Figure 4.

Graphs showing (a) the effect of EA-2X/3, sham EA-2X/3 and twice weekly non-acupoint electroacupuncture (N/A-2X/3) treatment on tumor-induced hyperalgesia in the mice used for this experiment and ((b) to (e)) the effects of EA-2X/3 on neutrophils and macrophages at the osteosarcoma tumor site of male (M) mice at PID 7 and 14 (n = 5/group). EA-2X/3, but neither Sham EA-2X/3 nor N/A-2X/3, significantly decreased tumor-induced hyperalgesia at PID 7, 10 and 14 ((a); n = 10–15/group). Data in “(a)” shown as mean % response ± SEM; *P < 0.05, ****P < 0.0001 for N/A-2X. Sections were immunostained with NIMP-R14 an antibody specific for neutrophils (b) and (c) or with MOMA-2 for macrophages (d) and (e). The average percent Pixel Density (PD) of neutrophils (b), (c) and macrophages (d), (e) within the tumor were calculated from the 40 μm thick immunostained sections as described in Section 2. Each bar represents the average % PD (mean ± SEM) of neutrophils or macrophages at the tumor site for animals treated with no EA, EA-2X, Sham-EA-2X, or EA applied to a non-acupoint (M N/A-2X). Images were analyzed with ImageJ software (NIH, Bethesda, MD)—see Figure 5. EA-2X/3 treatment produced a significant reduction in the average neutrophil density within the tumor periphery at PID 7 as compared to tumor-no EA (****P < 0.0001), Sham-2X/3 (**P < 0.01), and N/A-2X/3 (***P < 0.001), returning to control densities by PID 14; P > 0.05 for all groups. Average densities of macrophages remained comparable in all groups regardless of the time point measured (P > 0.05).