which had received an intravenous injection of hypertonic saline or two subcutaneous injections of mannitol. The plasma clearance and the urinary excretion of HRP were not altered significantly after injection of hypertonic saline, but the plasma clearance was decreased and the urinary excretion increased after injection of mannitol. When the dose of injected HRP was varied, the reabsorption of HRP by the renal cortex was proportional to the dose in the experimental and the control animals. Cytochemical staining for peroxidase activity also showed that the phagosomes and phagolysosomes of the proximal tubule cells contained much less peroxidase in the experimental rats than in the control rats. After injection of mannitol, large vacuoles appeared in the proximal tubule cells. The vacuoles often contained peroxidase-positive granules (phagosomes) which varied in diameter from the limit of microscopic visibility up to several microns. Most of the vacuoles did not react for acid phosphatase activity, but lysosomes were often aggregated around the vacuoles and seemed to release acid phosphatase into the cytoplasm. Certain analogies between the reabsorption of protein and that of water by the proximal tubule cells are discussed.     

Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction

The aim of this study was to investigate the efficacy and mechanism of action of a noninvasive remote ischemic preconditioning (IPC) technique for the protection of multiple distant skeletal muscles against ischemic necrosis (infarction). It was observed in the pig that three cycles of 10-min occlusion and reperfusion in a hindlimb by tourniquet application reduced the infarction of latissimus dorsi (LD), gracilis (GC), and rectus abdominis (RA) muscle flaps by 55%, 60%, and 55%, respectively, compared with their corresponding control (n = 6, P < 0.01) when they were subsequently subjected to 4 h of ischemia and 48 h of reperfusion. This infarct-protective effect of remote IPC in LD muscle flaps was abolished by an intravenous bolus injection of the nonselective opioid receptor antagonist naloxone (3 mg/kg) 10 min before remote IPC and a continuous intravenous infusion (3 mg/kg) during remote IPC and by an intravenous bolus injection of the selective delta 1-opioid receptor antagonist 7-benzylidenealtrexone maleate (3 mg/kg). However, this infarct-protective effect of remote IPC was not affected by an intravenous bolus injection of the ganglionic blocker hexamethonium chloride (20 mg/kg) or the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (10 mg/kg) or by a local intra-arterial injection of the adenosine1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (3 mg/muscle flap) given 10 min before remote IPC. It was also observed that this remote IPC of skeletal muscle against infarction was associated with a slower rate of muscle ATP depletion during the 4 h of sustained ischemia and a reduced muscle neutrophilic myeloperoxidase activity after 1.5 h of reperfusion. These observations led us to speculate that noninvasive remote IPC by brief cycles of occlusion and reperfusion in a pig hindlimb is effective in global protection of skeletal muscle against infarction. This infarct-protective effect is most likely triggered by the activation of opioid receptors in the skeletal muscle, and remote IPC is associated with an energy-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.