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. 2021 Jul 31;158(5):1110–1130. doi: 10.1111/jnc.15468

FIGURE 1.

FIGURE 1

The following flow chart includes all the study designs. A total of 355 rats were used in this experiment. Figure 1a corresponds to the experimental steps in Figure 2. To verify whether the bone cancer pain (BCP) model was successfully established, the baseline pain threshold was determined before the operation, and the behaviour test was performed 3, 6, 9, 12, 15 and 18 days after the operation. CT scanning and haematoxylin–eosin staining were performed on the 12th day after the operation in the sham group and BCP groups. Figure 1b corresponds to the experimental steps in Figures 3, 4, 7 and 8a. The dynamic expression of nuclear factor erythroid 2 (NFE2)‐related factor 2 (Nrf2), heme oxygenase‐1 (HO‐1) and nuclear factor‐kappa B (NF‐κB) in the spinal cord was detected by western blot (WB) analysis at 0, 6, 12 and 18 days after tumour inoculation. The spinal cords of sham and BCP12 rats were collected for immunofluorescence (IF) to detect the cellular localization of Nrf2, HO‐1 and NF‐κB. Figure 1c corresponding to Figure 5a, c–f, and illustrates effects of intrathecal injection of sulforaphane (SFN) and Nrf2 siRNA on mechanical hyperalgesia in rats with BCP. The spinal cord of sham and BCP12 rats was collected for WB and IF to detect molecular expression and cell localization. Figure 1d corresponds to the experimental steps in Figure 5b. Single‐dose SFN was administered intrathecally to rats on day 12 after surgery in this experiment. The paw withdrawal threshold (PWT) was measured 1, 2, 4, 6, 8 and 12 hr after drug administration. Figure 1e HO‐1 is involved in the regulation of BCP. The experimental steps correspond to results shown in Figure 6a–e, g. Effects of hemin or HO‐1 siRNA treatment on mechanical hyperalgesia in rats with BCP. Rats were killed after 12 days of BCP to collect spinal cord and tibia tissues for WB, real‐time quantitative PCR and haematoxylin–eosin staining. Figure 1f corresponds to Figure 8b–g. The rats in the model group received the corresponding drug intervention from days to 6–12 after the operation. The spinal cord was collected 12 days after the operation to determine the expression of NF‐κB nuclear protein and related inflammatory factors. BL, baseline. ELISA, enzyme‐linked immunosorbent assay. The number of experimental animals was indicated in parenthesis