Effects and Mechanisms of Electroacupuncture on Chronic Inflammatory Pain and Depression Comorbidity in Mice

Abstract

Comorbidity of chronic pain and major depression disorder (MDD) are common diseases. However, the mechanisms of electroacupuncture (EA) and the responses of N-methyl-D-aspartate receptors in the brain remain unclear. Three injections of complete Freund's adjuvant (CFA) were administered to induce chronic inflammatory pain (CIP). EA was then performed once every other day from days 14 to 28. Behavior tests of chronic pain and depression were evaluated to make sure of the successful induction of comorbidity. We used Western blotting to analyze brain tissue from the prefrontal cortex (PFC), hippocampus, and hypothalamus for levels of phosphorylated N-methyl-D-aspartate receptor subunit 1 (pNR1), NR1, pNR2B, NR2B, and calcium/calmodulin-dependent protein kinase type II alpha isoform (pCaMKIIα). The mechanical hyperalgesia, thermal hyperalgesia, and depression were observed in the CIP group. Furthermore, decreased levels of N-methyl-D-aspartate receptors (NMDARs) were also noted. Not Sham EA but EA reversed chronic pain and depression as well as the decreased levels of NMDA in the signaling pathway. The CFA injections successfully induced a significant comorbidity model. EA treated the comorbidity by upregulating the NMDA signaling pathway in the PFC, hippocampus, and hypothalamus. Our results indicated significant mechanisms of comorbidity of chronic pain and MDD and EA-analgesia that involves the regulation of the NMDAR signaling pathway. These findings may be relevant to the evaluation and treatment of comorbidity of chronic pain and MDD.

Figure 1

EA significantly attenuated mechanical, thermal hyperalgesia, and depression induced by CFA. (a) Changes in the withdraw threshold of mice in the von Frey test. (b) Changes in the withdraw latency of mice in the radial heat test. Figures 1(c) and 1(d) present the day 28 OFT results. After accepting EA, the time mice stayed in the center area longer. (c) The distance mice stayed in the central area. (d) The percentage of time mice spent in the center area. Day 28 FST results are presented in Figures 1(e) and 1(f). EA reduced the immobility of mice. (e) Duration of immobility spent in the FST. (f) Frequency of immobility counted in the FST. ^∗p < 0.05 for CIP versus control. ^#p < 0.05 for EA versus CIP.

Figure 2

Expression levels of pCaMKIIα in the mPFC, hippocampal CA1, and hypothalamus for all four groups. (a) Representative immunofluorescence staining of pCaMKIIα (green) in the mice mPFC; (b) representative immunofluorescence staining of pCaMKIIα (green) in the mice hippocampal CA1; (c) representative immunofluorescence staining of pCaMKIIα (green) in the mice hypothalamus. Arrows indicate immunopositive neurons.

Figure 3

Protein levels of (a) pNR1 (b) NR1, (c) pNR2B, (d) NR2B, and (e) pCaMKIIα, measured through Western blotting in mice mPFC. ^∗p < 0.05 versus control. The Western blot bands at the top indicate the cropped target protein. The lower bands indicate the cropped internal controls (β-actin or α-tubulin).

Figure 4

Protein levels of (a) pNR1 (b) NR1, (c) pNR2B, (d) NR2B, and (e) pCaMKIIα, measured using Western blotting, in mice hippocampus. ^∗p < 0.05 versus control. Western blot bands at the top indicate the cropped target protein. Lower bands indicate the cropped internal controls (β-actin or α-tubulin).

Figure 5

Protein levels of (a) pNR1 (b) NR1, (c) pNR2B, (d) NR2B, and (e) pCaMKIIα, measured using Western blotting in mice hypothalamus. ^∗p < 0.05 versus control. Western blot bands at the top indicate the cropped target protein. Lower bands indicate the cropped internal controls (β-actin or α-tubulin).