July 14, 2004

CXC Chemokine Expression and Synthesis In Skeletal Muscle During Ischemia Reperfusion

Hong T Hua, Michael C Stoner, Hassan Al-Badawi, Fateh Entabi, Michael T Watkins
Massachusetts General Hospital, Boston, MA

Introduction: The CXC chemokines, KC and MIP-2, have been implicated in neutrophil activation and infiltration following ischemia reperfusion (IR) in myocardial, renal, and retinal tissue. These studies were designed to determine whether KC mRNA and protein and/or MIP-2 protein are increased in a murine model of acute hindlimb skeletal muscle ischemia and IR.

Methods: Mice were subjected to a defined interval of 3 hours of unilateral hindlimb ischemia alone, or 3 hours of ischemia followed by 4 hours or 24 hours reperfusion using a controlled tension tourniquet. After ischemia or IR, experimental and non-ischemic contralateral hindlimbs were harvested. Quantitative real time RT-PCR was performed to analyze relative expression of KC mRNA. Protein analyses for KC and MIP-2 were performed using ELISA.

Results: Ischemia alone did not increase KC mRNA levels. In contrast, IR resulted in a 15-fold increase in KC mRNA at 4 hours reperfusion (p<0.001) and a 10-fold increase at 24 hours reperfusion (p<0.05) compared to sham. Both KC and MIP-2 protein levels were unchanged in contralateral, sham and ischemia-only limbs; however, they were significantly elevated at 24 hours reperfusion (p<0.001).

Conclusions: Murine hindlimbs subjected to ischemia alone do not increase tissue levels of KC or MIP-2. Ischemia followed by reperfusion significantly increases tissue KC and MIP-2 protein levels. The increase in KC protein levels appears to be transcriptionally regulated. The CXC chemokines, KC and MIP-2, may be specific markers or targets for therapeutic intervention following ischemia reperfusion.