Electroacupuncture activates corticotrophin-releasing hormone-containing neurons in the paraventricular nucleus of the hypothalammus to alleviate edema in a rat model of inflammation

Abstract

Background: Studies show that electroacupuncture (EA) has beneficial effects in patients with inflammatory diseases. This study investigated the mechanisms of EA anti-inflammation, using a rat model of complete Freund's adjuvant (CFA)-induced hind paw inflammation and hyperalgesia.

Design: Four experiments were conducted on male Sprague-Dawley rats (n = 6-7/per group). Inflammation was induced by injecting CFA into the plantar surface of one hind paw. Experiment 1 examined whether EA increases plasma adrenocorticotropic hormone (ACTH) levels. Experiments 2 and 3 studied the effects of the ACTH and corticotropin-releasing hormone (CRH) receptor antagonists, ACTH(11-24) and astressin, on the EA anti-edema. Experiment 4 determined whether EA activates CRH neurons in the paraventricular nucleus of the hypothalammus. EA treatment, 10 Hz at 3 mA and 0.1 ms pulse width, was given twice for 20 min each, once immediately post and again 2 hr post-CFA. Plasma ACTH levels, paw thickness, and paw withdrawal latency to a noxious thermal stimulus were measured 2 h and 5 h after the CFA.

Results: EA significantly increased ACTH levels 5 h (2 folds) after CFA compared to sham EA control, but EA alone in naive rats and CFA alone did not induce significant increases in ACTH. ACTH(11-24) and astressin blocked EA anti-edema but not EA anti-hyperalgesia. EA induced phosphorylation of NR1, an essential subunit of the N-methyl-D-aspartic acid (NMDA) receptor, in CRH-containing neurons of the paraventricular nucleus.

Conclusion: The data demonstrate that EA activates CRH neurons to significantly increase plasma ACTH levels and suppress edema through CRH and ACTH receptors in a rat model of inflammation.

Figure 1

Effects of EA treatment on plasma ACTH levels (% changes vs baseline, n = 6 per group, mean ± SEM). EA treatment induced no significant changes of plasma ACTH in uninflamed rats. CFA-induced inflammation alone resulted in no significant changes in plasma ACTH levels. EA treatment in inflamed rats significantly increased ACTH levels compared to sham EA. *P < 0.05 compared to sham EA.

Figure 2

Effects of ACTH_(11–24), an ACTH receptor antagonist, on EA-produced inhibition of edema (n = 7 per group, mean ± SEM). All rats were CFA-inflamed. Note that ACTH_(11–24) prevented EA-produced inhibition of edema 5 h post CFA. Edema was determined by increased paw thickness (mm). * P < 0.05 compared to vehicle plus sham EA.

Figure 3

Effects of ACTH_(11–24) on EA-produced inhibition of hyperalgesia (n = 7 per group, mean ± SEM). Note that EA significantly inhibited hyperalgesia 2 h post-CFA, and ACTH_(11–24) did not prevent EA-produced inhibition of hyperalgesia. * P < 0.05 compared to vehicle plus sham EA.

Figure 4

Effects of astressin, a CRH receptor antagonist, on EA-produced inhibition of edema (n = 7 per group, mean ± SEM). All rats were CFA-inflamed. Note that astressin prevented EA-produced inhibition of edema 5 hr post CFA. * P < 0.05 compared to vehicle plus sham EA.

Figure 5

Effects of astressin on EA-produced inhibition of hyperalgesia (n = 7 per group, mean ± SEM). Note that EA significantly inhibited hyperalgesia 2 h post-CFA, and astressin partially prevented EA-produced inhibition of hyperalgesia. * P < 0.05 compared to vehicle plus sham EA.

Figure 6

Photomicrographs showing co-localization of CRH and p-NR1 in the PVN. A-C: Sections from EA-treated naive rats were double-labeled with anti-CRH (red) and anti-p-NR1 (green). A: CRH-immunoreactive neurons in the PVN. B: p-NRI-immunoreactive neurons in the PVN. C: Merged graphs of A and B. Small arrows indicate examples of double-labeled CRH/p-NR1 neuron (yellow); Arrowheads point to single-labeled CRH and p-NR1. The insets in A, B and C are higher magnification of the square areas in A, B and C, respectively. D: Sections from untreated naive rats were signle-labeled with anti-p-NR1 and showed no labeling of P-NR1. Scale bars represent 50 μm in A, B, C, and 250 μm in D.