In the present study, we reproduced the critical limb syndrome in a murine model subjected to 3 h ischemia followed by 4 h reperfusion. A clinically significant infarct size of about 40 % in gastrocnemius muscles () was achieved with this model. Mitochondrial respiratory function, measured by complex I through IV activities, was markedly reduced in the ischemia-reperfusion group compared to the sham group (). Concurrently, superoxide production in gastrocnemius muscles was significantly elevated () and protein expression and activity of MnSOD, the main mitochondrial superoxide scavenger, were decreased in the ischemia-reperfusion group (). Pretreatment with tempol (SOD mimetic) or co-enzyme Q10, significantly inhibited superoxide production, improved the mitochondrial function, and decreased the infarct size in tourniquet-induced ischemia-reperfusion gastrocnemius muscles. These results strongly suggest that superoxide overproduction mediates the ischemia-reperfusion injury including cellular injury and mitochondrial dysfunction in skeletal muscles.
Since exsanguination is the most common cause of preventable trauma deaths, hemorrhage control usually takes precedence over fluid resuscitation (Bickell et al., 1994
). A controversial topic for decades (Klenerman, 1962
), arterial tourniquet has become one of the first-line treatments of exsanguinating hemorrhage in civilian and military casualties who incur severe penetrating and blunt extremity injuries, either as an isolated injury or a part of multisystem trauma. While beneficial, prolonged applications of tourniquet can lead to ischemia-reperfusion injuries of the underlying skeletal muscles. These injuries range from minor skeletal muscle dysfunction to muscle necrosis to even limb loss and systemic life-threatening shock (Mabry, 2006
). Evidence from animal experiments showed muscle injury begins after 2 h of tourniquet-induced ischemia, as evidenced by the elevated levels of lactic acid and creatine phosphokinase (Heppenstall et al., 1979
). Most clinical studies support the traditional 60–90 minutes as the upper limits of safe tourniquet time in the operative theaters (Doyle and Taillac, 2008
). The safety of these shorter tourniquet times is also re-affirmed for pre-hospital patients in recent military experience (Beekley et al., 2008
; Lakstein et al., 2003
; Sebesta, 2006
). Under certain tactical conditions in the civilian and military pre-hospital setting, however, longer tourniquet times may sometimes be necessary or unavoidable.
The timing and choice of revascularization method for acute limb ischemia in critical limb syndrome depends on patient’s level of limb ischemia. Using the clinical tools of motor, sensory assessment and Doppler study, a popular staging method is to categorize acute limb ischemia into Category I (Viable – No immediate threat), Category IIA (marginally threatened – Salvageable if promptly treated), Category IIB (Immediately threatened – Salvageable if immediately revascularized), and Category III (Irreversible – Major tissue loss, permanent nerve damage) (Ouriel et al., 1994
). This animal model utilized in this study was chosen to simulate acute limb ischemia IIB to III - severe enough to be clinically relevant, but that is not too severe to go past the point of non-viability and death for the entire limb. A rubber band tourniquet was applied to the hind limbs of mice for 3 h and released to begin the reperfusion. Hind limb muscles were harvested and examined at the end of 4 h of reperfusion. Our data for 1 and 2 h of ischemia (unpublished) showed that 3 h of ischemia gave the optimal and clinically relevant muscle infarction of 40%. The reperfusion time of 4 h was selected based on clinical and animal models showing stable pathology after 4 h of reperfusion (Blaisdell, 2002
; Hua et al., 2005
). Our preliminary data also found there is no significant difference between 4 h reperfusion injury and 24 h reperfusion injury (data not shown). During the application of the rubber band tourniquet, ischemia was confirmed by the near complete occlusion of blood flow to gastrocnemius muscle (), consistent with data in a previous study (Crawford et al., 2007
). The reperfusion was marked by post-ischemic hyperemia and partially recovered reflow (), consistent with previous clinical observations (Blaisdell, 2002
Consistent with previous studies using cardiac and other types of tissue (Solaini and Harris, 2005
), results in this study suggest that superoxide anion in skeletal muscles significantly contribute to ischemia-reperfusion-induced mitochondrial and cellular injury in skeletal muscles. First, the elevated superoxide production in the ischemia-reperfusion group in this study parallels the low reflow (), with previous studies having shown that these microcirculatory changes are caused by ROS-mediated endothelial damage (Menger et al., 1992
). Second, the elevated superoxide production in the ischemia-reperfusion group in this study is associated with 40% skeletal muscle necrosis and significant skeletal muscle mitochondrial dysfunction ( and ), an association that heretofore was not causally established in skeletal muscle. Third, tempol (a SOD mimetic) or co-enzyme Q10
(an endogenous antioxidant) scavenges the superoxide and prevents the gastrocnemius against the ischemia-reperfusion injuries (low blood flow, mitochondrial dysfunction and necrosis).
A number of papers have identified mitochondria as the principal source of superoxide in striated muscle (Sadek et al., 2003
). While < 2% of reactive oxygen speicies in the form of superoxide anion is produced by mitochondria under physiologic condition, superoxide production by mitochondria is significantly elevated in ischemia-reperfusion (Becker et al., 1999
). Low levels of reactive oxygen species during ischemia cause the electron transport chain damage, leading to inefficient transfer of electrons, and in turn, increased production of reactive oxygen species (Lesnefsky et al., 2004
; Sack, 2006
). At the onset of reperfusion, the additional burst of reactive oxygen species causes further damage to the electron transport chain and membrane damage via lipid peroxidation, leading to further oxidative damage to the cell and loss of cell viability (Kim et al., 2006
). In our present study, the activities of mitochondrial complex I, II, III, and IV were decreased in ischemia-reperfusion (); and complex I and III inhibitors significantly elevated the superoxide production in sham gastrocnemium muscles (). Tempol and co-enzyme Q10
conversely improved the mitochondrial dysfunction () accompanied by inhibiting ischemia-reperfusion-induced superoxide production () and mitochondrial complex inhibitor (rotenone or antimycin A)-enhanced superoxide production (). Based on these results, it is reasonable to assume that mitochondria also serve as producers of superoxide besides serving as targets for reactive oxygen species-induced injury in the pathophysiological conditions.
Superoxide anion in mitochondria is normally scavenged under physiologic conditions by MnSOD, which converts superoxide into hydrogen peroxide (H2
), and H2
is subsequently reduced to water by mitochondrial glutathione peroxidase. MnSOD protein expression and activities were reduced in the ischemia-reperfusion group as compared to shams (). Similar to the study by Kim (Kim et al., 2002
), in which a deficiency in MnSOD was found to exacerbate cerebral infarction after focal cerebral ischemia-reperfusion in mice, the harmful effects of superoxide production in this study were probably magnified by the inability of the endogenous antioxidant MnSOD (). These results suggest the endogenous cellular compensatory antioxidant mechanism failed to offset the increase in oxidative stress, either because the cellular antioxidant defenses were being overwhelmed by reactive oxygen species or there existed a post-translational modification of MnSOD induced by the ischemia-reperfusion injury.
Even though pretreatment of tempol or co-enzyme Q10
significantly reduced superoxide overproduction and normalized the activities of mitochondrial complex enzymes, tempol or co-enzyme Q10
did not increase MnSOD activity. Although the present study cannot explain this phenomenon, one possible explanation is that tempol and co-enzyme Q10
did not stop the ischemia-reperfusion-induced MnSOD nitration because some studies have shown that MnSOD nitration decreases the MnSOD activity (Filipovic et al., 2007
; Redondo-Horcajo et al., 2010
; Tangpong et al., 2008
), which needs further study. Nevertheless, this lack of effect by tempol or co-enzyme Q10
on MnSOD activity may explain the partial improvement in the size muscle infarction by tempol or co-enzyme Q10
in the ischemia-reperfusion, and it is possible that other factors (such as calcium overload) also contribute to the ischemia-reperfusion injuries besides superoxide.
In conclusion, using a murine model that simulates clinical acute limb ischemia caused by prolonged tourniquet application, we have shown that superoxide overproduction may be associated with mitochondrial dysfunction, cellular injury and cellular death in the skeletal muscle, and antioxidants can partially prevent the skeletal muscle from the ischemia-reperfusion injuries.