Protective effect of edaravone for tourniquet-induced ischemia-reperfusion injury on skeletal muscle in murine hindlimb

Ischemia-reperfusion (I/R) injury refers to tissue damage caused when blood returns to a tissue after a period of ischemia[1]. Reperfusion triggers a cascade of acute inflammatory events, leading to cellular death and resulting in tissue dysfunction and necrosis[1]. Most research concerning I/R injury has focused on the heart, kidney, liver, brain, lung and intestines since these tissues are considered vital organs[2‐4]. I/R injury to skeletal muscles is also a serious problem however, and can cause local disturbances as well as systemic problems such as crush syndrome due to leakage from muscle cells into circulating blood. I/R injuries of skeletal muscles are commonly seen in a variety of trauma and other injuries including muscle damage, transplantations, plastic surgery and limb surgery with extended tourniquet application[5‐7].

Although the pathophysiology of I/R injuries has not been fully elucidated due to complex interactions of inflammatory and immunologic signaling pathways[8], it is clear that accumulated free radicals and anaerobic metabolites during ischemia cause the migration of white blood cells and the release of inflammatory factors such as interleukin and free radicals in the reperfusion phase[9‐13]. These compounds eventually damage muscle tissues, resulting in irrecoverable muscle damage and general complications[14‐16]. One of the strategies of treatment for I/R injury likely to be considered is scavenging free radicals, because I/R injury can activate pathways generating reactive oxygen species with the ability to break down cell membranes[14, 17]. In fact, there have been several reports in which a scavenger of free radicals was effective in reducing I/R injuries. Elmali et al. stated that resveratrol, which was previously shown to have free radical scavenging and antioxidant properties in various tissues, protected the skeletal muscles against I/R injuries[18]. Ozyurt et al. described how caffeic acid phenethyl ester (CAPE) protected skeletal muscles from reperfusion injury, and how this protective effect can probably be ascribed to CAPE’s free radical scavenging activity[19].

Edaravone (3-methy-1-pheny1-2-pyrazolin-5-one) is a potent and novel synthetic scavenger of free radicals. This drug inhibits both nonenzymatic lipid peroxidation and the lipoxygenase pathway, in addition to having potent antioxidant effects against ischemia reperfusion-induced vascular endothelial cell injury, delayed neuronal death, brain edema, and concomitant neurological deficits[20‐23]. In Japan, edaravone was approved in 2002 for use in treating acute brain infarctions and has been reported to have anti-stroke neuro-protective effects[20‐23]. Therefore, we hypothesized that edaravone could protect skeletal muscles from I/R injury in an in vivo model.

The purposes of this study were 1) to assess the histological findings of gastrocnemius muscle (GC) with many slow fibers and tibialis anterior muscles (TA) with many fast fibers in using the Crawford model of I/R injury[5], 2) to histologically analyze whether a single pretreatment of edaravone inhibits I/R injury on skeletal muscle in these mouse models, and 3) to evaluate the effect of oxidative stress on these muscles.

I/R injuries are defined as the paradoxical exacerbation of cellular dysfunction and death following the restoration of blood flow to previously ischemic tissues[1]. Re-establishment of blood flow is essential to salvage ischemic tissues; however reperfusion itself causes further damage to the ischemic tissue, threatening function and viability of the organ. I/R injuries occur in a wide range of organs including the heart, kidney, liver, brain and intestines heart, liver, brain, kidney, colon and skeletal muscles[2‐4, 31]. Examples of I/R injuries of skeletal muscles in this study can arise as a consequence of a range of vascular events including thrombolytic therapy, organ transplantation, limb trauma, or aortic clamping during repair of abdominal aortic aneurysms. The resulting tissue destruction and edema can lead to further worsening of the physiology and increased microvessel constriction leading to compartment syndrome[32‐34]. In addition, if a large volume of tissue falls into ischemia, subsequent revascularization and hence reperfusion of the ischemic tissue will also induce systemic effects on distant organs, leading to a multi-system organ failure called crush syndrome[35, 36]. Effective therapies counteracting these complications are not yet available in clinical settings, though there have been a number of reports regarding treatment for I/R injuries of skeletal muscles[37‐39].

Despite such studies devoted to examining I/R injury of skeletal muscle and its underlying mechanisms, no universally accepted model for inducing skeletal muscle I/R injury has evolved. The Crawford model was used for the tourniquet-induced hindlimb I/R injury in this study[5]. Another model put forth by McGivney, using a hemorrhoidal ligator (MHL) band has been widely used as tourniquet-induced hindlimb I/R injury model in mice[37]. The MHL model, however, has been criticized for its inability to control for nonspecific neuromuscular damage due to the crushing force of the rubber band on the underlying tissue. Additionally, the tension gradually decreases in this band. On the other hand, the model using an orthodontic rubber band (ORB) reported by Crawford appears to be superior to the MHL band model because ORBs produce complete, consistent, and reproducible ischemia in murine models of I/R using significantly less tension than the MHL band. Our histological analysis also showed that there was little difference in the degree of injury of muscle fibers, indicating that this simple model was likely to be suitable for evaluation of the effects of treatment for skeletal muscles of I/R injury.

While there have been several clinical strategies designed to reduce the effects of I/R injuries, none of them have been broadly successful in attenuating detrimental effects. These strategies include pharmacological intervention, hyperbaric oxygen, hypothermia, and pre- and post-injury conditioning[38‐40]. The potential therapeutic benefits of edaravone has been previously recognized and investigated for I/R injuries such as cerebral infarctions as there have been a number of reports that oxygen free radicals are involved in the pathogenesis of I/R injury[20, 22, 41]. A recent Japanese multicenter randomized clinical trial has demonstrated that edaravone, administered within 72 hours after onset of ischemic stroke significantly reduces brain infarct volume and produces sustained benefits in the functional outcomes of patients compared with placebo treatment[23]. These results led to our hypothesis that edaravone, approved in Japan as a neuro-protective agent, could protect skeletal muscles from I/R injury not only in the animal model, but also in clinical situations. Furthermore, it has been reported that application of other antioxidants has therapeutic potential for I/R injury of skeletal muscle. The present study demonstrates that pretreatment of edaravone significantly protects against I/R injury of skeletal muscles in the hindlimb of mice. This study is particularly significant for showing the therapeutic benefits of a single-dose administration of edaravone 30 minutes prior to the onset of ischemia. This preischemic treatment protocol mimics the commonly seen clinical conditions of long duration tourniquet use for extremity surgery and plastic surgeries such as free muscle transfer.

Infiltration of inflammatory cells was also inhibited by scavenging free radicals with edaravone. The final common pathway of I/R injury is edema, attraction of activated leukocytes, and the formation of membrane-attack complexes, all of which lead to disruption of cell membranes resulting cell death[17]. The oxygen free radicals initiate lipid peroxidation of cell membranes, which causes cellular dysfunction and tissue necrosis. Our results showed that one dose of pretreatment edaravone efficiently reduced lipid peroxidation and neutrophil infiltration into the ischemic muscles. Therefore, the scavenging of oxygen free radicals induced immediately after ischemia-reperfusion may not only maintain tissue viability but also enable sufficient contractile function of skeletal muscle.

Furthermore, expression of iNOS, which was shown to play an important role in the pathophysiology of I/R injury in other organs as well as inflammatory cytokine, was inhibited in the skeletal muscles treated by edaravone[20, 42]. The skeletal muscles were also reported to be influenced by NO generated by inducible form of NOS[43]. Barker et al. demonstrated that the area of necrosis was greater in wild-type than in iNOS-knockout mice, the difference being significant after 90 minutes of ischemia[43]. NO is formed as a metabolic product the stepwise conversion of L-arginine guanidine nitrogen atoms[44]. The presence of an unpaired electron in the NO radical makes it highly reactive, with a half-life of only a few seconds and readily capable of reacting with other species such as superoxides. The reaction is catalyzed by the NOS enzyme, of which three isoforms have been identified, each having specific localizations and functions[29]. Three NOS isoforms has been characterized, each encoded by different chromosomes. Two enzyme isoforms are constitutively expressed (endothelial eNOS and neuronal nNOS), whereas one isoform is an inducible enzyme (iNOS), initially found in macrophages. iNOS levels are markedly increased in inflammatory conditions, such as autoimmune inflammatory myopathies. The inducible isoform of NOS becomes up regulated in response to inflammatory stimuli such as endotoxins, cytokines and lipid mediators. Surges in NO production mediated by iNOS are cytotoxic and have been implicated in the inflammatory destruction of tissues such as heart, kidney, liver, brain, intestine and skeletal muscle[45]. In our experiments expression of iNOS was inhibited in the skeletal muscles treated by edaravone. Therefore, directly inhibiting lipid peroxidation of the myocyte by free radicals in skeletal muscles may reduce the secondary edema and inflammatory infiltration incidence of oxidative stress on tissues.

The injury severity score in the I/R + Ed group compared to the I/R group showed a significantly a lower rate both in GC and TA samples. While edaravone may have multiple effects, our data showed that pretreatment with edaravone protected muscle tissue from damage due to ischemia-reperfusion. We speculate that pretreatment of edaravone directly inhibits lipid peroxidation of myocytes by free radicals in skeletal muscles after I/R injury and indirectly protects myocytes from secondary damage due to edema, inflammatory infiltration and transduction of oxidative stress. Further studies are warranted to confirm the novel therapeutic potential edaravone may have for I/R injuries of skeletal muscle.