Effect of MMTL pretreatment on the protein level of CGRP in the Sp5C after NTG injection. (A) Representative immunofluorescent staining images of CGRP in the rostral Sp5C-V3 and caudal V1 area from sham and MMTL groups on day 8 after surgery. (B) Quantification of integrated densities of CGRP in the Sp5C (n = 3 mice per group). (C,E) Representative immunofluorescent staining images of CGRP in the caudal Sp5C-V1 from sham or MMTL plus the higher dose (10 mg/kg) of NTG groups (C), and sham or MMTL plus the lower dose (1 mg/kg) of NTG groups (E). (D,F) Quantification of integrated densities of CGRP in the caudal Sp5C-V1 (n = 3 mice per group). Note that MMTL significantly increased CGRP in the ipsilateral rostral Sp5C-V3, but had no effect on the CGRP protein level in the caudal Sp5C-V1 compared with the sham-treated group (A,B), and that MMTL pretreatment significantly increased CGRP in the ipsilateral caudal Sp5C-V1 after NTG injection of both higher dose (10 mg/kg, E, F) and lower dose (1 mg/kg, C and D). * p < 0.05 vs. the respective control groups. Ftreatment (1, 8) = 3.725, p = 0.90; Fside (1, 8) = 6.384, p < 0.05; Finteraction (1, 8) = 4.951, p = 0.057 for the rostral Sp5C-V3. Ftreatment (1,8) = 1.472, p = 0.260; Fside (1,8) = 0.225, p = 0.648; Finteraction (1,8) = 0.838, p = 0.387 for the caudal Sp5C-V1 in (B). Ftreatment (3,16) = 15.100, p < 0.001; Fside (1,16) = 11.28, p < 0.005; Finteraction (3,16) = 6.426, p < 0.05 for (D). Ftreatment (3,16) = 10.750, p < 0.001; Fside (1,16) = 20.07, p < 0.005; Finteraction (3,16) = 5.746, p < 0.05 for (F). CGRP: calcitonin gene-related peptide; MMTL: masseter muscle tendon ligation; NTG: nitroglycerin; NTG(1): 1 mg/kg of nitroglycerin; NTG(10): 10 mg/kg of nitroglycerin.