Amputated tissue maintained in a hypothermic environment can endure prolonged
ischemia and improve
replantation success. The authors hypothesized that local tissue
hypothermia during the early reperfusion period may provide a protective effect against
ischemia-reperfusion injury similar to that seen when
hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local
hypothermia during
ischemia only (p = 18), reperfusion only (p = 18), and both
ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to
hypothermia of 10 degrees C during 4 hours of
ischemia, the initial 3 hours of reperfusion, or both periods.
Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent
nitroblue tetrazolium staining), local
edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil
integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-
rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion.
Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (
ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and
ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle
edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and
ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the
ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and
ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the
ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and
ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the
ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local
hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue
hypothermia during the early reperfusion period (reperfusion-only and
ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local
hypothermia to ischemic muscle tissue during the early reperfusion period.