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

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

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

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

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

Establishment of CCL2-induced hyperalgesia following restoration of defective ROS generation in Dark Agouti (DA) rats.

<p>Peritoneal macrophages from Wistar and DA rats were isolated and treated for 1 h with 0.2 µg CCL2 or solvent control. ROS production was quantified by flow cytometry using the phagoburst assay. Representative histograms are shown for Wistar rats (<b>A</b>) and DA rats (<b>B</b>) with solvent (grey), CCL2 (black line) or positive assay control PMA (dotted line). (<b>C</b>) Results were analyzed statistically (n = 6/group, CCL2 i.pl. (white bars) or solvent (black bars), *p<0.05, t-test). (<b>D</b>) Wistar and DA rats were i.pl. injected with CCL2 i.pl. (lower panel) or solvent (upper panels) for 3 h, subcutaneous paw tissue was representative immunohistochemistry is shown for HNE (red), ED1⁺ macrophages (green) and merge of both together with nuclei (DAPI-blue). (<b>E</b>) Wistar and DA rats were i.pl. injected with CCL2 i.pl. (white bars) or solvent (black bars) for 3 h, subcutaneous paw tissue was prepared and HNE adducts were quantified by ELISA (n = 6/group, *p<0.05, t-test). (<b>F</b>) DA rats were s.c. injected with phytol for 5 days. Mechanical nociceptive thresholds were quantified before and after i.pl. injection of CCL2 (0.3 µg – open circle, 1 µg – filled triangle, 3 µg – open triangle). Control animals were treated 5 days with 0.9 % NaCl before i.pl. injection of 3 µg CCL2 (filled circle), (*p<0.05 versus time point 0 h, Two way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM.</p

Comparable nociceptive thresholds and DRG currents in Wistar and Dark Agouti (DA) rats.

<p>Rats received an i.pl. injection of CFA (<b>A–D</b>; ipsilateral injected paw  =  open circle, contralateral, non injected paw  =  filled circle). Mechanical (<b>A, B</b>) and thermal nociceptive (<b>C, D,</b>) thresholds were quantified (*p<0.05 versus time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM of raw values (<b>A, B</b>) or % maximal possible effects (MPE) (<b>C, D</b>). (<b>E</b>) DRG-neurons from naïve DA rats were obtained and cultured for 48 h. Representative traces of whole cell recordings are shown after application of capsaicin (500, 1000 nM, left). Bar graph quantifies capsaicin-activated inward currents in DRG neurons from Wistar and DA rats (right) (n = 9, means ± SEM, p>0.05, t- test). In addition DA rats received i.pl. injection of capsaicin (<b>F</b>; solvent – filled circle, 30 µg capsaicin – open circle) and thermal nociceptive thresholds were quantified (*p<0.05 versus time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM of % maximal possible effects (MPE).</p

Reconstitution of CCL2-induced hyperalgesia by adoptive transfer of macrophages derived from Wistar, but not from Dark Agouti (DA) rats.

<p>(<b>A</b>) Mechanical nociceptive thresholds were quantified after i.pl. injection of 3 µg CCL2 with prior leukocyte depletion by systemic injection of CTX (filled triangles), 3 µg CCL2 with prior systemic injection of solvent (cyclo-phosphamid (CTX) treatment (open circles), or solvent of CCL2 without CTX treatment (filled circles). CTX injections were performed 3 d with 10 mg/kg and 1d 50 mg/kg before cell and/or CCL2 treatment. (<b>B</b>) Representative dot blots (x-axis: forward scatter (FSC) – cell size; y-axis: sideward scatter (SSC) – cell granularity) for leukocyte subpopulations in peripheral blood of untreated (left) and CTX-treated Wistar rats (right) are shown by flow cytometry. Gates were set on beads (for quantification), neutrophils, lymphocytes and monocytes. (<b>C–F</b>) Wistar and DA rats were leukocyte-depleted by i.p. injection with CTX followed by i.pl. injection of different numbers of macrophages from either Wistar or DA rats, 30 min later, by i.pl. injection of 3 µg CCL2. Injection of 2×10⁶ macrophages without concomitant injection of CCL2 served as a negative control. (<b>C</b>) Wistar rats reconstituted with cells from Wistar rats. I.p. injections of solvent only served as a negative control for CTX treatment (open triangles) with the same injection pattern. (<b>D</b>) Wistar rats reconstituted with cells from DA rats. (<b>E</b>) DA rats reconstituted with cells from Wistar rats. (<b>F</b>) DA rats reconstituted with cells from DA rats. (*p<0.05 compared to time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM.</p

CCL2-induced hyperalgesia in Wistar, but not in Dark Agouti (DA) rats.

<p>Mechanical (<b>A, B</b>) and thermal (<b>C, D</b>) nociceptive thresholds were quantified before and after i.pl. injection of CCL2 (0.3 µg – open circle, 1 µg – filled triangle, 3 µg – open triangle) or 0.9 % NaCl (solvent  =  control – filled circle) into Wistar (<b>A, C</b>) and DA (<b>B, D</b>) rats (*p<0.05 versus time point 0 h, Two Way RM ANOVA, Student-Newman-Keuls, n = 6). Data are presented as means ± SEM of raw values (<b>A, B</b>) or % maximal possible effects (MPE) (<b>C, D</b>).</p