Electroacupuncture in conscious free-moving mice reduces pain by ameliorating peripheral and central nociceptive mechanisms

Abstract

Integrative approaches such as electroacupuncture, devoid of drug effects are gaining prominence for treating pain. Understanding the mechanisms of electroacupuncture induced analgesia would benefit chronic pain conditions such as sickle cell disease (SCD), for which patients may require opioid analgesics throughout life. Mouse models are instructive in developing a mechanistic understanding of pain, but the anesthesia/restraint required to administer electroacupuncture may alter the underlying mechanisms. To overcome these limitations, we developed a method to perform electroacupuncture in conscious, freely moving, unrestrained mice. Using this technique we demonstrate a significant analgesic effect in transgenic mouse models of SCD and cancer as well as complete Freund's adjuvant-induced pain. We demonstrate a comprehensive antinociceptive effect on mechanical, cold and deep tissue hyperalagesia in both genders. Interestingly, individual mice showed a variable response to electroacupuncture, categorized into high-, moderate-, and non-responders. Mechanistically, electroacupuncture significantly ameliorated inflammatory and nociceptive mediators both peripherally and centrally in sickle mice correlative to the antinociceptive response. Application of sub-optimal doses of morphine in electroacupuncture-treated moderate-responders produced equivalent antinociception as obtained in high-responders. Electroacupuncture in conscious freely moving mice offers an effective approach to develop a mechanism-based understanding of analgesia devoid of the influence of anesthetics or restraints.

Figure 1. Electroacupuncture results in varied antinociceptive responses in three separate pain models.

Mechanical hypealgesia in (A) male (n = 8–10/group) and (B) female sickle mice (n = 18–20 per group), (C) female transgenic C3TAg mice with painful tumors (n = 8–10 per group), and (D) male FVB/N mice with complete Freund’ adjuvant (CFA)–induced inflammatory pain (n = 8–10 per group) were treated with four consecutive electroacupuncture (EA, frequency: 4–10 Hz, intensity: #3 out of 10, pulse width: 100 μm, duration: 30 min) at day 0 (baseline: BL) and days 3, 6, and 9. Pain measures were obtained before starting electroacupuncture treatments on day 0 (baseline, BL), and daily until day 12 before each electroacupuncture treatment. Similar pain measures were also performed on sham treated (Sham-EA) or untreated mice (Untreated). Paw withdrawal frequency (PWF) threshold response to mechanical allodynia was determined (see Methods) and expressed as the % change in pain threshold. (E–H) Based on the percent change in pain threshold, mice were grouped as high- (nociceptive threshold increase >200%), moderate (nociceptive threshold increase 100~200%) and non-responders (nociceptive threshold increase ≤100%) for days 7–12. *p < 0.05 vs BL of matching group; ^$p < 0.05 vs untreated; ^#p < 0.05 vs sham-EA (two-way ANOVA, Tukey); ^§p < 0.05 vs EA moderate-responders; ^†p < 0.05 vs EA non-responders. Data are mean ± SEM of values normalized to BL for (A–C) and normalized to day 1 post-CFA injection and electroacupuncture treatment for (D). Abbreviations: BL: baseline; complete Freund’s adjuvant: CFA; EA, electroacupuncture; i.pl.: intraplantar; PWF: paw withdrawal frequency.

Figure 2. Electroacupuncture attenuates chronic hyperalgesia in sickle mice.

Male (A,B) and female (C,D) sickle mice (HbSS) were treated with four consecutive electroacupuncture (EA) treatments as described in Fig. 1. Measures of (A,C) thermal sensitivity to cold (PWF), and (B,D) deep tissue pain (grip force) are shown. *p < 0.05 vs BL of matching group; ^$p < 0.05 vs Sickle untreated; ^#p < 0.05 vs Sickle + sham-EA (two-way ANOVA, Tukey). Each value is the mean ± SEM from 8–10 mice with 3 observations per mouse. Abbreviations: BL: baseline; EA, electroacupuncture; PWF, paw withdrawal frequency.

Figure 3. Electroacupuncture reduces systemic inflammation in sickle mice.

Levels of (A) serum amyloid P (SAP), (B) IL-6, (C) IL-1beta, (D) TNF-alpha, and (E) substance P (SP) in plasma of HbSS-BERK sickle mice were measured on day 13 after the last nociceptive assessment. Electroacupuncture (EA)-treated sickle mice were grouped in high- (n = 9–10), moderate- (n = 6–7), and non-responders (n = 4–5) according to the varied antinociceptive responses observed in Fig. 1. Levels in untreated (n = 7–9) or sham treated (n = 7–9) sickle mice were were also assesed. ^$p < 0.05 vs Sickle untreated; ^†p < 0.05 vs Sickle + EA high-responders (one-way ANOVA, Tukey). Data are presented as mean ± SEM. Abbreviations: EA, electroacupuncture; SAP, serum amyloid P; SP: substance P.

Figure 4. Electroacupuncture reduces peripheral inflammation and mast cell activation in sickle mice.

Levels of (A) IL-1beta, (B) substance P (SP), (C) IL-6, and (D) TNF-alpha from cultured skin biopsies were compared between untreated (n = 6–11) or sham treated (n = 7–9) sickle mice, as well as electroacupuncture (EA)-treated high- (n = 6–9), moderate- (n = 5–8) and non-responder (n = 3–5) sickle mice. (E) Representative images of toluidine blue-stained mast cells in skin for untreated or sham treated sickle mice, as well as electroacupuncture-treated high-, moderate-, and non-responder sickle mice (n = 5 for each group). Scale bar, 10 μm. (F) Degranulated mast cells as percentage of total mast cells (n = 5 for each group). ^$p < 0.05 vs Sickle untreated; ^#p < 0.05 vs Sickle + sham-EA; ^†p < 0.05 vs Sickle + EA high-responders (one-way ANOVA, Tukey for D and F, Dunnett’s for C). Data are presented as mean ± SEM. Abbreviations: EA, electroacupuncture; SP: substance P.

Figure 5. Electroacupuncture ameliorates neurogenic inflammation evoked by substance P or capsaicin in sickle mice.

(A,B) Evans blue leakage evoked by substance P (SP), capsaicin (CS), or vehicle (Veh) in untreated or sham treated sickle mice, as well as electroacupuncture (EA)-treated high-, moderate-, and non-responder sickle mice. Each image represents reproducible images from 5 mice each, and each value is the mean ± SEM from 5 male mice. ^$p < 0.05 vs Sickle untreated; ^†p < 0.05 vs Sickle + EA high-responders; ^¶p < 0.05 vs vehicle of matching group (one-way ANOVA, Tukey). Abbreviations: EA, electroacupuncture; CS, capsaicin; SP: substance P; Veh, vehicle.

Figure 6. Electroacupuncture attenuates spinal cord nociception in sickle mice.

Levels of (A) IL-6, (B) TNF-alpha, (C) IL-1beta, and (D) SP were measured in spinal cord lysates of HbSS-BERK sickle mice collected after the last day of nociceptive assessment. Electroacupuncture-treated sickle mice were grouped as high- (n = 6–8), moderate- (n = 6–10), or non-responders (n = 2–5). Analysis of untreated (n = 6–10) or sham treated (n = 5–10) sickle mice is also shown. (E,F) Phosphorylation level of p38 MAPK relative to total p38 MAPK in the spinal cord of untreated (n = 7), sham treated (n = 6), and EA-treated high- (n = 5), moderate- (n = 6), or non-responder (n = 4) sickle mice. (G–J) Phosphorylation level of p44/42 MAPK or STAT3 relative to total p44/42 MAPK or STAT3 in the spinal cord of untreated, sham treated, or EA-treated high-responder sickle mice (n = 3–5 in each group). ^$p < 0.05 vs Sickle untreated; ^#p < 0.05 vs Sickle + sham-EA; ^§p < 0.05 vs Sickle + EA moderate-responders; ^†p < 0.05 vs Sickle + EA high-responders (one-way ANOVA, Tukey). Data are presented as the mean ± SEM. Abbreviations: EA, electroacupuncture; SP: substance P.

Figure 7. Morphine enhances electroacupuncture-induced attenuation of chronic hyperalgesia in sickle mice.

HbSS-BERK sickle mice were treated with three consecutive electroacupuncture treatments as described in Fig. 1, and grouped according to their nociceptive response as in Fig. 1. At day 7, morphine (MS, 10 mg/kg) or vehicle (Veh) solution was administered to electroacupuncture-treated (A,B) moderate- (n = 6) or (C,D) non-responder (n = 5) sickle mice. (E,F) Non-responders also received 20 mg/kg morphine (n = 5). (A,C,E) PWF threshold response to mechanical allodynia is expressed as % change of pain threshold, and (B,D,F) deep tissue pain is measured by grip force. (G) Measurements were taken at BL (before electroacupuncture treatment) and day 7; on day 7 multiple measurements were taken: time 0 (before MS/Veh administration) and 0.5, 1, 2, 4, and 24 h after MS/Veh injection. *p < 0.05 vs BL of matching group; ^¶p < 0.05 vs Veh at same time point (two-way ANOVA, Tukey). Data are presented as the mean ± SEM. Abbreviations: BL: baseline; EA, electroacupuncture; MS, morphine; Veh, vehicle.