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Methods to Protect from Skeletal Cardiovascular Insufficiency: Improving Soldier Performance in Adverse Environments

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Annual rept. 1 Nov 2004-31 Oct 2005

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The authors compared two methods of determining the extent of skeletal muscle injury. The first method used nitroblue tetrazolium dye NBT to distinguish between live and injured muscles. The muscle flap was sectioned transversely into 1 mm thick segments before staining in 0.033 dye solution in the presence of 1.9 mM NADH. The viable area stained bluepurple as the mitochondrial enzyme activity yielded a blue reaction product and the nonviable area did not stain. The areas were quantified using an automated image analysis program Image-Pro Plus, version 5.0. Infarct size was calculated as amount of infracted area divided by the total area in percentage. Results showed that NBT dye can distinguish between live versus injured areas. The second method of quantifying skeletal muscle injury was the use of Evans Blue Dye EBD. EBD is taken up by injured muscle cells due to permeabilization of the plasma membrane during ischemiareperfusion. They used the EBD method to investigate the protective anti-ischemic effect of adenosine. Results showed that the adenosine A1 receptor-selective agonist CCPA 2-chloro-N-cyclopentyladenosine, when administered 2 hours before a 90-minute ischemia6 hour or a 90-minute24-hour reperfusion period, was able to decrease the percentage of necrosis. Data were plotted as a percentage of EBD-positive cells. The reduced infarction size of the mouse hindlimb skeletal muscle by CCPA remained significant 24 hours after reperfusion in this model. In the next series of experiments, another adenosine receptor agonist, R-phenyl-2-propyladenosine R-PIA, was tested. Adenosine receptor agonists significantly reduced the infarction size 6 hours after reperfusion in the mouse hindlimb ischemia reperfusion model. The data establish a novel model of skeletal muscle ischemiareperfusion injury that allows reproducible quantitation of muscle injury and show that adenosine receptor activation can exert a potent cytoprotective effect in skeletal muscle.

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  • Biochemistry
  • Medicine and Medical Research

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