The Effects And Mechanisms Of Electroacupuncture On Chemotherapy-induced And Inflammatory Pain

BackgroundAcupuncture, a traditional therapeutic modality, has been used in China and other Asiancountries for thousands of years to treat a variety of diseases and symptoms, including pain;Researches including our own support the effectiveness of electroacupuncture (EA) forconditions such as chronic low back and knee pain. In a five-patient pilot study the modalityalso improved the symptoms of chemotherapy-induced neuropathic pain. The underlyingmechanisms of acupuncture analgesia are still not fully understood. It is well documented thatEA's analgesic effects on acute pain are mediated by the endogenous opioids. Further, it hasbeen demonstrated that spinal μ and δ, but not κ opioid receptors are involved inEA-produced anti-hyperalgesia in complete Freund's adjuvant (CFA)-and capsaicin-inducedinflammatory and caudal trunk injury-induced neuropathic pain models. So, opioid receptorsmay also mediate EA suppressing chemotherapy-induced neuropathic pain. On the other hand,researches support the effectiveness of EA on inflammatory pain, and, the effect may partlybe mediated by spinal glial cells related cytokine; Also, it is well known that glial cellsincluding astrocytes and microglia are activated in the lumbar spinal cord in inflammatorypain. The Interleukin-17(IL-17) cytokine family which has recently emerged as a criticalplayer in immune system and inflammatory diseases is known mainly produced by TH17cells. Recent studies showed that glial cells of the central nervous system also express IL-17and studies also showed peripheral IL-17play a role in arthritis pain and neuropathic pain.So, spinal glial cell may also express IL-17, if so, it may be involved in the inflammatory pain.Our previous study demonstrated that EA's analgesic effects on pain are mediated by glialrelated cytokine such as IL-1β. So, IL-17may be also involved in the EA suppressinginflammatory pain.ObjectiveThe study was designed to, first, investigate whether EA can suppresschemotherapy-induced neuropathic pain and whether spinal opioid receptors are involved inEA action on paclitaxel-evoked peripheral neuropathy; second, investigated whether spinalcells express IL-17during inflammatory pain and if so, whether IL-17is involved in theinflammatory pain and whether IL-17is involved in the EA suppressing inflammatory pain. MethodsSeven sets of experiments were conducted:1) effect of EA on hyperalgesia-andallodynia-like behavior in rats with paclitaxel-evoked peripheral neuropathy,2) effect ofopioid sub-receptor antagonists on EA action.3) immunostaining to determineIL-17-immunopositive cells, and IL-17R/NMDAR NR1co-localization,4) completeFreund's adjuvant (CFA)-or saline-injected rats plus IL-17antiserum,5) naive rats plusIL-17protein,6) effects of EA on CFA induced inflammatory pain and IL-17relatedmechanism,7) effects of EA on IL-17induced pain and related mechanism.In Experiment1, rats were divided into the following four groups (n=7per group):paclitaxel+10Hz EA, paclitaxel+100Hz EA, paclitaxel+sham EA, and vehicle+sham EA.Mechanical allodynia/hyperalgesia was assessed on baseline before paclitaxel injection, day13post-paclitaxel injection and30min after each EA treatment on day14,16,18and20dayspost-paclitaxel injection using von Frey filaments with bending forces of2,4,6and15g.In Experiment2, paclitaxel-injected rats were randomly divided into the followinggroups (n=7per group):(1) intrathecal (i.t.) μ opioid receptor antagonist CTOP (12.5nmolin10μl, Sigma) plus10Hz EA;(2) CTOP plus sham EA;(3) i.t. δ opioid receptorantagonist NTI (10nmol in10μl, Sigma) plus10Hz EA;(4) NTI plus shamEA;(5) i.t. κopioid receptor antagonistBNI (10nmol in10μl, Sigma) plus10Hz EA;(6) BNI plus shamEA;(7) saline plus10Hz EA; and (8) saline plus sham EA. All antagonists were dissolved insaline and administered10min before each of EA treatment on day14,16,18and20dayspost-paclitaxel injection. Behavior tests were the same as Experiment1.In Experiment3, double immunofluorescence was used to identify the cell type thatproduces IL-17during inflammatory pain. This experiment comprised two sub-experiments.In sub-experiment1, CFA-inflamed rats were randomly divided into2h and24h post-CFAinjection groups (n=3per group). Another3rats,24h post-saline injection, were used ascontrol. The lumbar spinal cord was used for IL-17immunostaining and doubleimmunofluorescence staining of IL-17with glial fibrillary acidic protein (GFAP), OX-42andNeuN, the respective markers of astrocytes, microglia and neurons. In sub-experiment2,spinal cord sections from the24h group were double stained for IL-17RA and NR1todetermine whether IL-17RA is localized in NMDAR-containing neurons.In Experiment4, antiserum against IL-17was administered to the CFA-orsaline-injected rats to determine whether it alleviates pain. CFA or saline was subcutaneouslyinjected into the plantar surface of one hind paw. CFA-injected rats were randomly divided(n=7per group) into a control group and two IL-17antiserum groups,0.2and2μg/rat (10μl). I.t. IL-17antiserum was given three times, i.e.24h before CFA to block the action of basalIL-17and2h prior to each of two hyperalgesia tests to block the CFA-induced IL-17. Thecontrol group received saline (10μl, i.t.) on the same schedule. Saline-injected rats weresimilarly treated in three sub-groups. Paw withdrawal latency (PWL) tests were conductedbefore CFA (-48h) for baseline and2and24h after CFA to measure thermal hyperalgesia.Spinal cord was removed after PWL testing24hr post-CFA injection. The relative levels ofIL-17RA and NR1phosphorylation was measured with western blot.In Experiment5, naive rats were divided into five groups (n=7per group), which wererespectively treated with saline,10ng,100ng,400ng IL-17or400ng IL-17plus2μg ofantiserum against IL-17. PWL was measured at-48h prior to i.t. injection and2,24and48hpost the i.t. injection. Since IL-17induced the most significant decrease of PWL24hrpost-IL-17injection, spinal cord was removed24hr post-IL-17injection in5groups (n=4per group) of rats treated as in the experiment3. And The relative levels of IL-17RA and NR1phosphorylation was measured with western blot.In Experiment6, rats were divided into the following three groups (n=7per group):CFA+EA, CFA+sham EA, and vehicle+sham EA. PWL was measured at-48h prior to CFAinjection and2and24h post the injection. Immunofluorescence was used to measure the levelof IL-17expression in lumbar spinal cord and the relative levels of IL-17RA and NR1phosphorylation was measured with western blot.In Experiment7, rats were divided into the following three groups (n=8per group):Saline+Sham, IL-17+Sham, and IL-17+EA. PWL was measured at-48h prior to IL-17(400ng, i.t.) injection and2and24h post the i.t. injection and the relative levels of IL-17RA andNR1phosphorylation was measured with western blotResults1. From days13to20post-paclitaxel injection, drug-injected rats showed significantlyhigher mechanical response frequencies than did vehicle-injected ones when both groups weregiven sham EA (P<0.05). At stimulation of4,8, and15g,10Hz EA treatment significantly(P<0.05) decreased response frequency compared to sham EA while100Hz EA treatmentonly decreased response frequency at15g.2. EA plus vehicle (i.t.) significantly decreased mechanical response frequency comparedto sham EA plus vehicle (P<0.05), EA plus CTOP did not significantly decrease responsefrequency compared to sham EA plus vehicle; Sham EA plus CTOP did not significantly change mechanical response frequency compared to sham EA plus vehicle. Similarly, EA pluseither the δ or κ opioid receptor antagonist did not significantly decrease mechanicalresponse frequency compared to sham EA plus vehicle; Sham EA plus NTI or BNI did notsignificantly change mechanical response frequency compared to sham EA plus vehicle.3. Double immunofluorescence labeling demonstrated that IL-17immunoreactivity wasco-localized with the astrocyte marker GFAP. In laminae I-II, the number of ipsilateral IL-17immunoreactive cells2and24hr post-CFA was significantly increased compared to that insaline control rats (P<0.01). The number of contralateral IL-17immunoreactive cells wasslightly but not significantly higher than that in saline control rats(P>0.05). The number ofipsilateral IL-17immunoreactive cells was also significantly higher than that of contralateralIL-17immunoreactive cells24hr post CFA (P=0.039). In laminae V-VI, IL-17immunoreactive cells were significantly increased both ipsilaterally and contralaterally2hrpost-CFA compared to those of saline control rats(P<0.05), as were ipsilateral cells24hrpost-CFA(P=0.034). In lamina X, IL-17was significantly elevated2hr (P=0.049) but not24hr post-CFA compared to that in saline control rats (P=0.167). In laminae III-IV, nosignificant differences were observed (P>0.05).Double immunofluorescence labeling demonstrated that NR1and IL-17RA co-localizein spinal cord neurons.4. In CFA-injected rats, the IL-17antiserum pretreatment dose-dependently alleviatedthe CFA-induced hyperalgesia assessed by the PWL test. Repeated-measures ANOVArevealed the main effect of drug treatment (P<0.05), time (P<0.001), and the interaction ofdrug treatment and time (P=0.84). Post hoc means comparisons revealed that i.t. IL-17antiserum at2μg/rat significantly increased PWL compared to saline treatment (P<0.05).Contralateral PWL did not change after the IL-17antiserum treatment in CFA-injected rats,nor did PWL in saline-injected rats.P-NR1levels were significantly higher in vehicle-treated CFA rats compared tosaline-injected control. The levels were significantly lower in CFA-injected rats given2μgIL-17antiserum than those in rats given vehicle (P<0.05).IL-17RA levels were significantly higher in CFA-injected rats than those insaline-injected control rats (P<0.05) and significantly lower in rats given2μg IL-17antiserum than those in rats given vehicle or0.2μg IL-17antiserum (P<0.05).5. IL-17dose-dependently induced a significant decrease in PWL of both hind paws. At400ng, the decrease began2h and peaked24h after administration; PWL normalized48h after the injection. Repeated-measures ANOVA revealed the main effect of drug treatment(P<0.001), time (P<0.0001), and the interaction of drug treatment and time (P<0.05). Posthoc means comparisons revealed that i.t. IL-17at100ng and400ng/rat significantlydecreased PWL from2-24hours post-CFA compared to saline control (P<0.01and P<0.0001,respectively). IL-17plus its antiserum in naive rats did not differ from saline control.IL-17dose-dependently induced NR1phosphorylation, paralleling the behavioralhyperalgesia induced by i.t. IL-17. IL-17antiserum pretreatment significantly blockedIL-17-induced p-NR1.IL-17dose-dependently increased IL-17RA. IL-17antiserum pretreatment significantlyblocked such up-regulation.6. EA significantly alleviated the CFA-induced hyperalgesia at2-24hr post-CFAinjection assessed by the PWL test. Repeated-measures ANOVA revealed the effect of EA onthe PWL of ipsilateral,main treatment (P<0.01), time (P<0.01), and the interaction of EAtreatment and time (P<0.01). Post hoc means comparisons revealed that CFA significantlydecreased PWL from2-24hours post-CFA compared to saline control (P<0.01). EAsignificantly block the decrease (P<0.05)。Contralateral PWL did not change after EAtreatment in CFA-injected rats, nor did PWL in saline-injected rats。IL-17, P-NR1and IL-17RA levels were significantly higher in sham-treated CFA ratscompared to saline-injected control. The levels were significantly lower in EA treated thanSham treated CFA rats (P<0.05).7. EA significantly alleviated the IL-17-induced hyperalgesia at2-24hr post-IL-17injection assessed by the PWL test (average of both side)。Repeated-measures ANOVArevealed the effect of EA main treatment (P<0.01), time (P<0.01), and the interaction of EAtreatment and time (P<0.01). Post hoc means comparisons revealed that IL-17significantlydecreased PWL from2-24hours post-IL-17compared to saline control (P<0.01). EAsignificantly block the decrease (P<0.05)。P-NR1and IL-17RA levels were significantly higher in sham-treated CFA rats comparedto saline-injected control. The levels were significantly lower in EA treated than Sham treatedCFA rats (P<0.05).Conclusion1. EA can inhibit chemotherapy-induced neuropathic pain through three subtypes ofspinal opioid receptor and EA may be a useful treatment for neuropathic pain patients. 2. Spinal IL-17, produced by astrocytes, enhances NR1phosphorylation to facilitatepersistent inflammatory pain.3. EA alleviates inflammatory pain, at least in part by suppressing spinal IL-17expresssion.4. The results support the clinical use of EA in the treatment of chemotherapy-inducedand inflammatory pain...