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Background: The efficacy of acupuncture at Zusanli (ST36) in alleviating lower-limb pain is widely acknowledged in clinical practice, while its underlying mechanism remains incompletely elucidated. Our previous research had revealed that the prompt analgesia induced by needling-ST36 was accompanied by expression alterations in certain exco-nucleotidases within the sciatic nerve. Building upon this finding, the current work focused on NTPDase1, the primary ecto-nucleotidase in the human body, which converts ATP into AMP. Methods: A 20-min acupuncture was administered unilaterally at the ST36 on rats with acute ankle arthritis. The pain thresholds of the injured hind paws were determined. Pharmacological interference was carried out by introducing the corresponding reagents to the sciatic nerve. ATP levels around the excised nerve were measured using a luciferase-luciferin assay. Live calcium imaging, utilizing the Fura 2-related-F340/F380 ratio, was conducted on Schwann cells in excised nerves and cultured rat SCs line, RSC96 cells. Results: The analgesic effect induced by needling-ST36 was impaired when preventing ATP degradation via inhibiting NTPDase1 activities with ARL67156 or Ticlopidine. Conversely, increasing NTPDase1 activities with Apyrase duplicated the acupuncture effect. Similarly, preventing the conversion of AMP to adenosine via suppression of NT5E with AMP-CP hindered the acupuncture effect. Unexpectedly, impeded ATP hydrolysis ability and diminished NTPDase1 expression were observed in the treated group. Agonism at P2Y2Rs with ATP, UTP, or INS365 resulted in anti-nociception. Contrarily, antagonism at P2Y2Rs with Suramin or AR-C 118925xx prevented acupuncture analgesia. Immunofluorescent labeling demonstrated that the treated rats expressed more P2Y2Rs that were predominant in Schwann cells. Suppression of Schwann cells by inhibiting ErbB receptors also prevented acupuncture analgesia. Finally, living imaging on the excised nerves or RSC96 cells showed that agonism at P2Y2Rs indeed led to [Ca2+]i rise. Conclusion: These findings strongly suggest that the analgesic mechanism of needling-ST36 on the hypersensation in the lower limb partially relies on NTPDase1 activities in the sciatic nerve. In addition to facilitating adenosine signaling in conjunction with NT5E, most importantly, NTPDase1 may provide an appropriate low-level ATP milieu for the activation of P2Y2R in the sciatic nerve, particularly in Schwann cells.

The efficacy of acupuncture at Zusanli (ST36) in alleviating lower-limb pain is widely acknowledged in clinical practice, while its underlying mechanism remains incompletely elucidated. Our previous research had revealed that the prompt analgesia induced by needling-ST36 was accompanied by expression alterations in certain exco-nucleotidases within the sciatic nerve. Building upon this finding, the current work focused on NTPDase1, the primary ecto-nucleotidase in the human body, which converts ATP into AMP. A 20-min acupuncture was administered unilaterally at the ST36 on rats with acute ankle arthritis. The pain thresholds of the injured hind paws were determined. Pharmacological interference was carried out by introducing the corresponding reagents to the sciatic nerve. ATP levels around the excised nerve were measured using a luciferase-luciferin assay. Live calcium imaging, utilizing the Fura 2-related-F 340 /F 380 ratio, was conducted on Schwann cells in excised nerves and cultured rat SCs line, RSC96 cells. The analgesic effect induced by needling-ST36 was impaired when preventing ATP degradation via inhibiting NTPDase1 activities with ARL67156 or Ticlopidine. Conversely, increasing NTPDase1 activities with Apyrase duplicated the acupuncture effect. Similarly, preventing the conversion of AMP to adenosine via suppression of NT5E with AMP-CP hindered the acupuncture effect. Unexpectedly, impeded ATP hydrolysis ability and diminished NTPDase1 expression were observed in the treated group. Agonism at P2Y2Rs with ATP, UTP, or INS365 resulted in anti-nociception. Contrarily, antagonism at P2Y2Rs with Suramin or AR-C 118925xx prevented acupuncture analgesia. Immunofluorescent labeling demonstrated that the treated rats expressed more P2Y2Rs that were predominant in Schwann cells. Suppression of Schwann cells by inhibiting ErbB receptors also prevented acupuncture analgesia. Finally, living imaging on the excised nerves or RSC96 cells showed that agonism at P2Y2Rs indeed led to [Ca2+] i rise. These findings strongly suggest that the analgesic mechanism of needling-ST36 on the hypersensation in the lower limb partially relies on NTPDase1 activities in the sciatic nerve. In addition to facilitating adenosine signaling in conjunction with NT5E, most importantly, NTPDase1 may provide an appropriate low-level ATP milieu for the activation of P2Y2R in the sciatic nerve, particularly in Schwann cells. [Display omitted] • Acupuncture (AP) at ST36 generated immediate analgesia in adjuvant arthritis rats. • NTPDase1 and Nt5E actively contributed to AP analgesia. • While AP suppressed the sciatic nerve to hydrolyze ATP and express NTPDase1. • ATP-sensitive P2Y2Rs in the sciatic nerve were actively involved in AP analgesia. • Schwann cells expressed NTPDase1 and P2Y2Rs, and also contributed to the AP effect. [ABSTRACT FROM AUTHOR]

• Acupuncture analgesia is based on ATP release from (sub)cutaneous/muscular tissue. • ATP and its degradation product adenosine participate in acupuncture analgesia. • Peripheral and central mechanisms participate in the effect of acupuncture analgesia. • A1/P2X3,4,7 receptors appear to mediate acupuncture analgesia. Purinergic signaling has recently been suggested to constitute the cellular mechanism underlying acupuncture-induced analgesia (AA). By extending the original hypothesis on endogenous opioids being released during AA, Geoffrey Burnstock and Maiken Nedergaard supplied evidence for the involvement of purinoceptors (P2 and P1/A1 receptors) in the beneficial effects of AA. In view of certain pain states (e.g. neuropathic pain) which respond only poorly to therapy with standard analgesics, as well as with respect to the numerous unwanted effects of opioids and non-steroidal anti-inflammatory drugs, it is of great significance to search for alternative therapeutic options. Because clinical studies on AA yielded sometimes heterogeneous results, it is of eminent importance to relay on experiments carried out on laboratory animals, by evaluating the data with stringent statistical methods including comparison with a sufficient number of control groups. In this review, we summarize the state of the art situation with respect to the participation of P2 receptors in AA and try to forecast how the field is likely to move forward in the future. [ABSTRACT FROM AUTHOR]

Abstract: Hypothalamic supraoptic nucleus (SON) has been demonstrated to involve in pain modulation. Acupuncture analgesia is a very useful clinical skill for pain relief, which has over 2500-year history in China. The present study investigated the effect of SON on acupuncture analgesia in the rat. Electrical stimulation of the SON or microinjection of a small dose l-glutamate sodium into the SON enhanced acupuncture analgesia in a dose-dependent manner, while cauterization of the SON weakened acupuncture analgesia. Pituitary removal did not influence the effect of l-glutamate sodium that enhanced acupuncture analgesia in the SON. The data suggested that the neurons and not the nerve fibers in the SON played an important role in acupuncture analgesia, which effect might be through the central nervous system rather than the hypothalamo-neurohypophyseal system. [Copyright &y& Elsevier]

Abstract: Our previous study proved that hypothalamic paraventricular nucleus (PVH) plays an important role in acupuncture analgesia. The effect of acupuncture on the concentrations of arginine vasopressin (AVP), oxytocin (OXT), leucine-enkephaline (L-Ek), β-endorphin (β-Ep) and dynorphinA1–13 (DynA1–13) was investigated in rat PVH. Electrical acupuncture of “Zusanli” points (St. 36) 30min increased the AVP, not OXT, L-Ek, β-Ep and DynA1–13 concentrations in PVH tissue using micropunch and radioimmunoassay, which showed a negative relationship between the pain threshold and AVP concentrations in PVH tissue. Electrical acupuncture could elevate the AVP concentrations in PVH perfuse liquid during acupuncture, and then reduce the AVP concentrations in PVH perfuse liquid after acupuncture. But no change in OXT, L-Ek, β-Ep and DynA1–13 concentrations was detected in PVH perfuse liquid. Electrical acupuncture decreased the number of AVP, not OXT, L-Ek, β-Ep and DynA1–13 immunoreactive cells in PVH using immunocytochemistry. The results suggested that only AVP, not OXT and endogenous opiate peptides in PVH involved acupuncture analgesia in the rat. [Copyright &y& Elsevier]

Characteristics of the acupuncture point in producing acupuncture analgesia (AA) were examined by the inhibition of noxious responses in the brain stem reticular formation, potentials, and neuronal activity in the dorsal periaqueductal central gray (D-PAG), and analgesia caused by low frequency stimulation of the acupuncture point. As a result, stimulation of the muscle beneath the acupuncture point was found to be effective in producing AA. AA measured by tail flick, vocalization, and writhing tests was abolished by hypophysectomy, and by antiserum of beta-endorphin administered into the 3rd ventricle. The pathway from the D-PAG to the anterior hypothalamus (AA-AH) in the AA afferent pathway from the acupuncture point to the pituitary gland was determined. The lateral hypothalamus, lateral septum, cingulate bundle, dorsal-hippocampus, and habenulo-interpeduncular tract were found, in addition to regions previously found, to belong to the AA afferent pathway. A network of divergence and convergence in their rostral and caudal relations was observed. The AA afferent pathway diverges from the D-PAG, converges to the HP, and then projects to the AA-AH.

The descending pain inhibitory system (DPIS) associated with acupuncture analgesia (AA), caused by low frequency stimulation of an acupuncture point, was identified by the results of lesion and stimulation procedures previously determined to differentiate the afferent and efferent paths in rats. The DPIS starts in the posterior arcuate nucleus and descends to the hypothalamic ventromedian nucleus (HVM) from whence it divides into two pathways: one path, the serotonin mediated path, descends through the ventral periaqueductal central gray (V-PAG) and then to the raphe magnus (RM). The other, the noradrenaline mediated path, descends through the reticuloparagigantocellular nucleus (NRPG) and part of the reticulogigantocellular nucleus (NRGC). The afferent and efferent paths are both present in the RM and NRGC, and were separately identified by means of the analgesia (SPA) produced by stimulation of the separate regions in AA responders and nonresponders, because SPA of these regions in nonresponders produced only efferent pathway mediated analgesia.

Potentials in the final sector of the afferent pathway from the acupuncture point (AP) were enhanced by intraperitoneal 0.5 mg/kg morphine without changing the threshold of AP stimulation and greatly decreased by hypophysectomy. The decreased potentials were restored to the control level by morphine (0.5 mg/kg, IP). Potentials evoked in the final sector of the afferent pathway from the nonacupuncture point (NAP) by NAP stimulation after lesion of the analgesia inhibitory system were greatly enhanced by corticotropin (ACTH) (0.25 mg/kg, IP) and greatly decreased by hypophysectomy. Diminished potentials were restored to the control level by ACTH (0.25 mg/kg, IP). Both morphine (0.5 mg/kg, IP) and ACTH (0.25 mg/kg, IP) produced analgesia, but morphine did not affect acupuncture analgesia (AA) and ACTH did not affect nonacupuncture point stimulation-produced analgesia (NAA). All analgesia, that due to 0.5 mg/kg morphine or 0.25 mg/kg ACTH, AA, and NAA were abolished by hypophysectomy. The abolished AA and NAA were restored by 0.5 mg/kg morphine and 0.25 mg/kg ACTH, respectively. Hence, beta-E and ACTH liberated from the pituitary gland by stimulation of an AP and NAP may act as positive feedback on the AA and NAA afferent pathways, respectively.

Lesion of the preoptic area (POA) or medial arcuate nucleus (M-HARN) abolished acupuncture analgesia (AA). Potentials in the median eminence (ME) evoked by stimulation of the acupuncture point (AP) were not affected by lesion of either the POA or M-HARN alone, but were abolished by concurrent lesion of both. No analgesia was produced by stimulation of the POA. Analgesia produced by stimulation of the M-HARN was abolished by lesion of the POA, and the abolished analgesia was restored by concurrent stimulation of the POA and M-HARN, hence POA and M-HARN outputs might converge in the ME to produce AA. Similar convergence from the anterior arcuate nucleus (A-HARN) and POA to the ME was observed in analgesia (NAA) produced by stimulation of a nonacupuncture point (NAP). Two pathways diverged from the lateral hypothalamus in the AA afferent pathway and two from the lateral periaqueductal central gray (L-PAG) in the NAA afferent pathway. POA potentials evoked by stimulation of the AP were reversed by naloxone, and those evoked by stimulation of the AP were reversed by dexamethasone. ACTH sensitive sites were found in both the L-PAG and the anterior hypothalamus.

Stimulation of a nonacupuncture point (NAPS) does not normally produce analgesia in the same way that stimulation of an acupuncture point does. However, NAPS did produce dexamethasone reversible analgesia after intraperitoneal treatment with D-phenylalanine (DPA) or proglumide, or after microinjection of these compounds into such parts of the analgesia inhibitory system (AIS) as the lateral centromedian nucleus of the thalamus and part of the posterior hypothalamus. Inhibition of acupuncture analgesia (AA), or of morphine analgesia (MA) by 0.5 mg/kg, IP, which is equivalent to AA after AIS lesion, and of potentials in the lateral periaqueductal central gray evoked by repetitive NAPS or stimulation of the AIS, were antagonized by DPA. Disappearance of AA and MA in morphine tolerant acupuncture responder and nonresponder rats was reversed to reproduce the same magnitude of analgesia after proglumide application. The reproduced AA and MA were antagonized by dexamethasone. These results indicate that DPA and proglumide antagonized the AIS and unmasked the dexamethasone reversible AA and MA.

Acupuncture analgesia (AA) caused by low frequency stimulation of the acupuncture point (AP) was abolished by hypophysectomy and adrenalectomy. Termination of the AA producing pathway from the AP to the pituitary gland was in the medial hypothalamic arcuate nucleus (M-HARN). The origin of the descending pain inhibitory system associated with AA was in the posterior HARN (P-HARN). AA in the hypophysectomized rats, and enhanced neuronal activity in the P-HARN that were abolished during acupuncture stimulation, were both restored by intraperitoneal microinjection of 0.5 mg/kg morphine or 0.1 micrograms beta-endorphin into the P-HARN during acupuncture stimulation. Of the analgesia produced by dopamine or beta-endorphin injected into the P-HARN, that caused by beta-endorphin disappeared after denervation of the M-HARN. The P-HARN neurons that responded to acupuncture stimulation also responded to iontophoretic dopamine, but not to iontophoretic morphine nor ultramicroinjected beta-endorphin. The transmission between the M-HARN and P-HARN may be dopaminergic, and beta-endorphin might presynaptically modulate this transmission. Reduction of sodium ions may have been the reason for abolition of AA after adrenalectomy.