Fig. 1
Repeated injection of NTG induced hyperalgesia and upregulated c-Fos expression. A. Experimental design and timeline. B. Schematic diagrams of injection sites. a-c. Injection sites and TNC location (red circles) are shown in coronal and sagittal sections according to the rat brain atlas adapted from Paxinos and Watson. d-e. Coronal sections of the rat brain with methylene blue injection and HE staining to verify the correct placement of guide cannulas (arrow). C-E. The basal mechanical pain threshold of periorbital (C) and hind paw (D) and the thermal pain threshold (E) of the hind paw in each group. Mean ± SEM, n = 6/group. Two-way ANOVA with the Bonferroni post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001 compared with saline group. F. The protein expression of c-Fos was assessed by western blot and was markedly higher in the NTG group than in the saline group. G. The white dotted line frame indicates the TNC regions. Scale bar: 100 μm. H-I. Representative images (H) and quantitative analysis (I) of c-Fos-positive cells (red) in TNC of different groups. Immunofluorescence staining showed that the average numbers of c-Fos-positive cells in the NTG group were significantly increased compared to the saline group. Scale bar: 50 μm. FOV = 1.02 × 106 µm3. Mean ± SEM, n = 3/group, two-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the saline group. Abbreviations: PACAP, pituitary adenylate cyclase-activating peptide; NTG, nitroglycerin; CM: chronic migraine