NTPDase1-ATP-P2Y2Rs axis in the sciatic nerve contributes to acupuncture at "Zusanli" (ST36)-induced analgesia in ankle arthritis rats.

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.

Mast Cell-Associated Serotonin in Acupoint Contributes to Acupuncture Analgesia in Arthritis Rats by Mediating ATP Release.

BACKGROUND: The activation of subcutaneous mast cells (MCs) helps to trigger the analgesic effect induced by acupuncture (AP), a traditional oriental therapy, that has been gradually accepted worldwide. This work aimed to reveal whether the serotonin (5-hydroxytryptamine, 5-HT) released from MCs plays an important role in this process, which has a controversial effect in the mechanism of pain. METHODS: In vivo tests, a 20-min session of AP was applied at Zusanli acupuncture point (acupoint) of acute ankle arthritis rats. Pain thresholds of the injured hindpaw were assessed to reflect the pain state, and the targeting substances in the interstitial space of the treated acupoint were sampled by microdialysis. In vitro experiments, exogenous 5-HT (exo-5-HT) was introduced to mediate adenosine triphosphate (ATP) release from cultured MCs. RESULTS: Needling promoted 5-HT accumulation at the Zusanli acupoint, which was prevented by sodium cromolyn. AP's analgesic effect was suppressed by the inhibition of 5-HT receptors at the acupoint, especially 5-HT1A subtype. In vitro tests, mechanical perturbation mimicking needling stimulation induced MCs to release 5-HT. 1 µM and 10 µM of exo-5-HT facilitated ATP release, which was restrained by blocking of 5-HT1 receptors rather than 5-HT3 receptors. As 5-HT, ATP and adenosine were also transiently accumulated in the treated acupoint during needling. Promoting ATP hydrolysis or activation adenosine A1 receptors duplicated AP analgesic effect. Finally, the inhibition of ATP receptors by suramin or pyridoxal phosphate-6-azo tetrasodium salt hydrate (PPADS) prevented AP analgesic effect. CONCLUSIONS: Our results suggest that MC-associated 5-HT release at acupoints contributes to AP analgesia, and the mediation of ATP secretion through 5-HT1A receptors might be the underlying mechanism at play. ATP could facilitate adenosine production or the propagation of needling signals.

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.

Activation of Subcutaneous Mast Cells in Acupuncture Points Triggers Analgesia.

This review summarizes experimental evidence indicating that subcutaneous mast cells are involved in the trigger mechanism of analgesia induced by acupuncture, a traditional oriental therapy, which has gradually become accepted worldwide. The results are essentially based on work from our laboratories. Skin mast cells are present at a high density in acupuncture points where fine needles are inserted and manipulated during acupuncture intervention. Mast cells are sensitive to mechanical stimulation because they express multiple types of mechanosensitive channels, including TRPV1, TRPV2, TRPV4, receptors and chloride channels. Acupuncture manipulation generates force and torque that indirectly activate the mast cells via the collagen network. Subsequently, various mediators, for example, histamine, serotonin, adenosine triphosphate and adenosine, are released from activated mast cells to the interstitial space; they or their downstream products activate the corresponding receptors situated at local nerve terminals of sensory neurons in peripheral ganglia. The analgesic effects are thought to be generated via the reduced electrical activities of the primary sensory neurons. Alternatively, these neurons project such signals to pain-relevant regions in spinal cord and/or higher centers of the brain.

[Subcutaneous Metabolites Involving Acupoint Sensitization Induced by Myocardial Ischemia and Acupuncture Stimulation in Rabbits].

OBJECTIVE: The present study aimed at observing the profile of metabolites in sensitized acupoints induced by myocardial ischemia (MI) and the effect of acupuncture intervention on the changes of the metabolites so as to explore the material basis of acupoint sensitization. METHODS: A total of 20 New Zealand rabbits were randomly and equally divided into a control group and a model group. The MI model was established by occlusion of the anterior descending branch of the left coronary artery with a controllable air balloon inflation method for 5 min/time, twice a day (4-hours' interval) for continuous 5 days (the first stage of MI). After one day's rest, another 5 days' occlusion was conducted again (the second stage of MI) in the same way. The rabbits of the control group were treated with the same procedures but without occlusion. Subcutaneous microdialysis fluid samples were collected from "Neiguan" (PC 6), "Shenmen" (HT 7), "Xinshu" (BL 15), and "Taixi" (KI 3) regions on day 8(after recovery from operation), 14 (the first stage of MI), and 20 (the second stage of MI), as well as collected from PC 6 region during and post-acupuncture stimulation of PC 6, respectively. Manual acupuncture stimulation was applied to the right PC 6 for 30 min. Partial least squares -linear discriminant analysis (PLS-DA) was used to identify different metabolism patterns of the microdialysis fluid sample between groups and at different time-points in the same one group, and the distinct metabolites as the potential markers between groups were weighted via the values of variable importance in the projection (VIP) in combination with t-test analysis. An area under the curve (AUC) >1.0 indicated a test exhibiting good discrimination between groups. RESULTS: Six metabolites identified to be significantly different between the control and model groups were L-glutamic acid, phenylalanine and 3-hydroxyisobutyric acid (which were significantly increased relevant to the control group), and L-histidine, octadecanedioic acid and 9-keto palmitic acid (significantly decreased relevant to the control group) in the microdialysate of PC 6, HT 7 and BL 15 regions. In the microdialysate of PC 6, 4 metabolites including L-glutamic acid, octadecanedioic acid and 8-isohydroxy PGF 2 α (significantly increased), as well as L-histidine (markedly decreased) were identified to be considerably different between the model and control groups. After acupuncture for 30 min, the AUC level of L-glutamic acid was further significantly increased (P<0.05), that of L-histidine obviously decreased, and those of octadecanedioic acid and 8-isohydroxy PGF 2 α turned back nearly to the level of pre-MI. CONCLUSION: L-glutamic acid, phenylalanine, 3-hydroxyisobutyric acid, L-histidine, octadecanedioic acid and 9-keto palmitic acid from PC 6, HT 7 and BL 15 regions may be used as the material biomarker for MI-induced sensitization of these acupoints. Manual acupuncture intervention of PC 6 induces a significant change of L-histidine and L-glutamic acid in the local subcutaneous tissues.

An investigation of the distribution and location of mast cells affected by the stiffness of substrates as a mechanical niche.

The distribution and location of mast cells are closely related to their physiological and pathological functions, such as allergic responses, immunity, and fibrosis, and are used in acupuncture. In this study, the distribution of mast cells in vivo was observed, and mechanical clues for understanding their distribution based on mechanical niches were explored. By toluidine blue staining and immunohistochemical staining, we examined the distribution and location of mast cells in rat skin and found that mast cells are distributed in a spatially nonuniform manner, preferring to locate at regions in the tissue and extracellular matrix with stiffness changes. In vitro experiments for studying the distribution of rat basophilic leukemia (RBL-2H3) mast cell line on poly-di-methyl-siloxane (PDMS) substrates with stiffness variations were performed. It was found that RBL-2H3 cells migrate and tend to remain in the areas with stiffness variations. The present research suggests that changing the stiffness of local tissues may stimulate mast cell recruitment, which may be the method by which some traditional Chinese medicine treatments, such as acupuncture. On the basis of the origin of mast cells and our experimental results, we predict that mast cells exist in tissues that contain permeable capillaries and prefer regions with stiffness changes. We discussed this prediction using examples of specific tissues from some cases.

[Observation on Acupoint Sensitization Phenomenon in Experimental Myocardial Ischemia Rabbits].

OBJECTIVE: To develop an animal model suitable for the study of acupoint sensitization in myocardial ischemia(MI) animals by observing changes of the mechanical withdrawal threshold (pain threshold, PT). METHODS: Twenty New Zea-land rabbits were randomly divided into control and model groups (n=10 in each group). The controllable MI model was set up by installing a balloon occluder at the left anterior descending branch of the left coronary artery. Air inflation of the balloon was given for 5 min/time, twice a day (4-hours' interval) for continuous 5 days (the first stage of MI), with one day's rest, then, another 5 days' air inflation was conducted again (2nd stage of MI). Electrocardiogram (ECG) of the standard limb lead Ⅱ was recorded for assessing MI, and myocardial 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to verify the degree of ischemic myocardium. The PT of the skin areas of "Neiguan" (PC 6), "Shenmen" (HT 7),"Taixi"(KI 3),hind paw(HP)was detected under conscious conditions,before MI, on day 8(after recovery from MI), day 14 (the first stage of MI), and day 20 (the second stage of MI), respectively. RESULTS: The controllable MI model was successful due to a marked elevation of ECG ST Ⅱ after inflation of the implanted balloon and a distinct recovery after balloon deflation (P<0.05). The myocardial TTC staining showed that the area of the myocardial necrosis was about 10%. The PT values were significantly decreased in PC 6 area at the first stage of MI and in both PC 6 and HT 7 regions at the 2nd stage of MI (P<0.01), suggesting a close relationship between the acupoints of the Pericardium and Heart meridians and the heart in terms of the basic theory of Chinese medicine. CONCLUSIONS: MI may induce a reduction of PT of some related acupoints in MI rabbits, and the controllable MI model of the present study is helpful to the study of acupoint sensitization phenomenon after occurrence of visceral diseases.

[Relationship between regional mast cell activity and peripheral nerve discharges during manual acupuncture stimulation of "Zusanli" (ST 36)].

OBJECTIVE: To observe changes of discharges of the sciatic nerve branch and mast cell activities and collagen fibers in the acupoint area during manual acupuncture stimulation of "Zusanli"(ST 36), so as to reveal the relationship between peripheral nerve and mast cell activities. METHODS: A total of 30 male SD rats were divided into normal, acupuncture control (an acupuncture neidle was inserted into ST 36 without manipulation), manual acupuncture (MA), disodium cromoglycate (DSCG, suppressing mast cell activity) plus acupuncture (MA + DSCG) and col lagenase (dissolving the collagen fibers) plus acupuncture (MA+ collagenase) groups (6 rats/group). After dissection of a branch of the sciatic nerve innervating ST 36 region in the left hind-limb under anesthesia, the ipsilateral ST 36 was stimulated by manipulating the acupuncture needle for 20 min. Discharges of the sciatic nerve branch were recorded by using a pair of metal electrodes and data acquisition system (Power Lab). Skin and muscle tissues of ST 36 area were sampled, sliced and stained with Toluidine Blue for detecting the number of degranulated mast cells. RESULTS: Compared with the control group, the mean power spectrum of d ischarges of the sciatic nerve and the mean rates of the degranulated mast cells in "Zusanli" (ST 36) area in the MA group were significantly increased (P<0.01). Whereas the mean power spectrum of discharges of the sciatic nerve and the mean degranulation rates of mast cells were considerably lower in the MA + DSCG group and MA+ collagenase group than in the MA group (P<0.01). No significant differences were found between the normal and control groups, and between the MA+NDSCG and MA+collagenase groups in the mean power density and degranulation rates of mast cells (P>0.05). CONCLUSION: Manual acupuncture stimulation of Zuai"ST 36 can significantly potentiate the discharge activity of the sciatic nerve and induce degranulation of mast cells at the same time, suggesting an involvement of mast cells in initiating acupuncture signals by peripheral sensory nerve.

[Development of studies on bioeffects of ultrasound-acupuncture therapy and its underlying mechanism].

The so-called ultrasound acupuncture is a therapeutic approach for clinical problems and health care by applying the ultrasound energy to the acupoints of the human body directly or indirectly. It has been applied in clinic for about 30 years since 1980s. In the present paper, the authors review the development of both experimental and clinical researches in the past 30 years. Its clinical application includes allergic rhinitis, local pain, mastitis, angina pectoris of coronary heart disease, stroke, etc. Regarding the researches on the underlying mechanism of ultrasound and ultrasound acupuncture, the authors make a summary from 1) bioeffects (thermal and nonthermal effects) of ultrasound intervention; 2) cell lysis and nonlysis effects of ultrasound intervention; and 3) effects of ultrasound acupuncture on the degranulation of mast cells. Based on the idea that "inflammatory reaction caused by mast cell degranulation is one of the initial factors of acupuncture for inducing therapeutic effects", bioeffects including cellular changes, especially mast cell degranulation caused by ultrasound stimulation, are thought to be the main possible mechanisms underlying the favorable efficacy of ultrasound acupuncture intervention. However, the ultrasound metrology and the specific superiority of ultrasound acupuncture remain unknown up to now.

In adjuvant-induced arthritic rats, acupuncture analgesic effects are histamine dependent: potential reasons for acupoint preference in clinical practice.

This study investigated whether immediate acupuncture effects in the acupoint are histamine dependent. Both histamine injection and manual acupuncture stimulation increased the pain threshold (PT) after treatment compared with the model group (P < 0.01), producing an analgesic effect. After pretreatment with clemastine, an H1 receptor antagonist and an antipruritic, the increase in the animals' pain threshold after acupuncture was suppressed compared with the Acu group (P < 0.01); however, there was no interference with the acupuncture-induced degranulation of mast cells. Pretreatment with disodium cromolyn did not suppress the increase in PT induced by the histamine injection at Zusanli (ST-36). We conclude that in adjuvant-induced arthritic rats, acupuncture analgesic effects are histamine dependent, and this histamine dependence determines the acupoint preference of acupoints away from the target site in acupuncture practice.

Research on modern nonlinear dynamic model of five-elements theory.

This article studied on five elements system and set general and strict model expectations combining with Traditional Chinese Medicine Zang-fu organs theory, in which absolute stable state, conditional stable state or transient stability and instability in mathematical models were corresponding to human healthy state, sub-healthy state (pathological plateau phase) and health deterioration state respectively. Model parameters were set up according to the mutual generation and restriction relations among five elements. The dynamic model of Five-Elements System was built, of which impulse responses were corresponding to human response under TCM treatment. Analyses of initial value and excitation response were conducted by numerical simulation and results turned out to meet the requirements of general model expectation: five elements system dynamic model had self-organization function; there existed only one non-global stable point and instability region in the five-dimensional space consisting of variables, in which instability was corresponding to pathological deterioration; system stable region was an unbounded domain and it included the stable sub-regions of special straight line-type, ray-type and line segment-type. Among those ray-types, some contained "Regression Peak" were classed as conditional stable regions while others as absolute ones. The existence of this peak indicates that our body must exceed a "Regression Threshold" when transiting from sub-healthy state (pathological plateau phase) to healthy state through self-regulation mechanism. Impulse excitation can reduce certain threshold and then increase the system health level, which is complied with the operating principle of Five-Elements System and the empirical rule of TCM clinical practice. This model has revealed qualitatively the inherent movement law of Five-Elements System and thus provides a new analysis tool for basic theoretical study on TCM.

Suspended moxibustion relieves chronic visceral hyperalgesia and decreases hypothalamic corticotropin-releasing hormone levels.

AIM: To evaluate the effect of suspended moxibustion (SM) on rectal sensory thresholds and to analyze the possible mechanisms involved in SM treatment of chronic visceral hypersensitivity (CVH) in rats. METHODS: SM was administered once daily to 37-d-old CVH rats for 7 d. The two pairs of acupoints (ST25 and ST37, bilateral) were simultaneously treated with SM. Each treatment lasted for 30 min. Rats undergoing treatment with SM were not anesthetized. Untreated CVH rats and normal rats were used as controls. The abdominal withdrawal reflex was determined 30-90 min after the seven treatments. The hypothalamic corticotropin-releasing hormone (CRH) mRNA level was measured using real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: We found that SM treatment significantly decreased visceral sensitivity to colorectal distention in this rat model. In treated animals, SM also decreased the relative hypothalamic CRH mRNA expression level to control levels. CONCLUSION: Lower hypothalamic CRH levels may mediate the beneficial effects of SM in this rat irritable bowel syndrome model.

[Prospective study of Han's acupoint nerve stimulator for preventing relapse of detoxified heroin addicts].

OBJECTIVE: To assess the efficacy of transcutaneous electrical acupoint stimulation (TEAS) in preventing the relapse of detoxified heroin users in a period of 12 months. METHODS: A total of 164 rehabilitating heroin users in Shanghai area were recruited after compulsory detoxification treatment for a period ranging from 3 months to 3 years. The TEAS was executed by the device named Han's acupoint nerve stimulator (HANS). The patients were treated with HANS for at least 3 months. All the subjects were then followed up for one year and relapse was monitored by monthly heroin/morphine urinalysis. RESULTS: Of 164 rehabilitating former heroin addicts, 53 remained drug-free at the end of 12 months observation period as judged by negative urinalysis while 35 become relapsed as documented by positive urine tests. The rest 76 dropped off due to various reasons and were all counted into the category of "relapsed". Thus, our data indicated 32.3% rehabilitating heroin users could stay sober for at least one year, which is significantly higher than the existing reports that less than 5% detoxified abusers would stay drug free for one year with no further intervention. No obvious correlation was found between the susceptibility of relapse and the duration of compulsory detoxification. CONCLUSION: Compared to the existing literatures, our results indicate that HANS could produce a marked decrease of the relapse rate of rehabilitating heroin users after compulsory detoxification.

[Acupoint-injection of histamine induced analgesic effect in acute adjuvant-induced-arthritis rats].

OBJECTIVE: To observe the activation effect of histamine on mast cells in the acupoint area and its analgesic effects similar to acupuncture stimulation, so as to reveal the underlying mechanism of acupoint injection therapy. METHODS: A total of 60 SD rats were divided into control, model, normal saline (NS), histamine-injection and acupuncture groups (12 rats/group). Acute adjuvant-induced-arthritis (AIA) model was duplicated by injection of complete Freunds' Adjuvant (0.05 mL) into the left ankle articular cavity. For rats of saline and histamine groups, normal saline (50 microL) and histamine (50 miroL, 100 microg/mL) were injected into the left "Zusanli" (ST 36) acupoint area. For rats of acupuncture group, manual acupuncture (lifting-thrusting and twisting needle intermittently) was applied to ST 36 for 20 min. Paw withdrawal (pain threshold, PT) was detected by radiant heat irradiation of the hindpaw. Skin and muscle tissues of ST 36 area were sampled to be fixed in formalin, then, sliced (5 microm), and stained with Toluidine Blue, separately, followed by counting the degranulation rate of mast cells under microscope. RESULTS: Compared with normal control group and pre-modeling, PT of model, NS, acupuncture and histamine groups decreased significantly (P < 0.01), while in comparison with model group, PT of acupuncture and histamine groups increased considerably (P < 0.01), suggesting an induction of analgesic effect after acupuncture and histamine injection. The degranulation rates of acupuncture and histamine groups were remarkably higher than those of model and NS groups (P < 0.01), suggesting an activation of mast cells following regional acupuncture stimulation and histamine injection. CONCLUSION: Both manual acupuncture and histamine injection of ST 36 can relieve pain in AIA rats, which may be closely related to its effect in activating mast cells in acupoint area. It suggests that the mast cells are a new target for acupoint injection therapy.

[Relationship between laser acupuncture analgesia and the function of mast cells].

OBJECTIVE: To observe the analgesic effects of single-and combined-laser irradiation with low-intensity applied at "Zusanli" (ST 36) in rats, and their relation to degranulation of mast cells. METHODS: Sixty-six SD rats were randomly divided into 6 groups: normal control group (Group NC), model control group (Group MC), sham irradiation group (Group SI), 10.6 microm laser irradiation group (Group 10.6 microm LI), 650 nm laser irradiation group (Group 650 nm LI) and combined (10.6 microm + 650 nm) laser irradiation group (Group CLI). Complete Freund's Adjuvant (0.05 mL) was injected into the left ankle joints of all the rats except those in Group NC to cause acute adjuvant-induced arthritis. In treatment, laser irradiation was applied at "Zusanli" (ST 36) for 30 minutes in all the rats except those in Group NC and Group MC. The paw withdrawal latency (PWL) to radian heat was used to compare analgesic effects among the groups. By means of toluidine blue, dyed slices of local tissues of "Zusanli" (ST 36) were used to observe changes of mast cell degranulation before and after laser irradiation. RESULTS: The pain thresholds to irradiation of the rats in Group 650 nm LI and Group CLI were significantly higher than those in Group MC and Group SI (P < 0.01), and the mast cell degranulation rate in Group 650 nm LI and Group CLI were also significantly higher than that in Group MC and Group SI (P < 0.001). The pain threshold and mast cell degranulation rate in Group 10. 6 microm LI were not significantly different from those in Group MC and Group SI. There was a linear correlation between mast cell degranulation rate and PWL with 0. 737 in coefficient (P < 0.001). CONCLUSION: Single 650 nm laser and combined 650 nm + 10.6 microm laser with low intensity irradiated at "Zusanli" (ST 36) in acute adjuvant rats can provide remarkable analgesic effects, and there was a positive correlation between mast cell degranulation rate and analgesic effects, which plays an important part in laser irradiation-induced analgesia.

[Effects of acupoint-nerve block on mast cell activity, manual acupuncture- and electroacupuncture-induced analgesia in adjuvant arthritis rats].

OBJECTIVE: To observe the effects of acupoint-nerve block on manual-acupuncture (MA) and electroacupuncture (EA) analgesia and mast cells degranulation in adjuvant arthritis (AA) rats in order to analyze the underlying peripheral mechanism of MA and EA analgesia. METHODS: A total of 80 SD rats were randomized into control, AA model (M), normal + lidocaine (NL), EA, EA-ST36 + Lidoc (L + EA), Dubi (ST 35) + Lidoc + EA-ST36 (DL + ZEA), Xiajuxu (ST 39) + Lidoc + EA-ST36 (XL+ ZEA), MA, Lidoc+ MA-ST36 (L + MA), and ST35 + Lidoc + MA-ST36 (DL + ZMA) groups, with 8 cases in each. AA model was established by injection of complete Freund's adjuvant (0.05 ml) in the rat's right hind-paw under anesthesia. Lidoc hydrochloride (2%, 0.05 ml) was injected into ST36 or ST35 separately for blocking the local nerve. Paw withdrawal latency (pain threshold, PT) and mast cell degranulation ratios (DGR) were detected. EA (2/100 Hz, 0.5 mA, 1 mA, 1.5 mA) or MA was applied to ST36. RESULTS: Compared with model group, PT values of EA and MA groups increased significantly (P<0.05, P<0.01) and mast cell DGR of EA and MA groups were significantly higher (P<0.01). Both PT and DGR of NL group were basically not affected by ST36-Lidoc-injection in comparison with model group (P>0.05). PT values of L + EA and DL+ ZEA groups were significantly lower than those of control and EA groups (P<0.01), and comparable to those of model and NL groups (P>0.05). PT of XL + ZEA group was significantly higher than those of model and NL groups (P<0.01), and comparable to that of EA group (P>0.05). Similar situations were found in MA-related groups. Mast cell DGR of ST36 area in L + EA, DL + ZEA and XL + ZEA groups were comparable to that of EA group (P>0.05), and such was the case in MA-related groups. CONCLUSION: After nerve block of the local and proximal acupoints rather than the distal acupoint innervated by the same nerve, the analgesic effect of both MA and EA of ST36 is obviously attenuated; but no marked influences on MA- and EA-induced mast cell degranulation are found.

[Skin slice used as a model for investigating acupuncture effects].

OBJECTIVE: To establish an acupoint-connective tissue model for studying the mechanism of acupuncture by using in vitro patch clamp technique in the rat skin slices. METHODS: The local connective tissue under the corium of "Housanli" (ST 36) area from SD rat was acutely and bluntly separated and fixed in a chamber filled with artificial incubation solution. Mast cells in the prepared connective tissue slice were labeled by toluidine blue (TB) or neutral red (NR). The whole-cell current of mast cells responding to pressure stimulation applied through a patch pipette was recorded in rat slices derived from acupoint ST 36 area by using in vitro patch-clamp technique. RESULTS: 1) After staining with TB and NR, the labeled mast cells were found to distribute in the extracellular matrix of the connective tissue samples, and their degranulation phenomenon could be seen clearly. 2) The whole-cell current of mast cells in response to mechanical stress stimulation was successfully recorded in the connective tissue slices of the rat acupoint ST 36 area. The cellular membrane currents increased evidently when pressure gradients of -30, -60 or -90 cmH2O were applied to the recorded mast cells. CONCLUSION: The connective tissue slice from the rat ST 36 area may be used as a model for investigating the peripheral mechanism of acupuncture by combining the microtechniques and electrophysiological techniques. The results obtained in this model prove for the first time by electrophysiology that the mast cells in the connective tissue are probably involved in the transduction process of the mechanical signal from acupuncture stimulation. This new model provides a base for investigating the characters of the cells, collagen fibers, proteoglycans, etc. and their interactions in the acupoint connective tissue in the future.

[Effect of multiple paralleled acupuncture needles stimulation of scalp points on hemodynamics and blood flow energy of the common carotid artery in stroke patients].

OBJECTIVE: To observe the changes of carotid hemodynamic and hydraulic power indices after paralleled scalp-point acupuncture therapy in post-stroke patients. METHODS: Twenty post-stroke volunteer patients were enlisted in the present study. Aseptic filiform needles were inserted into Baihui (GV 20), bilateral Motor Area (MS 8), etc. with the needles retained for 5 min. The treatment was given one time. Hemodynamic indices: mean velocity of blood flow (Vmean), maximum velocity (Vmax), minimum velocity (Vmin), mean blood flow (Qmean), maximum blood flow (Qmax), minimum blood flow (Qmin), natural impedance (Zc), peripheral resistance (Rc), dynamic resistance (Dr), and critical pressure (Pc) before and after the treatment were detected with KF-3000 Brain Circulation Analyzer. The hydraulic power indices:stable potential energy (Ws), oscillation potential energy (Wo), total potential energy (Wc), stable kinetic energy (Ks), success kinetic energy (Ko), total kinetic energy (Kc), stable total energy (Ts), oscillation total energy (To), total energy (Tc), To/Tc, Kc/Tc, Wo/Wc and Ks/Kc were obtained through calculation. RESULTS: After the therapy, Vmin, Vmean, Qmin and Qmean increased significantly (P<0.001), while Rc decreased evidently (P<0.001). Ws, Ks, Ts, Wc, Kc, Tc and Kc/Tc increased obviously (P<0.001, P<0.05), and Ks/Kc declined significantly (P<0.01). The rest indices had no significant changes after the treatment. CONCLUSION: Multiple paralleled scalp acupuncture needles stimulation can effectively raise the blood flow volume of the common caroitid artery, leading to an increase of the energy supply of the cerebral blood circulation, but has no apparent influence on the total oscillatory energy/total energy in stroke patients.

[Analysis on the difference of afferent mechanism of analgesic signals from manual acupuncture and electroacupuncture of "Zusanli" (ST 36)].

OBJECTIVE: To observe the difference of signaling afferent mechanism of acupoints analgesic effect between manual acupuncture (MA) and electroacupuncture (EA) of "Zusanli" (ST 36) in order to analyze the initiating mechanism of these two commonly used clinic acupuncture methods. METHODS: A total of 64 SD rats were randomized into control, model, MA, EA, collagenase (type- I)-pretreatment (CP) + MA, CP + EA, disodium cromoglicate-pretreatment (DSCP) + MA and DSCP + EA groups with 8 cases in each. Arthritis model was established by intra-joint injection of complete Freunds' agent. MA(rotation, 20 min), and EA (21 min) were applied to bilateral "Zusanli" (ST 36) respectively in different groups. Pain threshold (PT, paw withdraw latency) was detected with 336 GT PAW/TAIL STIMULATOR and degranulated mast cells were counted under microscope separately. RESULTS: Compared with normal control group, PT values of model, MA, EA, CP + MA, CP + EA, DSCP + MA and DSCP + EA groups lowered significantly before treatment (P < 0.05). Comparison of the same one group showed that after the treatment, PT values of MA and EA groups increased considerably (P < 0.05). PT changing rates of CP + MA and DSCP + MA were significantly lower than that of MA group (P < 0.05), suggesting disappearance of MA analgesia (not EA analgesia) after lesion of collagen protein by CP in ST36 area. In comparison with control group, the degranulation ratios (DGR) of mast cells in MA and EA groups increased significantly (P < 0.05); while compared with MA and EA groups separately, DGR of the corresponding CP + MA and DSCP + MA, and CP + EA and DSCP + EA groups decreased markedly (P < 0.05), displaying an inhibition of mast cell degranulation following CP and DSCP in ST36 area. CONCLUSION: Both MA and EA can induce analgesia and concomitant increase of DGR of mast cells. The collagen fibers may contribute to MA initiated analgesic effect while the peripheral nerve receptors are probably involved in EA analgesia in rat.

Repeated electro-acupuncture attenuates chronic visceral hypersensitivity and spinal cord NMDA receptor phosphorylation in a rat irritable bowel syndrome model.

Acupuncture has been used in clinical trials for the treatment of abdominal pain in patients with irritable bowel syndrome (IBS). However, scientific evidence is still lacking and the underlying mechanism remains largely unexplored. The aim of this study was to examine the effects of repeated administration of electro-acupuncture (EA) on chronic visceral hypersensitivity and on the phosphorylation of spinal cord N-methyl-D-aspartic acid (NMDA) receptors in a rat model of IBS. The results showed that repeated administration of EA at bilateral points of Zu-san-li (ST-36) and Shang-ju-xu (ST-37) significantly attenuated chronic visceral hypersensitivity induced in young adult rats by neonatal colon irritation. Such an effect was not seen in either of the two controls: sham-EA at ST-36 and ST-37 without electrical stimulation and EA at control points (BL-62 and tail). Furthermore, rats with chronic visceral hypersensitivity exhibited high-level expression of phosphorylated NMDA receptor subunit 1 (pNR1) in the spinal cord (L4-L5 segments), which was markedly attenuated by EA treatment. In addition, EA at ST-36 and ST-37 neither altered the pain threshold of normal rats nor affected the expression of pNR1 in the lumbosacral spinal cord. Altogether, these data indicate that the EA-mediated attenuation of chronic visceral hypersensitivity is correlated with the down-regulation of NMDA receptors phosphorylation at the spinal level.