Opioid peptide–dependent sustained antinociception and increase opioid peptide expressed macrophages by repeated CXCL10 injection.

<p>Rats were i.pl. injected with CFA and daily with CXCL10 (0.2 ng) or solvent control. [A] Mechanical nociceptive thresholds were determined daily before each injection. Data were presented as mean ± SEM (n = 6 per group, *p<0.05, **p<0.01, CFA+CXCL10 versus CFA+solvent; Two-way RM ANOVA, Student-Newman-Keuls). [B] Anti-END (2 µg, anti-ENK (1.25 µg) or anti-DYN (1 µg) was locally injected (i.pl.) at 4 d post CFA on rats with repeated injection of CXCL10 (0.2 ng). Identical doses of anti-rabbit IgG were used as control. Data were presented as mean ± SEM (n = 6 per group, *p<0.05, **p<0.01, CFA+CXCL10+IgG versus CFA+CXCL10+anti-END/ENK/DYN; Two-way RM ANOVA, Student-Newman-Keuls). [C] Immunohistochemical staining of paw tissue was performed at 96 h with a mouse anti-ED1 (CD68) macrophage antibody (green) and with rabbit anti-END, anti-ENK or anti-DYN antibodies (all was marked red) as well as DAPI. Representative sections are shown. Arrows pointing at double positive cells. (scale bar: 50 µm). [D] The percentage of END/ENK/DYN+ and ED1+ was quantified. All the data are presented as mean ± SEM (n = 3 per group, *p<0.05, CFA+ solvent versus CFA+CXCL10; t-test).</p

Differential alterations in pro- and anti-inflammatory cytokines in inflamed paw tissue by electroacupuncture (EA).

<p>Rats were injected with CFA treated with (CFA+EA) or without (CFA) EA. Based on the results from pilot experiments for immune array of 29 cytokines (data not shown), [<b>A–F</b>] <i>pro- and anti-inflammatory</i> cytokines including TNF-alpha, IL-1alpha, IL-1beta, IFN-gamma, IL-4 and IL-13 in the paws were selectively quantified by ELISA after 96 h CFA. Data are presented as mean ± SEM (n = 5–10 per group, *p<0.05, CFA+EA versus CFA; t-test).</p

EA enhanced the recruitment of opioid-containing macrophages.

<p>Rats were injected with CFA with (CFA+EA), (CFA + sham) or without (CFA) EA treatment for 4 days. Immunohistochemical staining was performed for mouse anti-CD68 macrophages (green) and rabbit [<b>A</b>] anti-END, [<b>B</b>] anti-ENK or [<b>C</b>] anti-DYN antibodies respectively (red). DAPI (blue) was used to recognize cell nuclei (Representative sections are shown by arrows, scale bars: 50 µm). [<b>D</b>] The percentage of ED1 and opioid positive cells was quantified. All the data are presented as mean ± SEM (n = 3 per group, *p<0.05, one way ANOVA, Holm-Sidak method).</p

Upregulation of CXCL10 and an increase of CXCR3⁺macrophages in inflamed paw tissue by electroacupuncture (EA).

<p>Rats were injected with CFA and treated with (CFA+EA), (CFA+sham) or CFA only. On day 4 (96 h), CXCL10 was quantified by ELISA [<b>A</b>] and semi-quantitative RT-PCR (72 and 96 h) in subcutaneous paw tissue ([<b>B</b>] noninflamed contralateral paw (contra.) is only shown as a negative control). Data are presented as mean ± SEM (For ELISA: n = 6 per group, *p<0.05, one way ANOVA, Holm-Sidak method; For RT-PCR: n = 6 per group, *p<0.05, CFA+EA versus CFA; t-test). [<b>C</b>] Tissue sections were stained with rabbit anti-rat macrophage serum (red), mouse anti-rat CXCR3 antibody (green) and DAPI. The arrows are pointing at CXCR3 expressed macrophages. Representative sections are shown, arrows pointing on double positive cells (scale bar: 50 µm). [<b>D</b>] The percentage of macrophages and opioid positive cells was analyzed. All data are presented as mean ± SEM (n = 3 per group, *p<0.05, CFA+EA versus CFA; t-test).</p

Neutralization of CXCL10 fully reversed electroacupuncture (EA)-induced antinociception and increase of opioid-containing monocytes/macrophages.

<p>[A] Rats with CFA inflammation and EA treatment were daily i.pl. injected with an antibody against CXCL10. Controls were injected with anti-rabbit IgG antibody. Mechanical nociceptive thresholds were determined before (BL) and after injections. Data are presented as mean ± SEM (n = 6 per group, *p<0.05, **p<0.01, CFA+EA+IgG versus CFA+EA+anti-CXCL10; Two-way RM ANOVA, Student-Newman-Keuls). [B–D] Immunohistochemical staining was performed for mouse anti-ED1 monocytes/macrophages (green) and rabbit anti-END, anti-ENK or anti-DYN antibodies respectively (red). DAPI (blue) was used to recognize cell nuclei. Representative sections are shown, arrows pointing at double positive cells (scale bar: 50 µm). [E] Quantification for immunohistochemical staining showed the percentage of double positive ED1 and END/ENK/DYN cells. All the data are presented as mean ± SEM (n = 3 per group, *p<0.05, CFA+EA+IgG versus CFA+EA+anti-CXCL10; t-test).</p

The antinociceptive effect of electroacupuncture (EA) via opioid peptides at the site of inflammation.

<p>Wistar rats were injected with CFA i.pl. for 48–96 h and treated with CFA and electroacupuncture (EA) at GB30 at 0 and 24 h (day 0 and 1, 100 Hz, 20 min, 2–3 mA) (CFA+EA). [<b>A, E</b>] In previous studies, sham-EA rats did not show significant difference in both mechanical and thermal nociceptive thresholds measurements at 0, 48, 72 and 96 h. Data were presented as mean ± SEM (*p<0.05, CFA+EA versus CFA; ^$p<0.05, CFA+EA versus CFA+ sham; ^#p<0.05, CFA+ sham versus CFA; Two-way RM ANOVA, Student-Newman-Keuls). We therefore omitted sham-EA treatment in following studies. Anti-END (2 µg [<b>B, F</b>], anti-ENK (1.25 µg [<b>C, G</b>]) or anti-DYN (1 µg [<b>D, H</b>]) was locally injected (i.pl.) at 4 d post CFA and concomitant twice EA treatment (black circles). Two control groups were added: injection with identical doses of nonspecific anti-rabbit IgG (white circle) or for comparison CFA without EA (black triangle). Paw withdrawal latency (thermal nociceptive thresholds [<b>A–D</b>]) or paw pressure thresholds (mechanical nociceptive thresholds [<b>E–H</b>]) were determined before (BL: baseline) and 5 min after injection (treated). All the data are presented as mean ± SEM (n = 6 per group, *p<0.05, **p<0.01, CFA+EA+IgG versus CFA+EA+anti-END/ENK/DYN; Two-way RM ANOVA, Student-Newman-Keuls).</p