Figure 4
Involvement of reactive oxygen species (ROS) and TRPA1 in the hindlimb ischemia/reperfusion-evoked spontaneous licking.
(a) H2O2 production following the hindlimb ischemia/reperfusion. The H2O2 contents (nmol/mg protein) in the contralateral (contra) and ipsilateral (ipsi) paws 5 min after reperfusion following 60-min hindlimb ischemia were measured. n = 5, *P < 0.05, vs contralateral. (b) Effects of ROS scavengers on the hindlimb ischemia/reperfusion-evoked spontaneous licking. 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPOL; 250 mg/kg), α-phenyl-tert-butyl nitrone (PBN; 100 mg/kg), or vehicle was administered intraperitoneally (i.p.) 15 min before reperfusion following 60-min hindlimb ischemia or sham treatment. The time spent licking was measured for 40 min after reperfusion following hindlimb ischemia of 60 min. n = 6, ***P < 0.001. (c) Wild-type (WT) or TRPA1-knockout (KO) mice were subjected to hindlimb ischemia/reperfusion and blood flow was measured in both the contralateral and ipsilateral hindpaws. Blood flow in the ipsilateral hindpaw was normalised to that in the contralateral hindpaw and the value obtained just before ischemia served as 100%. n = 5. No difference in blood flow is observed between WT and TRPA1-KO mice. (d) The static tactile responses to a von Frey filament in WT and TRPA1- KO mice were measured during hindlimb ischemia and after reperfusion. The paw-withdrawal responses in the ipsilateral hindpaw were scored. n = 5–6. (e) The time spent licking in WT, TRPA1-KO, or TRPV1-KO mice was measured for 40 min following hindlimb ischemia/reperfusion. n = 5–9. **P < 0.01. N.S. = not significant. (f) The TRPA1 antagonist HC-030031 (10, 30 and 50 mg/kg) or vehicle was administered i.p. immediately before initiating ischemia or in sham-treated mice. The time spent licking was measured for 40 min following hindlimb ischemia/reperfusion. n = 5–7, **P < 0.01, ***P < 0.001.
