Adenosine A2A receptors in the rostral ventrolateral medulla participate in blood pressure decrease with electroacupuncture in hypertensive rats.

Acupuncture is increasingly used to manage high blood pressure (BP) as a complementary therapy. However, the mechanisms underlying its hypotensive effects remain unclear. Our previous studies have shown that electroacupuncture (EA) at the ST36-37 acupoints, overlying the deep peroneal nerve, attenuates pressor responses through adenosine A2A receptors (A2AR) in the rostral ventrolateral medulla (rVLM). However, it is uncertain whether rVLM A2AR contributes to EA's BP-lowering effect in sustained hypertension. We hypothesized that a course of EA treatment lowers BP, in part, through the activation of adenosine A2AR in the rVLM in hypertensive rats. To mimic essential hypertension in the clinic, we performed EA in conscious Dahl salt-sensitive hypertensive rats (DSHRs). EA (0.1-0.4 mA, 2 Hz) was applied at ST36-37 for 30 min twice weekly for four weeks, while sham-EA was conducted in a similar manner but without electrical input. In hypertensive rats, BP was reduced by EA (n = 14) but neither by sham-EA (n = 14) nor in the absence of needling (n = 8). Following four weeks of eight treatments and then under anesthesia, EA's modulatory effect on elevated BP was reversed by unilateral rVLM microinjection of SCH 58261 (1 mM in 50 nl; an A2AR antagonist; n = 7; P < 0.05) but not the vehicle (n = 5) in EA-treated DSHRs. Activation of rVLM A2AR in DSHRs treated with sham-EA by an A2AR agonist, CGS-21680 (0.4 mM in 50 nl; n = 8), decreased BP. Unilateral administration of SCH 58261 or CGS-21680 into the rVLM did not alter basal BP in Dahl salt-sensitive rats fed a regular diet with normal BP. The A2AR level in the rVLM after EA was increased compared to the sham-EA and untreated DSHRs (n = 5 in each group; all P < 0.05). These data suggest that a 4-week twice weekly EA treatment reduced BP in salt-sensitive hypertensive rats likely through adenosine-mediated A2AR in the rVLM.

Sympathoinhibitory electroacupuncture (EA) interacts positively with anti-inflammatory EA alleviating blood pressure in hypertensive rats.

Elevated sympathetic activity and chronic inflammation are known contributory factors observed in hypertension. We have observed that sympathoinhibitory electroacupuncture (SI-EA) at acupoints ST36-37 alleviates sympathetic activity and hypertension. Additionally, EA at acupoints SP6-7 exerts anti-inflammatory (AI-EA) effects. However, it is not known whether simultaneous stimulation of this combination of acupoints attenuates or enhances individual effects. A 2 × 2 factorial design was used to test the hypothesis that combining SI-EA and AI-EA (cEA) leads to greater reduction of hypertension by decreasing sympathetic activity and inflammation in hypertensive rats than either set of acupoints alone. Dahl salt-sensitive hypertensive (DSSH) rats were treated with four EA regimens including cEA, SI-EA, AI-EA, and sham-EA twice weekly for five weeks. A group of normotensive (NTN) rats served as control. Systolic and diastolic BP (SBP and DBP) and heart rate (HR) were measured non-invasively by tail-cuff. Plasma norepinephrine (NE), high-sensitivity C-reactive protein (hs-CRP) and interleukin 6 (IL-6) concentrations were determined with ELISA at the completion of treatments. DSSH rats on high salt diet progressively developed moderate hypertension within five weeks. DSSH rats treated with sham-EA showed continuous increase in SBP and DBP and elevations in plasma NE, hs-CRP, and IL-6 levels relative to NTN control. Both SI-EA and cEA decreased SBP and DBP, and had corresponding changes in biomarkers (NE, hs-CRP, and IL-6) compared with sham-EA. AI-EA prevented SBP and DBP elevation and decreased IL-6 and hs-CRP relative to sham-EA. Importantly in DSSH rats that received repetitive cEA treatment, SI-EA interacted positively with AI-EA leading to greater reduction of SBP, DBP, NE, hs-CRP, and IL-6 than SI-EA or AI-EA alone. These data suggest that by targeting both elevated sympathetic activity and chronic inflammation, cEA regimen results in a greater reduction of BP effects in treating hypertension compared to using individual SI-EA or AI-EA alone.

Neurogenic Hypotension and Bradycardia Modulated by Electroacupuncture in Hypothalamic Paraventricular Nucleus.

Electroacupuncture (EA) stimulates somatic median afferents underlying P5-6 acupoints and modulates parasympathoexcitatory reflex responses through central processing in the brainstem. Although decreases in blood pressure and heart rate by the neural-mediated Bezold-Jarisch reflex responses are modulated by EA through opioid actions in the nucleus tractus solitarius and nucleus ambiguus, the role of the hypothalamus is unclear. The hypothalamic paraventricular nucleus (PVN) is activated by sympathetic afferents and regulates sympathetic outflow and sympathoexcitatory cardiovascular responses. In addition, the PVN is activated by vagal afferents, but little is known about its regulation of cardiopulmonary inhibitory hemodynamic responses. We hypothesized that the PVN participates in the Bezold-Jarisch reflex responses and EA inhibits these cardiopulmonary responses through the PVN opioid system. Rats were anesthetized and ventilated, and their heart rate and blood pressures were monitored. Application of phenylbiguanide every 10 min close to the right atrium induced consistent depressor and bradycardia reflex responses. Unilateral microinjection of the depolarization blockade agent kainic acid or glutamate receptor antagonist kynurenic acid in the PVN reduced these reflex responses. In at least 70% of the rats, 30 min of bilateral EA at P5-6 acupoints reduced the depressor and bradycardia responses for at least 60 min. Blockade of the CCK-1 receptors converted the non-responders into EA-responders. Unilateral PVN-microinjection with naloxone reversed the EA inhibition. Vagal-evoked activity of the PVN cardiovascular neurons was reduced by 30 min EA (P5-6) through opioid receptor activation. These data indicate that PVN processes inhibitory cardiopulmonary reflexes and participates in EA-modulation of the neural-mediated vasodepression and bradycardia.

Relationships Between Diurnal Changes of Tongue Coating Microbiota and Intestinal Microbiota.

The oral cavity and the intestine are the main distribution locations of human digestive bacteria. Exploring the relationships between the tongue coating and gut microbiota, the influence of the diurnal variations of the tongue coating microbiota on the intestinal microbiota can provide a reference for the development of the disease diagnosis and monitoring, as well as the medication time. In this work, a total of 39 healthy college students were recruited. We collected their tongue coating microbiota which was collected before and after sleep and fecal microbiota. The diurnal variations of tongue coating microbiota are mainly manifested on the changes in diversity and relative abundance. There are commensal bacteria in the tongue coating and intestines, especially Prevotella which has the higher proportion in both sites. The relative abundance of Prevotella in the tongue coating before sleep has a positive correlation with intestinal Prevotella; the r is 0.322 (p < 0.05). Bacteroides in the intestine had the most bacteria associated with the tongue coating and had the highest correlation coefficient with Veillonella in the oral cavity, which was 0.468 (p < 0.01). These results suggest that the abundance of the same flora in the two sites may have a common change trend. The SourceTracker results show that the proportion of intestinal bacteria sourced from tongue coating is less than 1%. It indicates that oral flora is difficult to colonize in the intestine in healthy people. This will provide a reference for the study on the oral and intestinal microbiota in diseases.

Assessment Parameters for Arrayed Pulse Wave Analysis and Application in Hypertensive Disorders.

Study on the objectivity of pulse diagnosis is inseparable from the instruments to obtain the pulse waves. The single-pulse diagnostic instrument is relatively mature in acquiring and analysing pulse waves, but the pulse information captured by single-pulse diagnostic instrument is limited. The sensor arrays can simulate rich sense of the doctor's fingers and catch multipoint and multiparameter array signals. How to analyse the acquired array signals is still a major problem in the objective research of pulse diagnosis. The goal of this study was to establish methods for analysing arrayed pulse waves and preliminarily apply them in hypertensive disorders. While a sensor array can be used for the real-time monitoring of twelve pulse wave channels, for each subject in this study, only the pulse wave signals of the left hand at the "guan" location were obtained. We calculated the average pulse wave (APW) per channel over a thirty-second interval. The most representative pulse wave (MRPW) and the APW were matched by their correlation coefficient (CC). The features of the MRPW and the features that corresponded to the array pulse volume (APV) parameters were identified manually. Finally, a clinical trial was conducted to detect these feature performance indicators in patients with hypertensive disorders. The independent-samples t-tests and the Mann-Whitney U-tests were performed to assess the differences in these pulse parameters between the healthy and hypertensive groups. We found that the radial passage (RP) APV h1, APV h3, APV h4, APV h3/h1 (P < 0.01), and APV h4/h1 (P < 0.05) were significantly higher in the hypertensive group than in the healthy group; the intermediate passage (IP) APV h4, APV h3/h1 (P < 0.05), and APV h4/h1 (P < 0.01) and the mean APV h3, APV h3/h1 (P < 0.05), and APV h4/h1 (P < 0.01) were significantly higher in the hypertensive group than in the healthy group, and the ulnar passage (UP) APV h4/h1 (P < 0.05) was clearly elevated in the hypertensive group. These results provide a preliminary validation of this novel approach for determining the APV by arrayed pulse wave analysis. In conclusion, we identified effective indicators of hypertensive vascular function. Traditional Chinese medicine (TCM) pulses comprise multidimensional information, and a sensor array could provide a better indication of TCM pulse characteristics. In this study, the validation of the arrayed pulse wave analysis demonstrates that the APV can reliably mirror TCM pulse characteristics.

Role of opioid receptors in modulation of P2X receptor-mediated cardiac sympathoexcitatory reflex response.

Myocardial ischemia evokes powerful reflex responses through activation of vagal and sympathetic afferents in the heart through the release of ischemic metabolites. We have demonstrated that extracellular ATP stimulates cardiac sympathetic afferents through P2 receptor-mediated mechanism, and that opioid peptides suppress these afferents' activity. However, the roles of both P2 receptor and endogenous opioids in cardiac sympathoexcitatory reflex (CSR) responses remain unclear. We therefore hypothesized that activation of cardiac P2 receptor evokes CSR responses by stimulating cardiac sympathetic afferents and these CSR responses are modulated by endogenous opioids. We observed that intrapericardial injection of α,ß-methylene ATP (α,ß-meATP, P2X receptor agonist), but not ADP (P2Y receptor agonist), caused a graded increase in mean arterial pressure in rats with sinoaortic denervation and vagotomy. This effect of α,ß-meATP was abolished by blockade of cardiac neural transmission with intrapericardial procaine treatment and eliminated by intrapericardial A-317491, a selective P2X2/3 and P2X3 receptor antagonist. Intrapericardial α,ß-meATP also evoked CSR response in vagus-intact rats. Furthermore, the P2X receptor-mediated CSR responses were enhanced by intrapericardial naloxone, a specific opioid receptor antagonist. These data suggest that stimulation of cardiac P2X2/3 and P2X3, but not P2Y receptors, powerfully evokes CSR responses through activation of cardiac spinal afferents, and that endogenous opioids suppress the P2X receptor-mediated CSR responses.

Adenosine Receptor A2a, but Not A1 in the rVLM Participates Along With Opioids in Acupuncture-Mediated Inhibition of Excitatory Cardiovascular Reflexes.

Electroacupuncture (EA) can be used to lower high blood pressure (BP) in clinical practice. However, precise mechanisms underlying its effects on elevated BP remain unclear. Our previous studies have shown that EA at the P5-6 acupoints, overlying the median nerve, attenuates elevated BP induced by gastric distension (GD) through influence on rostral ventrolateral medulla (rVLM). Although adenosine is released during neuronal activation in the rVLM, its role in acupuncture-cardiovascular regulation is unknown. The purinergic system is involved in cardiovascular pressor and depressor responses, including via selective activation of A1 and A2 a rVLM receptors, respectively. The action of A2 a receptor stimulation in the central nervous system may be further regulated through an endogenous opioid mechanism. However, it is uncertain whether this putative action occurs in the rVLM. We hypothesized that adenosine in the rVLM contributes to EA modulation of sympathoexcitatory reflexes through an A2 a but not an A1 adenosine receptor-opioid mechanism. EA or sham-EA was applied at the P5-6 acupoints in Sprague-Dawley male rats subjected to repeated GD under anesthesia. We found that EA (n = 6) but not sham-EA (n = 5) at P5-6 significantly (P < 0.05) attenuated GD-induced elevations in BP. EA modulation of sympathoexcitatory cardiovascular reflexes was reversed significantly after rVLM microinjection (50 nl) of 8-SPT (10 mM; non-selective adenosine receptor antagonist; n = 7) or SCH 58261 (1 mM; A2 a receptor antagonist; n = 8; both P < 0.05), but not by DPCPX (3 mM; A1 receptor antagonist; n = 6) or the vehicle (5% dimethylsulfoxide; n = 6). Moreover, microinjection of an A2 a receptor agonist, CGS-21680 (0.4 mM; n = 8) into the rVLM attenuated GD-induced pressor responses without EA, which mimicked EA's inhibitory effects (P < 0.05). After blockade of opioid receptors with naloxone (1 mM) in the rVLM, SCH 58261's reversal of EA's effect on GD-induced pressor responses was blunted, and CGS-21680-mediated inhibitory effect on pressor responses was not observed. Furthermore, neurons labeled with adenosine A2 a receptors were anatomically co-localized with neurons stained with enkephalin in the rVLM. These data suggest that the involvement of rVLM adenosine A2 a receptors in EA modulation of GD-induced pressor reflexes is, at least in part, dependent on the presence of endogenous opioids.

Acupuncture activates a direct pathway from the nucleus tractus solitarii to the rostral ventrolateral medulla.

Our previous studies have shown that electroacupuncture (EA) at the Jianshi-Neiguan acupoints (P5-6, overlying the median nerve) attenuates sympathoexcitatory responses through its influence on neuronal activity in the rostral ventrolateral medulla (rVLM). The nucleus tractus solitarii (NTS) receives input from somatic nerve stimulation. Connections between the NTS and the rVLM during EA stimulation have not been investigated and thus were the focus of the present study. Seven to ten days after unilateral microinjection of a rhodamine-conjugated microsphere retrograde tracer (100 nl) into the rVLM, rats were subjected to EA or sham-EA without electrical stimulation. EA was performed for 30 min at the P5-6 acupoints bilaterally. Perikarya containing the microsphere tracer were found in the NTS of both groups. Compared to controls (needle placement without electrical stimulation, n = 7), c-Fos immunoreactivity and neurons double-labeled with c-Fos, an immediate early gene, and the tracer were significantly increased in the NTS of EA-treated rats (all P < 0.05; n = 8), particularly, in the medial and lateral subdivisions of NTS at subpostremal and obex levels. These results suggest that EA at the P5-6 acupoints activates NTS neurons. Furthermore, EA-activated NTS neurons directly project to the rVLM and likely influence the rVLM activity.

Modulation of Neurally Mediated Vasodepression and Bradycardia by Electroacupuncture through Opioids in Nucleus Tractus Solitarius.

Stimulation of vagal afferent endings with intravenous phenylbiguanide (PBG) causes both bradycardia and vasodepression, simulating neurally mediated syncope. Activation of µ-opioid receptors in the nucleus tractus solitarius (NTS) increases blood pressure. Electroacupuncture (EA) stimulation of somatosensory nerves underneath acupoints P5-6, ST36-37, LI6-7 or G37-39 selectively but differentially modulates sympathoexcitatory responses. We therefore hypothesized that EA-stimulation at P5-6 or ST36-37, but not LI6-7 or G37-39 acupoints, inhibits the bradycardia and vasodepression through a µ-opioid receptor mechanism in the NTS. We observed that stimulation at acupoints P5-6 and ST36-37 overlying the deep somatosensory nerves and LI6-7 and G37-39 overlying cutaneous nerves differentially evoked NTS neural activity in anesthetized and ventilated animals. Thirty-min of EA-stimulation at P5-6 or ST36-37 reduced the depressor and bradycardia responses to PBG while EA at LI6-7 or G37-39 did not. Congruent with the hemodynamic responses, EA at P5-6 and ST36-37, but not at LI6-7 and G37-39, reduced vagally evoked activity of cardiovascular NTS cells. Finally, opioid receptor blockade in the NTS with naloxone or a specific µ-receptor antagonist reversed P5-6 EA-inhibition of the depressor, bradycardia and vagally evoked NTS activity. These data suggest that point specific EA stimulation inhibits PBG-induced vasodepression and bradycardia responses through a µ-opioid mechanism in the NTS.

Role of TRPV1 in acupuncture modulation of reflex excitatory cardiovascular responses.

We have shown that acupuncture, including manual and electroacupuncture (MA and EA), at the P5-6 acupoints stimulates afferent fibers in the median nerve (MN) to modulate sympathoexcitatory cardiovascular reflexes through central regulation of autonomic function. However, the mechanisms underlying acupuncture activation of these sensory afferent nerves and their cell bodies in the dorsal root ganglia (DRG) are unclear. Transient receptor potential vanilloid type 1 (TRPV1) is present in sensory nerve fibers distributed in the general region of acupoints like ST36 and BL 40 located in the hindlimb. However, the contribution of TRPV1 to activation of sensory nerves by acupuncture, leading to modulation of pressor responses, has not been studied. We hypothesized that TRPV1 participates in acupuncture's activation of sensory afferents and their associated cell bodies in the DRG to modulate pressor reflexes. Local injection of iodoresiniferatoxin (Iodo-RTX; a selective TRPV1 antagonist), but not 5% DMSO (vehicle), into the P6 acupoint on the forelimb reversed the MA's inhibition of pressor reflexes induced by gastric distension (GD). Conversely, inhibition of GD-induced sympathoexcitatory responses by EA at P5-6 was unchanged after administration of Iodo-RTX into P5-6. Single-unit activity of Group III or IV bimodal afferents sensitive to both mechanical and capsaicin stimuli responded to MA stimulation at P6. MA-evoked activity was attenuated significantly ( P < 0.05) by local administration of Iodo-RTX ( n = 12) but not by 5% DMSO ( n = 12) into the region of the P6 acupoint in rats. Administration of Iodo-RTX into P5-6 did not reduce bimodal afferent activity evoked by EA stimulation ( n = 8). Finally, MA at P6 and EA at P5-6 induced phosphorylation of extracellular signal-regulated kinases (ERK; an intracellular signaling messenger involved in cellular excitation) in DRG neurons located at C7-8 spinal levels receiving MN inputs. After TRPV1 was knocked down in the DRG at these spinal levels with intrathecal injection of TRPV1-siRNA, expression of phosphorylated ERK in the DRG neuron was reduced in MA-treated, but not EA-treated animals. These data suggest that TRPV1 in Group III and IV bimodal sensory afferent nerves contributes to acupuncture inhibition of reflex increases in blood pressure and specifically plays an important role during MA but not EA.

Moxibustion Modulates Sympathoexcitatory Cardiovascular Reflex Responses Through Paraventricular Nucleus.

Electroacupuncture (EA) point specific (ST36-37) stimulation decreases cardiovascular reflex responses through supraspinal regions such as the hypothalamic paraventricular nucleus (PVN) while mechanical stimulation of acupoints decreases pressor responses through peripheral thermal transient receptor potential vanilloid type-1 (TRPV1). Moxibustion generating heat applied at acupoint in combination with antihypertensive drugs decreases elevated blood pressure. We hypothesized that moxibustion modulates sympathoexcitatory cardiovascular responses through the hypothalamic PVN and peripheral heat sensitive TRPV1 in the absence of antihypertensive drugs. Rats were anesthetized, ventilated, and heart rate and mean blood pressure were monitored. Gastric distention induced consistent pressor reflex responses every 10-min. Thirty-minutes of bilateral moxibustion at the acupoint ST36, overlying the deep peroneal nerves, reduced the gastric distention evoked elevation in blood pressure. Blood pressure reflex responses were not reduced by both EA and moxibustion at G39. The moxibustion inhibition but not EA inhibition of the cardiovascular responses was reversed with blockade of local heat sensitive TRPV1 at ST36. Accordingly, activation of thermal TRPV1 by moxibustion at an average of 44.2°C in contrast to 40°C reduced the pressor responses. Naloxone, an opioid receptor antagonist, microinjected into PVN inhibited transiently the effect of moxibustion. Thus, activation of peripheral heat sensitive TRPV1 mediated the moxibustion-inhibition, but not EA-inhibition, of sympathoexcitatory cardiovascular reflex responses through hypothalamic PVN opioid system.

Repetitive Electroacupuncture Attenuates Cold-Induced Hypertension through Enkephalin in the Rostral Ventral Lateral Medulla.

Acupuncture lowers blood pressure (BP) in hypertension, but mechanisms underlying its action are unclear. To simulate clinical studies, we performed electroacupuncture (EA) in unanesthetized rats with cold-induced hypertension (CIH) induced by six weeks of cold exposure (6 °C). EA (0.1 - 0.4 mA, 2 Hz) was applied at ST36-37 acupoints overlying the deep peroneal nerve for 30 min twice weekly for five weeks while sham-EA was conducted with the same procedures as EA except for no electrical stimulation. Elevated BP was reduced after six sessions of EA treatment and remained low 72 hrs after EA in 18 CIH rats, but not in sham-EA (n = 12) and untreated (n = 6) CIH ones. The mRNA level of preproenkephalin in the rostral ventrolateral medulla (rVLM) 72 hr after EA was increased (n = 9), compared to the sham-EA (n = 6), untreated CIH rats (n = 6) and normotensive control animals (n = 6). Microinjection of ICI 174,864, a δ-opioid receptor antagonist, into the rVLM of EA-treated CIH rats partially reversed EA's effect on elevated BP (n = 4). Stimulation of rVLM of CIH rats treated with sham-EA using a δ-opioid agonist, DADLE, decreased BP (n = 6). These data suggest that increased enkephalin in the rVLM induced by repetitive EA contributes to BP lowering action of EA.

Paraventricular Nucleus Modulates Excitatory Cardiovascular Reflexes during Electroacupuncture.

The paraventricular nucleus (PVN) regulates sympathetic outflow and blood pressure. Somatic afferent stimulation activates neurons in the hypothalamic PVN. Parvocellular PVN neurons project to sympathoexcitatory cardiovascular regions of the rostral ventrolateral medulla (rVLM). Electroacupuncture (EA) stimulates the median nerve (P5-P6) to modulate sympathoexcitatory responses. We hypothesized that the PVN and its projections to the rVLM participate in the EA-modulation of sympathoexcitatory cardiovascular responses. Cats were anesthetized and ventilated. Heart rate and mean blood pressure were monitored. Application of bradykinin every 10-min on the gallbladder induced consistent pressor reflex responses. Thirty-min of bilateral EA stimulation at acupoints P5-P6 reduced the pressor responses for at least 60-min. Inhibition of the PVN with naloxone reversed the EA-inhibition. Responses of cardiovascular barosensitive rVLM neurons evoked by splanchnic nerve stimulation were reduced by EA and then restored with opioid receptor blockade in the PVN. EA at P5-P6 decreased splanchnic evoked activity of cardiovascular barosensitive PVN neurons that also project directly to the rVLM. PVN neurons labeled with retrograde tracer from rVLM were co-labeled with µ-opioid receptors and juxtaposed to endorphinergic fibers. Thus, the PVN and its projection to rVLM are important in processing acupuncture modulation of elevated blood pressure responses through a PVN opioid mechanism.

elPBN neurons regulate rVLM activity through elPBN-rVLM projections during activation of cardiac sympathetic afferent nerves.

The external lateral parabrachial nucleus (elPBN) within the pons and rostral ventrolateral medulla (rVLM) contributes to central processing of excitatory cardiovascular reflexes during stimulation of cardiac sympathetic afferent nerves (CSAN). However, the importance of elPBN cardiovascular neurons in regulation of rVLM activity during CSAN activation remains unclear. We hypothesized that CSAN stimulation excites the elPBN cardiovascular neurons and, in turn, increases rVLM activity through elPBN-rVLM projections. Compared with controls, in rats subjected to microinjection of retrograde tracer into the rVLM, the numbers of elPBN neurons double-labeled with c-Fos (an immediate early gene) and the tracer were increased after CSAN stimulation (P < 0.05). The majority of these elPBN neurons contain vesicular glutamate transporter 3. In cats, epicardial bradykinin and electrical stimulation of CSAN increased the activity of elPBN cardiovascular neurons, which was attenuated (n = 6, P < 0.05) after blockade of glutamate receptors with iontophoresis of kynurenic acid (Kyn, 25 mM). In separate cats, microinjection of Kyn (1.25 nmol/50 nl) into the elPBN reduced rVLM activity evoked by both bradykinin and electrical stimulation (n = 5, P < 0.05). Excitation of the elPBN with microinjection of dl-homocysteic acid (2 nmol/50 nl) significantly increased basal and CSAN-evoked rVLM activity. However, the enhanced rVLM activity induced by dl-homocysteic acid injected into the elPBN was reversed following iontophoresis of Kyn into the rVLM (n = 7, P < 0.05). These data suggest that cardiac sympathetic afferent stimulation activates cardiovascular neurons in the elPBN and rVLM sequentially through a monosynaptic (glutamatergic) excitatory elPBN-rVLM pathway.

GABA in nucleus tractus solitarius participates in electroacupuncture modulation of cardiopulmonary bradycardia reflex.

Phenylbiguanide (PBG) stimulates cardiopulmonary receptors and cardiovascular reflex responses, including decreases in blood pressure and heart rate mediated by the brain stem parasympathetic cardiac neurons in the nucleus ambiguus and nucleus tractus solitarius (NTS). Electroacupuncture (EA) at P5-6 stimulates sensory fibers in the median nerve and modulates these reflex responses. Stimulation of median nerves reverses bradycardia through action of γ-aminobutyric acid (GABA) in the nucleus ambiguus, important in the regulation of heart rate. We do not know whether the NTS or the neurotransmitter mechanisms in this nucleus participate in these modulatory actions by acupuncture. We hypothesized that somatic nerve stimulation during EA (P5-6) modulates cardiopulmonary inhibitory responses through a GABAergic mechanism in the NTS. Anesthetized and ventilated cats were examined during either PBG or direct vagal afferent stimulation while 30 min of EA was applied at P5-6. Reflex heart rate and blood pressure responses and NTS-evoked discharge were recorded. EA reduced the PBG-induced depressor and bradycardia reflexes by 67% and 60%, respectively. Blockade of GABAA receptors in the NTS reversed EA modulation of bradycardia but not the depressor response. During EA, gabazine reversed the vagally evoked discharge activity of cardiovascular NTS neurons. EA modulated the vagal-evoked cardiovascular NTS cellular activity for 60 min. Immunohistochemistry using triple labeling showed GABA immunoreactive fibers juxtaposed to glutamatergic nucleus ambiguus-projecting NTS neurons in rats. These glutamatergic neurons expressed GABAA receptors. These findings suggest that EA inhibits PBG-evoked bradycardia and vagally evoked NTS activity through a GABAergic mechanism, likely involving glutamatergic nucleus ambiguus-projecting NTS neurons.

Electroacupuncture modulation of reflex hypertension in rats: role of cholecystokinin octapeptide.

Acupuncture or electroacupuncture (EA) potentially offers a nonpharmacological approach to reduce high blood pressure (BP). However, ~70% of the patients and animal subjects respond to EA, while 30% do not. EA acts, in part, through an opioid mechanism in the rostral ventrolateral medulla (rVLM) to inhibit sympathoexcitatory reflexes induced by gastric distention. CCK-8 opposes the action of opioids during analgesia. Therefore, we hypothesized that CCK-8 in the rVLM antagonizes EA modulation of sympathoexcitatory cardiovascular reflex responses. Male rats anesthetized with ketamine and α-chloralose subjected to repeated gastric distension every 10 min were examined for their responsiveness to EA (2 Hz, 0.5 ms, 1-4 mA) at P5-P6 acupoints overlying median nerve. Repeated gastric distension every 10 min evoked consistent sympathoexcitatory responses. EA at P5-P6 modulated gastric distension-induced responses. Microinjection of CCK-8 in the rVLM reversed the EA effect in seven responders. The CCK1 receptor antagonist devazepide microinjected into the rVLM converted six nonresponders to responders by lowering the reflex response from 21 ± 2.2 to 10 ± 2.9 mmHg (first vs. second application of EA). The EA modulatory action in rats converted to responders with devazepide was reversed with rVLM microinjection of naloxone (n = 6). Microinjection of devazepide in the absence of a second application of EA did not influence the primary pressor reflexes of nonresponders. These data suggest that CCK-8 antagonizes EA modulation of sympathoexcitatory cardiovascular responses through an opioid mechanism and that inhibition of CCK-8 can convert animals that initially are unresponsive to EA to become responsive.

Medullary GABAergic mechanisms contribute to electroacupuncture modulation of cardiovascular depressor responses during gastric distention in rats.

Electroacupuncture (EA) at P5-P6 acupoints overlying the median nerves typically reduces sympathoexcitatory blood pressure (BP) reflex responses in eucapnic rats. Gastric distention in hypercapnic acidotic rats, by activating both vagal and sympathetic afferents, decreases heart rate (HR) and BP through actions in the rostral ventrolateral medulla (rVLM) and nucleus ambiguus (NAmb), leading to sympathetic withdrawal and parasympathetic activation, respectively. A GABAA mechanism in the rVLM mediates the decreased sympathetic outflow. The present study investigated the hypothesis that EA modulates gastric distention-induced hemodynamic depressor and bradycardia responses through nuclei that process parasympathetic and sympathetic outflow. Anesthetized hypercapnic acidotic rats manifested repeatable decreases in BP and HR with gastric distention every 10 min. Bilateral EA at P5-P6 for 30 min reversed the hypotensive response from -26 ± 3 to -6 ± 1 mmHg and the bradycardia from -35 ± 11 to -10 ± 3 beats/min for a period that lasted more than 70 min. Immunohistochemistry and in situ hybridization to detect c-Fos protein and GAD 67 mRNA expression showed that GABAergic caudal ventral lateral medulla (cVLM) neurons were activated by EA. Glutamatergic antagonism of cVLM neurons with kynurenic acid reversed the actions of EA. Gabazine used to block GABAA receptors microinjected into the rVLM or cVLM reversed EA's action on both the reflex depressor and bradycardia responses. EA modulation of the decreased HR was inhibited by microinjection of gabazine into the NAmb. Thus, EA through GABAA receptor mechanisms in the rVLM, cVLM, and NAmb modulates gastric distention-induced reflex sympathoinhibition and vagal excitation.

Repetitive electroacupuncture causes prolonged increased met-enkephalin expression in the rVLM of conscious rats.

Enkephalinergic neurons in the rostral ventrolateral medulla (rVLM), an important presympathetic region in the brainstem, are activated by 30 min of low frequency (2 Hz) electroacupuncture (EA) at acupoints P5-P6, which overlie the median nerves. To more closely model the clinical application of acupuncture, we administered EA for 30 min twice over a 72 h period to unsedated conscious rats to examine its prolonged action. We hypothesized that repetitive EA would increase preproenkephalin mRNA and met-enkephalin in the rVLM of unsedated conscious rats. Rats received either EA (1-4 mA, 0.5 ms, 2 Hz) or sham stimulation (needle placement without electrical stimulation) twice at P5-P6 acupoints bilaterally. Preproenkephalin mRNA and its peptide met-enkephalin in the rVLM were measured 24 or 48 h after the final EA or sham procedure. Relative ratios of preproenkephalin mRNA levels (normalized with the 18S housekeeping gene) were almost doubled at 24h compared to sham (6.1 ± 0.79 vs. 3.1 ± 0.47). Met-enkephalin measured in rVLM tissue pooled from several rats exposed to the same treatment was increased by repeated EA by 68% after 24h and 51% after 48h, relative to sham. These findings suggest that repeated application of EA in the conscious rats enhances transcription and translation of enkephalin in rVLM for days. Since opioids in the rVLM contribute importantly to the action of EA on sympathetic outflow, this mechanism may contribute to the prolonged action of acupuncture on elevated blood pressure in patients.

Nucleus ambiguus cholinergic neurons activated by acupuncture: relation to enkephalin.

Acupuncture regulates autonomic function. Our previous studies have shown that electroacupuncture (EA) at the Jianshi-Neiguan acupoints (P5-P6, underlying the median nerve) inhibits central sympathetic outflow and attenuates excitatory cardiovascular reflexes, in part, through an opioid mechanism. It is unknown if EA at these acupoints influences the parasympathetic system. Thus, using c-Fos expression, we examined activation of nucleus ambiguus (NAmb) neurons by EA, their relation to cholinergic (preganglionic parasympathetic) neurons and those containing enkephalin. To enhance detection of cell bodies containing enkephalin, colchicine (90-100 µg/kg) was administered into the subarachnoid space of cats 30 h prior to EA or sham-operated controls for EA. Following bilateral barodenervation and cervical vagotomy, either EA for 30 min at P5-P6 acupoints or control stimulation (needle placement at P5-P6 without stimulation) was applied. While perikarya containing enkephalin were observed in some medullary nuclei (e.g., raphé), only enkephalin-containing neuronal processes were found in the NAmb. Compared to controls (n=4), more c-Fos immunoreactivity, located principally in close proximity to fibers containing enkephalin was noted in the NAmb of EA-treated cats (n=5; P<0.01). Moreover, neurons double-labeled with c-Fos and choline acetyltransferase in the NAmb were identified in EA-treated, but not control animals. These data demonstrate for the first time that EA activates preganglionic parasympathetic neurons in the NAmb. Because of their close proximity, these EA-activated neurons likely interact with nerve fibers containing enkephalin. These results suggest that EA at the P5-P6 acupoints has the potential to influence parasympathetic outflow and cardiovascular function, likely through an enkephalinergic mechanism.