Anti-nociceptive effects of adenosine A1 receptors. (a,b) Comparison of the effect of CCPA on mechanical allodynia (touch test, a) and thermal hyperalgesia (thermal test, b) in wild-type (WT, black) and A1 receptor knockout (A1RKO, gray) mice. CFA was administered in the right paw at day 0. The adenosine A1 receptor agonist CCPA (0.1 mM, 20 μl) was injected into the ipsilateral (right) Zusanli point (ST36) at day 4. All of the mice were evaluated ~10 min after the CCPA injection (**P < 0.01, Tukey-Kramer test compared with before CCPA; ^##P < 0.01, comparison of wild type and A1RKO at each time point; n = 5−9). (c,d) Effect of CCPA on mechanical (c) and thermal hypersensitivity (d) in wild-type and A1RKO mice with neuropathic pain evoked by partial ligation of the right leg ischias nerve at day 0 and CCPA administered at day 6 (n = 6). The sensitivity of the contralateral (control) leg to mechanical and thermal stimulation from these experiments is shown in Supplementary Figure 1. (e) The amplitude of fEPSP in left anterior cingulate cortex evoked by painful stimulation in the right foot. The effect of CCPA (0.1 mM, 20 μl, at 0 min) injected in the ipsilateral (right) or the contralateral (left) Zusanli point on fEPSP is plotted as a function of time in wild-type and A1RKO mice. (*P < 0.01, Tukey-Kramer test compared with the −18- to −12-min time point, n = 4−12). Error bars indicate s.e.m.

Pharmacological inhibition of deaminase activity enhances increases in adenosine and prolongs anti-nociception actions of acupuncture. (a) Schematic diagram outlining the two major pathways involved in extracellular enzymatic degradation of AMP. The nucleoside analog deoxycoformycin inhibits both AMP deaminase (AMPD) and adenosine deaminase (ADA). (b) Histogram comparing the production of adenosine, IMP and inosine when tissue sections harvested close to the Zusanli point were incubated in 1 mM AMP and an inhibitor of adenosine uptake, nitrobenzylthioinosine (100 μM), for 45 min. Deoxycoformycin (500 μM) increased accumulation of adenosine while inhibiting the production of IMP and inosine (*P < 0.05, **P < 0.01, t test comparison between control and deoxycoformycin, n = 5). (c) Analysis of microdialysis samples collected close to the Zusanli point in mice treated with deoxycoformycin (50 mg per kg, intraperitoneal) or vehicle (saline). Deoxycoformycin increased accumulation of adenosine, while inhibiting the production of IMP in vivo during and after acupuncture (n = 6−8). (d,e) Deoxycoformycin (50 mg per kg, intraperitoneal) prolonged the anti-nociceptive effect of acupuncture in wild-type mice with inflammatory pain in response to mechanical (d) and thermal (e) stimulation (^##P < 0.01, Tukey-Kramer test compared with before acupuncture, n = 6−10). (f,g) Deoxycoformycin prolonged the anti-nociceptive effect of acupuncture in wild-type mice with neuropathic pain induced by partial ligation of the ischias nerve to mechanical (f) and to thermal (g) stimulation (^#P < 0.05, ^##P < 0.01, Tukey-Kramer test compared with before acupuncture, n = 5). The sensitivity of the contralateral (control) leg to mechanical and thermal stimulation from these experiments is shown in Supplementary Figure 1. Error bars indicate s.e.m.

Acupuncture triggers an increase in the extracellular concentration of ATP, ADP, AMP and adenosine. (a) Representative HPLC chromatograms before, during and after acupuncture. The samples were collected by a microdialysis probe implanted in close proximity to the Zusanli point. Standards of adenosine, AMP, ADP and ATP (0.3 μM each) are shown on top. (b) Time course of purine release in response to acupuncture. (c) Histogram summarizing the mean concentrations of adenosine, AMP, ADP and ATP during baseline nonstimulated conditions, as well as during and following acupuncture (*P < 0.05, **P < 0.01, paired t test compared to 0 min, n = 8). Error bars indicate s.e.m.

Acupuncture fails to suppress pain in mice lacking adenosine A1 receptors. (a,b) Acupuncture reduced sensitivity to both mechanical (a) and thermal (b) stimulation in wild-type mice suffering from inflammatory pain after injection of CFA in the right paw, but not in A1RKO littermates tested at day 4. All of the mice were evaluated ~10 min after acupuncture (**P < 0.01, Tukey-Kramer test compared with before acupuncture; ^##P < 0.01, comparison of wild type and A1RKO at each time point, n = 5−9). (c,d) Acupuncture suppressed mechanical allodynia (c) and thermal hyperalgesia (d) in wild-type mice, but not in A1RKO mice, suffering from neuropathic pain. Neuropathic pain was induced by partial ligation of the right leg ischias nerve and the clinical effect of acupuncture tested at day 6 (n = 6). The sensitivity of the contralateral (control) leg to mechanical and thermal stimulation from these experiments is shown in Supplementary Figure 1. (e) The effect of acupuncture (0–30 min) on fEPSP amplitude in the left anterior cingulate cortex evoked by painful stimulation in the right leg. fEPSP amplitude is plotted as a function of time in wild-type and A1RKO mice (*P < 0.01, Tukey-Kramer test compared with the −18- to −12-min time points, n = 3−8). Error bars indicate s.e.m.