The real-time detection of acupuncture-induced extracellular ATP mobilization in acupoints and exploration of its role in acupuncture analgesia.

Our and in vitro studies had confirmed that mechanosensitive ATP release and accumulation in acupoints was elicited by acupuncture (AP), which might be a pivotal step for triggering AP analgesia. But to date, the dynamics of extracellular ATP (eATP) in the interstitial space during AP process was poorly known, mainly due to the low temporal resolution of the current detection approach. This study attempted to capture rapid eATP signals in vivo in the process of needling, and further explored the role of this eATP mobilization in initiating AP analgesic effect. Ipsilateral 20-min needling was applied on Zusanli acupoint (ST36) of complete Freund's adjuvant (CFA)-induced ankle arthritis rats. Pain thresholds were assessed in injured-side hindpaws. eATP in the interstitial space was microdialyzed and real-time quantified by luciferin-luciferase assay at 1-min interval with the aid of the microfluid chip. We revealed in behavioral tests that modulation of eATP levels in ST36 influenced AP analgesic effect on ankle arthritis. A transient eATP accumulation was induced by needling that started to mobilize at 4 min, climbed to the peak of 11.21 nM within 3.25 min and gradually recovered. Such AP-induced eATP mobilization was significantly impacted by ankle inflammation, needling depth, needle manipulation, and the presence of local ecto-nucleotidases. This work reveals that needling elicits a transient eATP mobilization in acupoints, which contributes to initiating AP analgesia. This study will help us better understand the peripheral mechanism of AP analgesia and guide clinicians to optimize the needle manipulations to improve AP efficacy.

Mechanosensitive TRPV4 Channel-Induced Extracellular ATP Accumulation at the Acupoint Mediates Acupuncture Analgesia of Ankle Arthritis in Rats.

(1) Background: Acupuncture (AP) is a safe and effective analgesic therapy. Understanding how fine needles trigger biological signals can help us optimize needling manipulation to improve its efficiency. Adenosine accumulation in treated acupoints is a vital related event. Here, we hypothesized that extracellular ATP (eATP) mobilization preceded adenosine accumulation, which involved local activation of mechanosensitive channels, especially TRPV4 protein. (2) Methods: AP was applied at the injured-side Zusanli acupoint (ST36) of acute ankle arthritis rats. Pain thresholds were assessed in injured-side hindpaws. eATP in microdialysate from the acupoints was determined by luminescence assay. (3) Results: AP analgesic effect was significantly suppressed by pre-injection of GdCl3 or ruthenium red in ST36, the wide-spectrum inhibitors of mechanosensitive channels, or by HC067047, a specific antagonist of TRPV4 channels. Microdialysate determination revealed a needling-induced transient eATP accumulation that was significantly decreased by pre-injection of HC067047. Additionally, preventing eATP hydrolysis by pre-injection of ARL67156, a non-specific inhibitor of ecto-ATPases, led to the increase in eATP levels and the abolishment of AP analgesic effect. (4) Conclusions: These observations indicate that needling-induced transient accumulation of eATP, due to the activation of mechanosensitive TRPV4 channels and the activities of ecto-ATPases, is involved in the trigger mechanism of AP analgesia.