Inhibition of chemokine CX3CL1 in spinal cord mediates the electroacupuncture-induced suppression of inflammatory pain.

PURPOSE: Chemokine CX3CL1 and its receptor CX3CR1 in the lumbar spinal cord play crucial roles in pain processing. Electroacupuncture (EA) is recognized as an alternative therapy in pain treatment due to its efficacy and safety. However, the analgesic mechanism of EA remains unclear. The aim of this study was to investigate whether EA suppressed complete Freund's adjuvant (CFA)-induced pain via modulating CX3CL1-CX3CR1 pathway. MATERIALS AND METHODS: Inflammatory pain was induced by intraplantar injection of CFA to the left hind paw of Sprague-Dawley rats. EA with 2 Hz for 30 mins was given to bilateral Zusanli acupoints (ST36) on the first and third day after CFA injection. Mechanical allodynia and thermal hyperalgesia were tested with von Frey tests and Hargreaves tests, respectively. The expressions of CX3CL1, CX3CR1 and p38 mitogen-activated protein kinase (MAPK) were quantified with Western blots. The release of IL-1ß, IL-6 and TNF-α were evaluated with ELISA. Recombinant CX3CL1 or control IgG were then injected through intrathecal catheters in the EA-treated CFA model rats. The behavioral tests, p38 MAPK activation and cytokine release were then evaluated. RESULTS: EA significantly inhibited inflammatory pain induced by CFA for 3 days. Meanwhile, EA downregulated the expression of CX3CL1 but not CX3CR1 in the lumbar spinal cord of the CFA rats. Besides, activation of p38 MAPK and the release of pain-related cytokines (IL-1ß, IL-6 and TNF-α) were inhibited by EA. Intrathecal injection of CX3CL1 largely reversed the analgesic effect of EA treatment and re-activated p38 MAPK signaling, and resulted in pro-inflammatory cytokines increase in acupuncture-treated rats. CONCLUSION: Our findings indicate that EA alleviates inflammatory pain via modulating CX3CL1 signaling in lumbar spinal cord, revealing a potential mechanism of anti-nociception of EA in inflammatory pain.

Spinal Cord Glycine Transporter 2 Mediates Bilateral ST35 Acupoints Sensitization in Rats with Knee Osteoarthritis.

The concept of "acupoint sensitization" refers to the functional status of acupoint switches from silent to active under pathological conditions. In clinic, acupoint sensitization provides important guidance for acupoints selection in different diseases. However, the mechanism behind this phenomenon remains unclear. We generated a model of knee osteoarthritis (KOA) by intra-articular injection of monosodium iodoacetate (MIA) into the left knee of rats. The paw withdrawal mechanical threshold (PWMT) and the total number of mast cells as well as mast cell degranulation rate (MCDR) of acupoint tissue were used to test whether the acupoints were sensitized. The results showed that KOA resulted in a reduced mechanical threshold and elevated total number of mast cell as well as mast cell degranulation rate at bilateral ST35 (Dubi) but not GB37 (Guangming) or nonacupoint area. The acupoint sensitization was accompanied by upregulation of glycine transporter 2 (GlyT2) and reduction of extracellular glycine levels in the bilateral dorsal horns of the spinal cord at L3-5. Selective inhibition of GlyT2 or intrathecal administration of glycine attenuated ST35 acupoint sensitization. The sensitization of bilateral ST35 was blocked after intraspinal GlyT2 short hairpin (sh) RNA (GlyT2-shRNA) microinjection to specifically downregulate GlyT2 expression in the left side (ipsilateral) L3-5 spinal cord dorsal horn before MIA injection. Moreover, electroacupuncture (EA) stimulation at ST35 ameliorated articular pathological lesions and improved KOA-related pain behaviors. GlyT2-shRNA injection reversed EA-induced pain relief but not EA-induced reduction of joint lesions. Overall, this study demonstrated that spinal GlyT2, especially elevated GlyT2 expression in the ipsilateral dorsal horn of the spinal cord, is a crucial mediator of ST35 acupoint sensitization in KOA rats.

Acupoint Sensitization is Associated with Increased Excitability and Hyperpolarization-Activated Current (Ih) in C- But Not Aδ-Type Neurons.

Under pathological conditions, acupoint sensitization is the phenomenon of acupoints transforming from the stable state to the dynamic state. Evidences suggest that hyperpolarization-activated current (Ih), conducted by the hyperpolarization-activated/cyclic nucleotide-gated (HCN) channel, greatly contributes to the peripheral and central sensitization. However, the role of the Ih current in acupoint sensitization has not been explained. In the present study, changes in excitability, Ih density and the HCN channel of dorsal root ganglion (DRG) nociceptive neurons were examined in the later phase of knee osteoarthritis (KOA) rats. To investigate the neuronal specificity of acupoint sensitization, retrograde dyes were injected into the acupoints ST35 and GB37. The results showed that acupoint sensitization occurred in bilateral ST35 but not GB37 acupoints. The excitability and Ih density of C- but not Aδ-type neurons innervating ST35 acupoint increased in bilateral L5 DRG of acupoint sensitized rats than that of sham rats. No obvious changes were found in the excitability or Ih density of C- and Aδ-type neurons innervating the GB37 acupoint in the bilateral L5 DRG. HCN channel subtype 2 (HCN2) expression levels significantly increased after acupoint sensitization. Furthermore, ZD7288, an HCN current (Ih) blocker, attenuated the acupoint sensitization of the ST35 acupoint. Taken together, our findings suggest that the increased excitability of C- but not Aδ-type neurons and the upregulation of Ih/HCN2 channels contribute to the formation of acupoint sensitization.

A rat model of knee osteoarthritis suitable for electroacupuncture study.

Acupuncture is widely used for knee osteoarthritis (KOA) treatment in clinical practice. In the present study, we aimed to set a standard KOA animal model for electroacupuncture (EA) study and provide an acupuncture recipe for further KOA studies. Rats intra-articularly administered monosodium iodoacetate (MIA, 0.3, 1 or 3 mg respectively, n=12 each) were evaluated for pain-like behavior: paw withdrawal mechanical threshold, weight bearing deficit, and joint pathological changes (OARSI score) until 28 days after injury. Then by using the suitable dose (1 mg MIA), therapeutic effects of EA treatment (bilateral ST36 and ST35 acupoints, 2/10 Hz, 30 min/d, 6d/w, 2w) were evaluated in 3 groups (n=16 each): Early-on EA, Mid-term EA and Delayed EA, in which EA was started on day 1, day 7 or day 14 after MIA injection. Both 1 mg and 3 mg MIA induced significant joint damage and persistent pain behavior. But animals accepted 3 mg MIA rapidly developed cartilage and bone damage within 14 days. Early-on EA treatment provided significant pain relief and joint structure preservation in KOA rats. Mid-term EA treatment only reduced pain, while delayed EA treatment resulted in no effects in both aspects. 1 mg of MIA produces steady pain behavior and progressive joint damage, which was suitable for EA treatment evaluation. Early-on EA treatment provided both joint protection and pain reduction, while Mid-term EA could only be used for studying EA-induced analgesia in KOA.