Inhibitory Effects of Scolopendra Pharmacopuncture on the Development and Maintenance of Neuropathic Pain in Rats: Possible Involvement of Spinal Glial.

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Inhibitory Effects of Scolopendra Pharmacopuncture on the Development and Maintenance of Neuropathic Pain in Rats: Possible Involvement of Spinal Glial Cells Chengjin Li, Byeong Uk Ji, Ji Eun Lee, Min Young Park, Sungchul Kim, Seung Tae Kim, Sungtae Koo Journal of Acupuncture and Meridian Studies Volume 8, Issue 5, Pages 236-244 (October 2015) DOI: 10.1016/j.jams.2015.01.001 Copyright © 2015 Terms and Conditions

Figure 1 Delayed (A) mechanical allodynia and (B) thermal hyperalgesia induced by spinal nerve ligation in rats treated with Scolopendra pharmacopuncture only once postoperatively. ∗p < 0.05 vs. spinal nerve ligation + saline group. Arrows indicate saline or Scolopendra pharmacopuncture injection time points. Journal of Acupuncture and Meridian Studies 2015 8, 236-244DOI: (10.1016/j.jams.2015.01.001) Copyright © 2015 Terms and Conditions

Figure 2 Downregulated ionized calcium binding adaptor protein (Iba-1) on postoperative Day 4 in the spinal cords of rats receiving one shot of Scolopendra pharmacopuncture (SPP) immediately after spinal nerve ligation (SNL). (A) Immunohistochemistry photomicrographs of the L5 spinal dorsal horn in SNL rats administered one shot of saline (SAL) and SPP and incubated with antibody recognizing Iba-1. Images (a–c) and (d–f) were viewed by using a 10 × and a 20 × lens, respectively. Scale bars are 200 μm for (a–c) and 100 μm for (d–f). (B) Bar graph presenting the number of Iba-1 positive microglia cells (##p < 0.001, SNL+SAL vs. naïve group; ∗p < 0.05, SNL+SPP vs. SNL+SAL group), n = 3. (C) Protein levels of Iba-1 and actin in the L5 spinal cord of rats undergoing different treatments, as obtained using western blotting. (D) Standardized ratio of the relative integrated densities (Iba-1/actin) of the SNL+SPP group to those of the SNL+SAL group (∗∗p < 0.001 vs. SNL+SAL group), n = 3. Iba-1 is the microglial marker. Journal of Acupuncture and Meridian Studies 2015 8, 236-244DOI: (10.1016/j.jams.2015.01.001) Copyright © 2015 Terms and Conditions

Figure 3 Unchanged glial fibrillary acidic protein (GFAP) on postoperative Day 4 in the spinal cords of rats receiving one shot of Scolopendra pharmacopuncture (SPP) immediately after spinal nerve ligation (SNL). (A) Immunohistochemistry photomicrographs of the L5 spinal dorsal horn in SNL rates administered one shot of saline (SAL) and SPP and incubated with antibody recognizing GFAP. Images (a–c) and (d–f) were viewed by using a 10 × and a 20 × lens, respectively. Scale bars are 200 μm for (a–c) and 100 μm for (d–f). (B) Bar graph presenting the number of GFAP positive astrocytes [#p < 0.05, SNL+SAL vs. naïve group; naïve (n = 3), SNL+SAL (n = 5), SNL+SPP (n = 5)]. (C) Protein levels of GFAP and actin in the L5 spinal cords of rats undergoing different treatments, as obtained using Western blotting. (D) Standardized ratio of the relative integrated densities (GFAP/actin) of the SNL+SPP group to those of the SNL+SAL group, n = 3. GFAP is the astrocytic marker. Journal of Acupuncture and Meridian Studies 2015 8, 236-244DOI: (10.1016/j.jams.2015.01.001) Copyright © 2015 Terms and Conditions

Figure 4 Attenuated late-phase neuropathic pain behaviors induced by spinal nerve ligation (SNL) in rats treated repetitively by using postligation with Scolopendra pharmacopuncture (SPP). SNL resulted in significant reductions in the hind-paw-withdrawal threshold and the latency to mechanical and thermal stimuli, respectively, compared to those prior to saline (SAL) or SPP was administered [+p < 0.05, ++p < 0.001 vs. the presurgical value (postoperative Day 0)]. Repeated daily injections of SPP suppressed SNL-induced (A) mechanical allodynia and (B) thermal hyperalgesia at late times in neuropathic rats (∗p < 0.05, ∗∗p < 0.001 vs. SNL+SPP group (postoperative Day 3). The difference between the SNL+SPP and the SNL+SAL groups was statistically significant (#p < 0.05, ##p < 0.001 vs. SNL+SAL group). Arrows indicate SAL or SPP injection time points. Journal of Acupuncture and Meridian Studies 2015 8, 236-244DOI: (10.1016/j.jams.2015.01.001) Copyright © 2015 Terms and Conditions

Figure 5 Downregulated ionized calcium binding adaptor protein (Iba-1) on postoperative Day 11 in the spinal cords of rats receiving repeated Scolopendra pharmacopuncture (SPP) after spinal nerve ligation (SNL). (A) Immunohistochemistry photomicrographs of the L5 spinal dorsal horn in SNL rats repeatedly administered saline (SAL) and SPP and incubated with antibody recognizing Iba-1. Images (a–c) and (d–f) were viewed by using a 10 × and a 20 × lens, respectively. Scale bars are 200 μm for (a–c) and 100 μm for (d–f). (B) Bar graph presenting the number of Iba-1 positive microglia cells [##p < 0.001, SNL+SAL vs. naïve group; ∗p < 0.05, SNL+SPP vs. SNL+SAL group; naïve (n = 3), SNL+SAL (n = 5), SNL+SPP (n = 5)]. (C) Protein levels of GFAP and actin in the L5 spinal cord of rats undergoing different treatments, as obtained using western blotting. (D) Standardized ratio of the relative integrated densities (Iba-1/actin) of the SNL+SPP group to those of the SNL+SAL group, n = 3. Iba-1 is the microglial marker. Journal of Acupuncture and Meridian Studies 2015 8, 236-244DOI: (10.1016/j.jams.2015.01.001) Copyright © 2015 Terms and Conditions

Figure 6 Downregulated glial fibrillary acidic protein (GFAP) on postoperative Day 11 in the spinal cord of rats receiving repeated Scolopendra pharmacopuncture (SPP) after spinal nerve ligation (SNL). (A) Immunohistochemistry photomicrographs of the L5 spinal dorsal horn in SNL rats repeatedly administered saline (SAL)and SPP and incubated with antibody recognizing GFAP. Images (a–c) and (d–f) were viewed by using a 10 × and a 20 × lens, respectively. Scale bars are 200 μm for (a–c) and 100 μm for (d–f). (B) Bar graph presenting the number of GFAP positive astrocytes [##p < 0.001, SNL+SAL vs. the naïve group; ∗p < 0.05, SNL+SPP vs. SNL+SAL group; naïve (n = 3), SNL+SAL (n = 5), SNL+SPP (n = 5)]. (C) Protein levels of GFAP and actin in the L5 spinal cords of rats undergoing different treatments, as obtained using western blotting. (D) Standardized ratio of the relative integrated densities (GFAP/actin) of the SNL+SPP group to those of the SNL+SAL group (∗p < 0.05 vs. SNL + SNL group), n = 3. GFAP is the astrocytic marker. Journal of Acupuncture and Meridian Studies 2015 8, 236-244DOI: (10.1016/j.jams.2015.01.001) Copyright © 2015 Terms and Conditions