Icing treatment in rats with crush syndrome can improve survival through reduction of potassium concentration and mitochondrial function disorder effect

Abstract

Crush syndrome (CS), a serious medical condition, which is characterized by damage to myocytes due to pressure and is associated with high mortality, even when patients receive fluid therapy. Icing therapy over the affected muscle has been reported to be effective in improving mitochondrial dysfunction and inflammation. These effects are thought to be secondary to improvements in the leakage of potassium and myoglobin from the damaged myocytes in the early stages of disease. However, their effects on the various symptoms of CS are unclear. It was hypothesized that treatment with icing will inhibit the influence of potassium by vasoconstriction, exert anti-inflammatory effects in the affected myocytes and improve mitochondrial function The CS model constructed by subjecting anesthetized rats to bilateral hindlimb compression with a rubber tourniquet for 5 h. The rats were then randomly divided into six groups: i) Sham; ii) CS without treatment (CS); iii) and iv) icing for 30 or 180 min over the entire hindlimb on CS rats (CI-30 and -180), respectively; and v) and vi) local icing for 30 or 180 min over the affected area on CS rats (CLI-30 and -180), respectively. Under continuous monitoring and recording of arterial blood pressures, blood and tissue samples were collected for biochemical analyses at designated time points prior to and following reperfusion. The survival rate, vital signs, and blood gas parameters in the CS group were lethal compared with the sham group. These were also improved in the CI-30 and CLI-30 groups compared with the CS group; however, they worsened in the CI-180 and CLI-180 groups due to hypothermia. The CI-30 and CLI-30 groups demonstrated tendencies of improvements compared with the CS group. Systemic inflammation and mitochondria dysfunction had improved in these groups compared with the CS group. We suggest icing therapy to temporarily prolong the viability after crush injury. Its effectiveness can be improved by combining it with other infusion therapies.

Keywords: anti-inflammation; crush syndrome; icing therapy; mitochondria.

Figure 1.

The impact of icing of varying durations on CS rat viability. Open circle: Sham group; closed circle: CS group; closed triangle: CI-30 group; open triangle: CI-180 group; closed diamond: CLI-30 group; and open diamond: CLI-180 group. *; vs. sham group (P<0.05), #; vs. CS group (P<0.05) (log-rank test). CS, crush syndrome; CI, icing over the entire hindlimb; CLI, local icing.

Figure 2.

Icing treatment improves vital signs. a) The relationship between BT and lactate levels, (B) the relationship between MBP and HR levels. The line graph is compares BT and MBP, while the bar graph lactate and HR levels. Open circle and white bar: Sham group; closed circle and black bar: CS group; closed triangle and shaded bar: CI-30 group; open triangle and gray bar: CI-180 group; closed diamond and diagonal bar: CLI-30 group; and open diamond and black dot bar: CLI-180 group. Values are showed as mean ± standard error of mean, *vs. sham group (P<0.05); ^#vs. CS group (P<0.05); †vs. CI-30 group (P<0.05); and ^ьvs. CLI-30 group (P<0.05) (Tukey's test). CS, crush syndrome; CI, icing on whole hindlimb of CS rat; CLI, icing on local hindlimb of CS rat; BT, body temperature; MBP, mean blood pressure and HR, hart rate.

Figure 3.

Attenuation of hyperkalemia secondary to icing treatments. (A) plasma K⁺ level, (B) plasma CPK level, (C) the relationship between blood vessel BPF and muscle BPF levels and (D) laser doppler image showing the changes in an affected hindlimb. Scale bar=1 cm. White bar: Sham group; black bar: CS group; shaded bar: CI-30 group; and diagonal bar: CLI-30 group. Values are presented as mean ± standard error in mean; *vs. sham group (P<0.05); ^#vs. CS group (P<0.05) (Tukey's test). CS, crush syndrome; CI, icing over the entire hindlimb of CS rats; CLI, local icing over the hindlimb of CS rats; K+, potassium; CPK, creatine phosphokinase; BPF, blood perfusion.

Figure 4.

Icing treatment was attenuated inflammation. (A) serum IL-6, (B) serum IL-10, (C) serum HMGB1 and (D) muscle tissue MPO activity. White bar: Sham group; black bar: CS group; shaded bar: CI-30 group; and diagonal bar: CLI-30 group. Values are showed as mean ± standard error in mean; *vs. sham group (P<0.05); ^#vs. CS group (P<0.05) (Tukey's test). CS, crush syndrome; CI, icing over the entire hindlimb of CS rats; CLI, local icing of hindlimb of CS rats; IL, interleukin; HMGB1, high mobility group box protein 1; MPO, myeloperoxidase.

Figure 5.

Improving ROS generation and prevention of mitochondrial dysfunction in the crushed limb. (A) TBARS level, (B) JC-l fluorescence, reflecting mitochondria membrane potential, (C) mitochondria cyt c content level and (D) cytoplasm cyt c content level in the crushed muscle tissue. White bar: Sham group; black bar: CS group; shaded bar: CI-30 group; and diagonal bar: CLI-30 group. Values are showed as mean ± standard error in mean; *vs. sham group (P<0.05) and ^#vs. CS group (P<0.05) (Tukey's test). CS, crush syndrome; CI, icing over the entire hindlimb of CS rats; CLI, local icing over the hindlimb of CS rats; TBARS, thiobarbituric acid reactive substance; cyt c, cytochrome c.