Unpredictability of Triphenyltetrazolium Chloride in Staining Irreversible Ischaemia-Reperfusion Injury in the Skeletal Muscle of Rats

Objective: To find out whether pale staining with triphenyltetrazolium chloride (TTC) in skeletal muscle of rat hindlimbs which had been subjected to ischaemia-reperfusion definitely indicated irreversible tissue damage. Design: Laboratory study. Setting: University hospital, Taiwan. Material: 77 Female Wistar rats. Interventions: Ischaemia of one hindlimb was caused by wrapping of a tourniquet above knee joint for 1.5 (n = 14), 2 (n = 15), 2.5 (n = 17), 3 (n = 17) or 4 (n = 14) hours. Each ischaemic group was divided into three subgroups to receive nil, 1 or 1.5 hours reperfusion, respectively. Gastrocnemius and soleus muscles were excised bilaterally. Main outcome measures: TTC reduction in the ischaemic limbs presented as a percentage of the opposite control limb measured by a spectrophotometric assay. Density of red formazan deposition in the ischaemic limbs assessed by microscopic examination of the TTC histochemical stain in the 3 hour ischaemic group. Results: In the 2, 2.5, and 3 hour ischaemic groups the TTC reduction in the ischaemic limbs after 1.5 hours reperfusion increased significantly as compared with that with one hour reperfusion (p < 0.01 in each case). The density of red formazan deposition in the muscle after 3 hours ischaemia and 1.5 hours reperfusion was significantly higher than that of only 1 hour of reperfusion. Conclusion: Lack of TTC staining does not necessarily represent irreversible ischaemia-reperfusion injury of the skeletal muscle in rats.