Rabbit anti-IL-17 (Santa Cruz Biotechnology) followed by Cy3-conjugated goat anti abbit IgG (Caltag Laboratories) or goat anti-bendorphin (Santa Cruz Biotechnology) followed by PE-conjugated anti-goat IgG (Abcam). Samples were tested using a FACSCalibur flow cytometer and data analyzed with Cell Quest Pro Software (BD Biosciences).Statistical AnalysisData are expressed as the mean 6 standard error (SE) of 4? observations. Statistical analyses were performed using the SPSS (Statistical Package for Social Sciences, Chicago, IL) software. One-way analysis of variance (ANOVA) was used to determine statistical differences between groups. Clinical scores were analyzed using the non-parametric Mann hitney U-test. P,0.05 was considered statistically significant.Results Effect of Naloxone Post Electroacupuncture MedChemExpress Gracillin treatment of EAEWe previously determined that electroacupuncture treatment of rats decreased lymphocyte infiltration into spinal cords and reduced EAE severity in rats (20). Similar results also showed that rats in the EAE group lost significant weight compared to animals in the EA group by days 12?4 days post-immunization (Fig. 1B, P,0.05) and rats treated with EA 13, 14 and, 15 days post EAE induction presented with reduced EAE clinical scores compared to untreated rats (Fig. 1B, P,0.05). We focused on analysis of the NAL group and demonstrated that despite electroacupuncture administration, EAE presentation in this group was similar to presentation in rats in the EAE group. These facts suggested acupuncture perhaps could enhance endogenous opioid peptides in EAE rats and the effects of beta-endorphins on EAE.Determination of Plasma and Hypothalamus bendorphin ConcentrationsRats from respective treatment groups were sacrificed 7, 14, and 21 d post-MBP68?6 immunization under ether anesthesia and blood from 5 rats/group was obtained by cardiac puncture and collected in heparinized tubes containing sodium EDTA. Plasma was separated by centrifugation at 4uC and stored at 280uC until use. b-endorphin samples from the hypothalamus were extracted as described by Javadi et al. (Javadi, 2003) and stored at 280uC until analyses were performed. Plasma and hypothalamus b-endorphin concentrations were determined using the b-endorphin 125I RIA kit (Tianjin Hope Year Medical Products Co., Ltd., China). The b-endorphin assay Calyculin A sensitivities were 3.5 pg/ml.Induced b-Endorphin Modulates Th Cell Responsescells were harvested from rats in the CFA, EAE, EA, and NAL group rats and proliferation assessed by measuring [3H] thymidine incorporation (Fig. 2). These data supported previous observations demonstrating that the proliferation MBP68?6-specific T cells harvested from EA-treated rats was significantly reduced (P,0.05). There were no differences in proliferation between T cells harvested from NAL and EAE group rats. We next evaluated T cell proliferation by measuring [3H] incorporation 14 days post EAE induction in response to stimulation with either MBP68?6 peptides, MBP68?6 peptides+b-endorphin, or MBP68?6 peptides+b-endorphin+naloxone. This analysis demonstrated that T cells from EAE rats proliferated significantly more compared to proliferation observed in T cells treated with b-endorphin (Fig. 3) and that this effect was reversed by the naloxone.Quantification of Lymphocyte ApoptosisFlow cytometric 1655472 assessment of apoptosis is summarized in Fig. 4 and Fig. 5. The percentage of apoptotic cells was significantly different between the EA and EAE groups 14.Rabbit anti-IL-17 (Santa Cruz Biotechnology) followed by Cy3-conjugated goat anti abbit IgG (Caltag Laboratories) or goat anti-bendorphin (Santa Cruz Biotechnology) followed by PE-conjugated anti-goat IgG (Abcam). Samples were tested using a FACSCalibur flow cytometer and data analyzed with Cell Quest Pro Software (BD Biosciences).Statistical AnalysisData are expressed as the mean 6 standard error (SE) of 4? observations. Statistical analyses were performed using the SPSS (Statistical Package for Social Sciences, Chicago, IL) software. One-way analysis of variance (ANOVA) was used to determine statistical differences between groups. Clinical scores were analyzed using the non-parametric Mann hitney U-test. P,0.05 was considered statistically significant.Results Effect of Naloxone Post Electroacupuncture Treatment of EAEWe previously determined that electroacupuncture treatment of rats decreased lymphocyte infiltration into spinal cords and reduced EAE severity in rats (20). Similar results also showed that rats in the EAE group lost significant weight compared to animals in the EA group by days 12?4 days post-immunization (Fig. 1B, P,0.05) and rats treated with EA 13, 14 and, 15 days post EAE induction presented with reduced EAE clinical scores compared to untreated rats (Fig. 1B, P,0.05). We focused on analysis of the NAL group and demonstrated that despite electroacupuncture administration, EAE presentation in this group was similar to presentation in rats in the EAE group. These facts suggested acupuncture perhaps could enhance endogenous opioid peptides in EAE rats and the effects of beta-endorphins on EAE.Determination of Plasma and Hypothalamus bendorphin ConcentrationsRats from respective treatment groups were sacrificed 7, 14, and 21 d post-MBP68?6 immunization under ether anesthesia and blood from 5 rats/group was obtained by cardiac puncture and collected in heparinized tubes containing sodium EDTA. Plasma was separated by centrifugation at 4uC and stored at 280uC until use. b-endorphin samples from the hypothalamus were extracted as described by Javadi et al. (Javadi, 2003) and stored at 280uC until analyses were performed. Plasma and hypothalamus b-endorphin concentrations were determined using the b-endorphin 125I RIA kit (Tianjin Hope Year Medical Products Co., Ltd., China). The b-endorphin assay sensitivities were 3.5 pg/ml.Induced b-Endorphin Modulates Th Cell Responsescells were harvested from rats in the CFA, EAE, EA, and NAL group rats and proliferation assessed by measuring [3H] thymidine incorporation (Fig. 2). These data supported previous observations demonstrating that the proliferation MBP68?6-specific T cells harvested from EA-treated rats was significantly reduced (P,0.05). There were no differences in proliferation between T cells harvested from NAL and EAE group rats. We next evaluated T cell proliferation by measuring [3H] incorporation 14 days post EAE induction in response to stimulation with either MBP68?6 peptides, MBP68?6 peptides+b-endorphin, or MBP68?6 peptides+b-endorphin+naloxone. This analysis demonstrated that T cells from EAE rats proliferated significantly more compared to proliferation observed in T cells treated with b-endorphin (Fig. 3) and that this effect was reversed by the naloxone.Quantification of Lymphocyte ApoptosisFlow cytometric 1655472 assessment of apoptosis is summarized in Fig. 4 and Fig. 5. The percentage of apoptotic cells was significantly different between the EA and EAE groups 14.
