- Email: [email protected]
Central and peripheral neural mechanisms of acupuncture in myocardial ischemia
International Congress Series 1238 (2002) 79 – 87
Central and peripheral neural mechanisms of acupuncture in myocardial ischemia John C. Longhurst* College of Medicine, Department of Medicine, Medical Sciences 1, Room C240, University of California, Irvine, CA 92697-4075, USA
Abstract Previous clinical and experimental studies suggest that acupuncture helps myocardial ischemia, hypertension and arrhythmias. To investigate mechanisms that underlie the cardiovascular influence of acupuncture, we have developed a feline model of reversible myocardial ischemia. Ischemia was induced by partial ligation of a coronary artery and stimulation of a sympathetic reflex induced by bradykinin applied to the gallbladder (GB). We superimposed low frequency (2 – 5 Hz), low intensity (2 – 5 mA) electroacupuncture (EA) to stimulate bilaterally the Neiguan acupoints, located on the pericardial meridian over the median nerves (MN) on each forelimb. EA for 30 min substantially reduced myocardial ischemia, measured as an improvement in regional myocardial wall thickening, a response requiring 10 – 20 min for onset and lasting for 60 – 90 min. Direct MN stimulation as well as stimulation from an acupuncture needle placed percutaneously at the Neiguan acupoints cause similar responses by stimulating Group III more than Group IV fibers in the MN. We also showed that EA works through the endogenous opioid system, since both intravenous and microinjected naloxone into the rostral ventral lateral medulla (rVLM) prevent the EA-related response. Finally, opioid A and y, but not n, receptors in the rVLM are responsible for the EA-related modulation of sympathetic outflow, suggesting that endorphins and enkephalins, but not dynorphins are the neuromodulators involved in this response. Current studies are focused on the cellular responses in rVLM neurons. Thus, EA stimulates mainly Group III fibers that ascend to the central nervous system and, through an opioid mechanism in the rVLM, particularly involving endorphins and enkephalins, EA inhibits sympathetic outflow to reduce the pressor response and resulting myocardial ischemia during reflex stimulation. Our studies provide an understanding of the physiological mechanisms underlying EA and assist in developing a framework of information that can be understood and, hopefully, accepted by scientists and clinicians in the Western countries. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Opioid receptors; Autonomic nervous system; Cardiovascular reflexes; Neiguan acupoint; Alternative medicine
*
Tel.: +1-949-824-8161; fax: +1-49-824-2200. E-mail address: [email protected] (J.C. Longhurst).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 4 1 5 - 6
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J.C. Longhurst / International Congress Series 1238 (2002) 79–87
1. Introduction Cardiovascular disease represents one of the more prevalent diseases affecting middleaged and older age individuals in both Western and Eastern societies. In particular, substantial morbidity and mortality result from hypertension and ischemic cardiovascular disease. We currently have available in our clinical armamentarium a number of approaches that can be used to treat patients with cardiovascular disorders, including risk factor reduction, pharmacologic therapy, invasive and interventional therapies as practiced by cardiologists and surgeons. Recently, there has been both profound interest and acceptance of a number of alternative therapies. These therapies have emerged because none of the more usual therapies is completely effective in eliminating either the symptoms or the adverse outcomes resulting from these diseases. Furthermore, many mainstay therapies are associated with side effects that surprising numbers of patients find unacceptable. Therefore, there has been a surge of interest in various alternative therapies. For example, alternative approaches in the medical management of cardiovascular disease have included spinal cord and electrical nerve stimulation as well as acupuncture [1]. Acupuncture as a therapy is almost 2000 years old. It has been used for a wide variety of treatments but probably it is most accepted for treatment of pain [2,3]. Increasing evidence indicates that acupuncture, acupressure, electroacupuncture (EA) and even moxibustion may be useful in treating patients with neurologic disease, including disorders of autonomic nervous system, hypertension and other forms of cardiovascular disease. For example, the World Health Organization has noted that acute infection and inflammation, dysfunction of autonomic nervous system, pain and peripheral central neurological diseases each represent conditions for which acupuncture may be indicated [4]. The National Institute of Health published a consensus statement in 1998, indicating that a number of issues related to acupuncture concerning its efficacy, sham effects, adverse reaction, acupuncture points, training and credentialing and future research need further exploration [4]. Recently, a workshop held by NHLBI and National Center for Complementary and Alternative Medicine identified areas of needed research in complementary medicine in general and acupuncture specifically. Areas of needed research in acupuncture include efficacy, controversies such as point specificity and side effects. Our laboratory has been interested in further defining the scientific basis of acupuncture. These studies began as a result of a collaboration between China and the United States, and included investigators who had expertise in peripheral sensory neurophysiology, central autonomic regulation, integrative cardiovascular neurophysiology, exercise physiology and clinical cardiology. The goal of these studies has been to gain a perspective of the mechanisms underlying acupuncture using an experimental animal preparation designed to mimic demand-induced ischemia in patients with symptomatic coronary heart disease.
2. Methods Adult cats anesthetized with ketamine (40 mg/kg), followed by bolus intravenous injection of a-chloralose (50 – 75 mg/kg) were used in these studies. The trachea was
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intubated and respiration maintained with a respirator. The physiological status of the animal was maintained by frequent sampling of arterial blood gases and correction of arterial blood PO2, PCO2 and pH. A femoral vein was cannulated for administration of drugs and fluids. A femoral artery was cannulated for monitoring arterial blood pressure. A ventral midline incision was used to expose the gallbladder (GB) for later chemical stimulation. In some experiments, the median nerves (MN) were isolated for direct stimulation, while in other experiments, acupuncture needles were placed percutaneously through the skin at the Neiguan acupoints. These acupoints are located over the MNs on the forelimbs. Subsequent electrical stimulation of the MNs or acupuncture needles was accomplished with a constant current stimulator and a stimulation isolation unit using pulses of 0.5-ms duration, 5 Hz, 1– 2 V, 1– 2 mA or 2 Hz, 1– 4 V, 1– 10 mA, respectively. In some experiments, the left chest was opened near the midline, two ribs were removed and the pericardium incised to expose the heart and coronary arteries. Subsequently, the proximal left anterior descending coronary artery (LAD) was isolated and partially occluded or a diagonal branch of the LAD was ligated. In some animals, a Doppler pulse flow transducer was positioned over the left anterior descending distal to the partially occluded LAD coronary artery to provide continuous measurement of coronary blood velocity, as an index of cardiac output. In other animals, regional left ventricular (LV) wall thickness was measured with a modified 20-MHz single-transducer sonomicrometer system [6]. This piezoelectric transducer was positioned in the region that would become ischemic as determined during a brief test occlusion of the LAD. In some experiments, a craniotomy was performed either by removing the basal occipital bone to expose the ventral medulla or a partial occipital craniotomy to expose the dorsal medulla. In both circumstances, the animal’s head was stabilized using a stereotaxic apparatus with an attached micropipette assembly [7,8]. Micropipettes were placed in the rostral ventral lateral medulla (rVLM), identified by the pressor response to electrical or chemical stimulations as well as subsequent anatomical localization of an injected dye spot. Thus, at the end of each microinjection experiment the brain stem was removed, fixed in 10% formalin and sectioned (50 m) with a cryostat microtome. After staining the slices with neutral red, the injection site was identified microscopically. Bradykinin (BK) was prepared for application to the GB using a 1-cm2 pledget soaked with BK solution (1 or 10 Ag/ml). The pledget was left on the GB for approximately 30 to 60 s, until the maximum pressor reflex response was attained. Recovery periods of approximately 15 min were provided between applications. In some animals, the area at risk as well as the extent of infarction in the LV were assessed by injecting patent blue violet dye (0.5%) into the left atrium after occlusion of the LAD or its diagonal branch at the end of the experiment [5]. The heart subsequently was removed, sliced in breadloaf fashion into four to six rings from apex to base. The risk area in the LV was defined as the nonblue region. The slices subsequently were weighed and placed in a solution of 1% triphenyltetrazolium chloride in potassium phosphate buffer for 20 min. Regions of viable myocardium stained brick red were distinguished from regions of necrosis within the area at risk. The percent area of infarction was converted to grams of tissue and used for calculation of risk and infarct regions expressed as percentages of total LV weight for each heart.
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3. Protocols 3.1. Effect of MN stimulation on myocardial ischemia [5] Animals were stabilized for 15 min after completion of surgery. Subsequently, BK was administered to the GB approximately every 15 min. After several control stimulations, partial occlusion of the LAD or complete occlusion of the diagonal branch was accomplished. The GB was stimulated twice more with BK while direct electrical stimulation of the MN was accomplished at an intensity sufficient to produce moderate paw twitches for 30 min. During this time BK was applied twice. The stimulus frequency and intensity used in these experiments is similar to that used in clinical EA [9]. After completion of MN stimulation, as a surrogate for EA, BK was applied every 15 min for the next hour. As a time control, five additional animals were studied following exposure of the MN and attachment of the electrodes but without electrical stimulation. Similar to the MN stimulation group, BK was applied at 15-min intervals for a total of 10 applications during the control protocol. 3.2. Afferent fibers activation by median nerve stimulation [5] To identify the fiber types activated by MN stimulation, single-unit afferent recording studies were performed in the desheathed MN isolated in the upper forelimb near the humerus. After splitting of the nerve into fine filaments, a unipolar recording electrode with a high impedance probe was used to record single unit activity. Fibers activated by the stimulus paradigm noted above were recorded. Relating the conduction time between the stimulus and recording electrodes to the conduction distance provided an estimate of conduction velocity. Group IV fibers were identified as axons with conduction velocity of < 2.5 m/s while Group III fibers as those with conduction velocity of z 2.5 m/s. 3.3. Influence of naloxone on EA response [7] In two protocols, the above procedure was repeated with the exception that naloxone was administered 5 –10 min after termination of EA, involving percutaneous needle stimulation of the Neiguan acupoints bilaterally. Naloxone was administered either intravenously (0.4 mg/kg) or by bilateral microinjection (10 nM in 0.1 Al) into the rVLM. Subsequently, the GB was stimulated every 15 min with BK for the next 30 min. 3.4. Effects of specific opioid antagonist and agonists on EA response [8] To identify the opioid receptor subtypes responsible for the EA-induced response, A, y or n receptor antagonists and agonists were administered with a picospritzer (0.1 Al) unilaterally into the rVLM through a micropipette. In this manner, artificial cerebrospinal fluid (CSF) as a control or CTOP (10 – 20 nM), ICI 174,864 (30 nM) or nor-BNI (50 nM), the A, y and n opioid antagonists, respectively, or DAGO (6– 12 nM), DADLE (3 nM), or U50,488 (3 –6 nM), the A, y and n opioid agonists were, administered. The sequence of studies included two to three applications of BK to the GB with a pledget every 15 min
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over a period of 30 –45 min, with percutaneously inserted needles EA at the Neiguan acupoints followed by microinjection of either the CSF control or one of the antagonists after 5 min of acupuncture and subsequent repeat stimulation of the GB with BK every 15 min for the next hour. Alternatively, after basal applications of BK to the GB were completed, one of the three opioid agonists was microinjected into the rVLM in lieu of EA and repeated GB stimulation with BK was conducted every 15 min for 45 min.
4. Data analysis Blood pressure, coronary blood velocity and regional wall motion were recorded on a polygraph. Data also were input to a PC-based computer with an A/D converter data interface card for data acquisition and analysis using a data reduction module (EGAA, R.C. Electronics). The double product was calculated as systolic blood pressure heart rate [10]. LV wall thickness (WTh) was calculated as regional wall motion according to the formula: WTh = 100 [(ESD EDD)/EDD], where ESD = end-systolic diameter, EDD = end-diastolic diameter. Percent wall thickness was calculated as the ratio of [(maximum WTh response to BK pre-BK WTh)/pre-BK WTh] 100 as previously described [11]. Data were presented as mean F SEM. Changes in hemodynamic function produced by administration of BK were analyzed by two-way repeated-measures ANOVA with Bonferroni or Student – Newman– Keuls post hoc test to test for significant differences between preselected comparisons. A statistical software package, SigmaStat (Jandel Scientific) was used for these analyses. The level of statistical significance was P V 0.05.
5. Results 5.1. Feline model of reflex-induced myocardial ischemia response [5] Application of BK to the GB caused a cardiovascular excitatory response including an increase in arterial blood pressure, double product and wall thickening. In the group in which the blood flow was measured, following partial LAD occlusion, the increases in wall thickening and double product were accompanied by an increase in coronary flow velocity. Basal and maximal stimulated coronary blood flows were substantially reduced by arterial occlusion. In contrast to the increase in wall thickening observed before partial coronary occlusion, the increase in coronary flow velocity after occlusion was accompanied by a reduction in regional wall thickening. This response was consistent with the appearance of regional myocardial ischemia during reflex activation of the cardiovascular system following application of BK to the GB. 5.2. Influence of MN stimulation on myocardial ischemia response [5] Direct stimulation of the MN for 30 min, as a surrogate for EA, reduced the reflex pressor response and the resulting increase in double product. In association with a reduced peak double product, we observed that wall thickening again increased (i.e.,
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returned towards normal) after 30 min of MN stimulation. The effect of MN stimulation lasted approximately 1 h. The region at risk for ischemia in the LV was 14% in the partial occlusion group and 2.8% in the complete occlusion group. There was no infarction in animals subjected to partial occlusion and only small infarction in two of the five animals that underwent complete occlusion of the diagonal branch of the LAD. In contrast to the response of the MN stimulation group, we observed no hemodynamic effect in the time control group in which the MN was isolated but not stimulated. 5.3. Influence of naloxone on EA response [7] Intravenous administration of naloxone reversed the blunted cardiovascular reflex pressor response to BK on the GB by percutaneous needle EA within a period of 15 min. Importantly, intravenous naloxone also reversed the beneficial effects that EA had on the myocardial ischemic response as shown by the reversal of wall thickening from a positive to a negative value following the administration of the drug. Microinjection of naloxone into the rVLM, like intravenous administration of the opioid antagonist, reversed the reflex change in blood pressure. This last observation contrasted with the continuing decline in the blood pressure response following EA and microinjection of saline into the rVLM. 5.4. Opioid receptors subtypes in EA response [8] Like naloxone microinjection of CTOP and ICI, the respective A and y opioid receptor antagonists reversed the EA response. This effect lasted for 30 min. Conversely, nor-BNI, the n-receptor antagonist, caused only a transient reversal of the EA-induced response. Microinjection of the A and n opioid agonists into the rVLM, like EA, reversed the reflex pressor response to application of BK on the GB. In contrast, administration of the nopioid agonists into the rVLM did not alter the reflex pressor response to stimulation of chemosensitive receptors on the GB. The effective sites to which the antagonists and agonists were delivered within the boundaries of rVLM in the cat [12 – 14].
6. Discussion Our studies utilized a feline model of demand-induced ischemia to demonstrate that either MN stimulation, as a surrogate for EA, or EA with percutaneous insertion of needles at the Neiguan acupoints bilaterally is capable of reducing the ischemia, as measured by changes in regional wall motion. Regional myocardial thickening has been shown to be a sensitive and accurate index of myocardial function and ischemia in the intact heart. The beneficial effects of the acupuncture began within 15 min and lasted for up to 1 h following its cessation. Potentially, acupuncture could improve cardiac performance by lessening ischemia either through an improvement in blood supply or through a reduction in oxygen demand by the myocardium. Our studies [5] suggest that, in this feline model of demand-induced
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ischemia, acupuncture reduced ischemia through a reduction in oxygen demand rather than an improvement in oxygen supply to the myocardium. This conclusion may be a function of the model in which there is little reflex-induced increase in sympathetic tone to the coronary arterial system [15]. Patients whose angina is precipitated by coronary vasoconstriction, e.g. during static exercise [16], may benefit by a reduction in sympathetic tone to the heart, leading to an increase in coronary flow, which could add to the reduction in oxygen requirements by the myocardium. A number of studies have suggested that acupuncture exerts its influence on the central nervous system through activation of finely myelinated Group III somatic sensory nerves [2]. These studies come primarily from investigations involving recording of multi-unit activity, as well as by sensations reported by patients during treatment. Individuals undergoing EA have reported a set of sensations that the Chinese call DeQi, which represents a heaviness, soreness or deep muscle ache. Our studies demonstrate that although two-thirds of the fibers activated by the stimulus parameters used in our study are Group III afferents, fully one-third of the fibers are Group IV unmyelinated somatic afferents. Thus, it seems reasonable to surmise that both Group III and Group IV fibers participate in the acupuncture-hemodynamic response. The mechanism by which EA or MN stimulation in the cat alters the hemodynamic response to chemosensitive receptor stimulation of the GB involves the opioid system, as revealed by our studies employing either intravenous or rVLM microinjection naloxone. Naloxone substantially reversed the beneficial effects of EA, both with respect to the reflex pressor response and the myocardial ischemic response following partial coronary artery occlusion. These data are consistent with studies by others [2] who have provided at least 10 different lines of experimental evidence implicating the opioid system in acupuncture analgesia. Thus, analgesic and reflex cardiovascular influences of low frequency electroacupuncture appear to operate through similar opioid neuromodulatory mechanisms. Recently, we extended our earlier observations by demonstrating that A and y-opioid receptors play a major role in the beneficial effects of EA on the reflex pressor response during stimulation of chemosensitive receptors by BK. Conversely, n-receptors appear to play little role in the EA-related effects. These data suggest that h-endorphins and enkephalins, but not dynorphins, which serve as the primary ligands for A, y and nreceptors, respectively, are the principle neuromodulators of sympathetic premotor outflow in the rVLM during EA stimulation [17]. We believe that studies like we have conducted are valuable in delineating the central neural physiological mechanisms underlying the autonomic influence of EA during cardiovascular activation. Clinically, EA has been used by traditional Chinese physicians to treat patients with cardiovascular disease. Preliminary reports have suggested that acupuncture may be beneficial in treating hypertension [18,19]. Other studies suggest that acupuncture may be used to treat patients with ischemic heart disease. For instance, patients with angina pectoris may derive benefit when they are treated with a several-week course of acupuncture [20,21]. However, there are a number of concerns relating to these previous clinical investigations. The foremost problem relates to the lack of adequate sham controls used in these early studies. A second concern is the small number of patients included in these investigations as well as the lack of blinding of patients or the
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investigators to the intervention. The issue of sham effects has been reviewed by Ernst and White [22] who noted a number of difficulties in rigorously demonstrating the clinical effectiveness of acupuncture. Furthermore, some studies have used acupuncture points, for instance, in the ear, the so-called auricular acupuncture [23], for which there is little carefully controlled clinical evidence of efficacy. Thus, it is clear that in the future, studies identifying both mechanisms and clinical utility of acupuncture need to be rigorously conducted. Specific questions that need to be addressed include: (1) What is the most appropriate sham control for acupuncture? (2) Which acupuncture point or set of acupuncture points are most efficacious? (3) What are the central neural pathways underlying the effect of acupuncture on sympathetic outflow and on the cardiovascular system? (4) What mechanisms are operative in the central nervous system during EA that reduce the sympathoexcitatory cardiovascular response? These and still other questions will have to be answered before scientists and physicians in all cultures will accept this potentially promising new therapy for the treatment of the cardiovascular disease. Acknowledgements The author acknowledges the collaborators: Peng Li, Stephanie Tjen-A-Looi, Stephen Rendig, Koullis Pitsillides, Hui-Lin Pan, Dong Chao, Lin Shen and the secretarial assistance of Sherry Ong. This work was supported by National Natural Science Foundation of China Grant no. 39610120955, National Heart, Lung, and Blood Institute Grants HL-36527, HL-52165, and HL-07682, Samueli Center/Basic, Samueli Center/ Clinical and DANA Foundation. References [1] J.C. Longhurst, Alternative approaches to the medical management of cardiovascular disease: acupuncture, electrical nerve and spinal cord stimuation, Heart Dis. (2001) 215 – 216. [2] B. Pomeranz, Scientific research into acupuncture for the relief of pain, J. Altern. Complement. Med. 2 (1996) 53 – 60. [3] D.J. Mayer, Acupuncture: an evidence-based review of the clinical literature, Annu. Rev. Med. 51 (2000) 49 – 63. [4] J.C. Longhurst, Acupuncture’s beneficial effects on the cardiovascular system, Prev. Cardiol. 1 (1998) 21 – 33. [5] P. Li, K.F. Pitsillides, S.V. Rendig, H.-L. Pan, J.C. Longhurst, Reversal of reflex-induced myocardial ischemia by median nerve stimulation: a feline model of electroacupuncture, Circulation 97 (1998) 1186 – 1194. [6] K.F. Pitsillides, J.C. Longhurst, An ultrasonic system for measurement of absolute myocardial thickness using a single transducer, Am. J. Physiol. 37 (1995) H1358 – H1367. [7] D.M. Chao, L.L. Shen, S. Tjen-A-Looi, K.F. Pitsillides, P. Li, J.C. Longhurst, Naloxone reverses inhibitory effect of electroacupuncture on sympathetic cardiovascular reflex responses, Am. J. Physiol. 276 (1999) H2127 – H2134. [8] P. Li, S. Tjen-A-Looi, J.C. Longhurst, Rostral ventrolateral medullary opioid receptor subtypes in the inhibitory effect of electroacupuncture on reflex autonomic response in cats, Autonomic Neuroscience: Basic & Clinical 89 (2001) 38 – 47. [9] B. Pomeranz, Scientific basis of acupuncture, in: G. Stux, B. Pomeranz (Eds.), Acupuncture Textbook and Atlas, Springer-Verlag, Berlin, 1987, pp. 1 – 34.
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[10] K. Kitamura, C.R. Jorgensen, F.L. Gobel, H.L. Taylor, Y. Wang, Hemodynamic correlates of myocardial oxygen consumption during upright exercise, J. Appl. Physiol. 32 (1972) 516 – 522. [11] J.D. Symons, K.F. Pitsillides, J.C. Longhurst, Chronic reduction of myocardial ischemia does not attenuate coronary collateral development in miniswine, Circulation 86 (1992) 660 – 671. [12] S.M. Barman, G.L. Gebber, Axonal projection patterns of ventrolateral medullospinal sympathoexcitatory neurons, J. Neurophysiol. 53 (1985) 1551 – 1566. [13] S.M. Barman, G.L. Gebber, Subgroups of rostral ventrolateral medullary and caudal medullary raphe neurons based on patterns of relationship to sympathetic nerve discharge and axonal projections, J. Neurophysiol. 77 (1997) 65 – 75. [14] P.G. Guyenet, Role of ventral medulla oblongata in blood pressure regulation, in: A.D. Loewy, K.M. Spyer (Eds.), Central Regulation of Autonomic Functions, Oxford Univ. Press, New York, 1990, pp. 145 – 167. [15] S.E. Martin, D.M. Pilkington, J.C. Longhurst, Coronary vascular responses to chemical stimulation of abdominal visceral organs, Am. J. Physiol. 256 (1989) H735 – H744. [16] R. Aung-Din, J.H. Mitchell, J.C. Longhurst, Reflex alpha-adrenergic coronary vasoconstriction during hindlimb static exercise in dogs, Circ. Res. 48 (1981) 502 – 509. [17] T. Reisine, G. Pasternak, Opioid analgesics and antagonists, in: J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon, A.G. Gilman (Eds.), The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, 1996, pp. 521 – 555. [18] Y.J. Chiu, A. Chi, I.A. Reid, Cardiovascular and endocrine effects of acupuncture in hypertensive patients, Clin. Exp. Hypertens. 19 (1997) 1047 – 1063. [19] T. Williams, K. Mueller, M.W. Cornwall, Effect of acupuncture-point stimulation on diastolic blood pressure in hypertensive subjects: a preliminary study, Phys. Ther. 71 (1991) 523 – 529. [20] S. Ballegaard, F. Pedersen, A. Pietersen, V.H. Nissen, N.V. Olsen, Effects of acupuncture in moderate, stable angina pectoris: a controlled study, J. Intern. Med. 227 (1990) 25 – 30. [21] A. Richter, J. Herlitz, A. Hjalmarson, Effect of acupuncture in patients with angina pectoris, Eur. Heart J. 12 (1991) 175 – 178. [22] E. Ernst, A.R. White, A review of problems in clinical acupuncture research, Am. J. Chin. Med. XXV (1997) 3 – 11. [23] H.-Q. Huang, Acupuncture at otoacupoint heart for treatment of vascular hypertension, J. Trad. Chin. Med. 12 (1992) 133 – 136.
Central and peripheral neural mechanisms of acupuncture in myocardial ischemia John C. Longhurst* College of Medicine, Department of Medicine, Medical Sciences 1, Room C240, University of California, Irvine, CA 92697-4075, USA
Abstract Previous clinical and experimental studies suggest that acupuncture helps myocardial ischemia, hypertension and arrhythmias. To investigate mechanisms that underlie the cardiovascular influence of acupuncture, we have developed a feline model of reversible myocardial ischemia. Ischemia was induced by partial ligation of a coronary artery and stimulation of a sympathetic reflex induced by bradykinin applied to the gallbladder (GB). We superimposed low frequency (2 – 5 Hz), low intensity (2 – 5 mA) electroacupuncture (EA) to stimulate bilaterally the Neiguan acupoints, located on the pericardial meridian over the median nerves (MN) on each forelimb. EA for 30 min substantially reduced myocardial ischemia, measured as an improvement in regional myocardial wall thickening, a response requiring 10 – 20 min for onset and lasting for 60 – 90 min. Direct MN stimulation as well as stimulation from an acupuncture needle placed percutaneously at the Neiguan acupoints cause similar responses by stimulating Group III more than Group IV fibers in the MN. We also showed that EA works through the endogenous opioid system, since both intravenous and microinjected naloxone into the rostral ventral lateral medulla (rVLM) prevent the EA-related response. Finally, opioid A and y, but not n, receptors in the rVLM are responsible for the EA-related modulation of sympathetic outflow, suggesting that endorphins and enkephalins, but not dynorphins are the neuromodulators involved in this response. Current studies are focused on the cellular responses in rVLM neurons. Thus, EA stimulates mainly Group III fibers that ascend to the central nervous system and, through an opioid mechanism in the rVLM, particularly involving endorphins and enkephalins, EA inhibits sympathetic outflow to reduce the pressor response and resulting myocardial ischemia during reflex stimulation. Our studies provide an understanding of the physiological mechanisms underlying EA and assist in developing a framework of information that can be understood and, hopefully, accepted by scientists and clinicians in the Western countries. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Opioid receptors; Autonomic nervous system; Cardiovascular reflexes; Neiguan acupoint; Alternative medicine
*
Tel.: +1-949-824-8161; fax: +1-49-824-2200. E-mail address: [email protected] (J.C. Longhurst).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 4 1 5 - 6
80
J.C. Longhurst / International Congress Series 1238 (2002) 79–87
1. Introduction Cardiovascular disease represents one of the more prevalent diseases affecting middleaged and older age individuals in both Western and Eastern societies. In particular, substantial morbidity and mortality result from hypertension and ischemic cardiovascular disease. We currently have available in our clinical armamentarium a number of approaches that can be used to treat patients with cardiovascular disorders, including risk factor reduction, pharmacologic therapy, invasive and interventional therapies as practiced by cardiologists and surgeons. Recently, there has been both profound interest and acceptance of a number of alternative therapies. These therapies have emerged because none of the more usual therapies is completely effective in eliminating either the symptoms or the adverse outcomes resulting from these diseases. Furthermore, many mainstay therapies are associated with side effects that surprising numbers of patients find unacceptable. Therefore, there has been a surge of interest in various alternative therapies. For example, alternative approaches in the medical management of cardiovascular disease have included spinal cord and electrical nerve stimulation as well as acupuncture [1]. Acupuncture as a therapy is almost 2000 years old. It has been used for a wide variety of treatments but probably it is most accepted for treatment of pain [2,3]. Increasing evidence indicates that acupuncture, acupressure, electroacupuncture (EA) and even moxibustion may be useful in treating patients with neurologic disease, including disorders of autonomic nervous system, hypertension and other forms of cardiovascular disease. For example, the World Health Organization has noted that acute infection and inflammation, dysfunction of autonomic nervous system, pain and peripheral central neurological diseases each represent conditions for which acupuncture may be indicated [4]. The National Institute of Health published a consensus statement in 1998, indicating that a number of issues related to acupuncture concerning its efficacy, sham effects, adverse reaction, acupuncture points, training and credentialing and future research need further exploration [4]. Recently, a workshop held by NHLBI and National Center for Complementary and Alternative Medicine identified areas of needed research in complementary medicine in general and acupuncture specifically. Areas of needed research in acupuncture include efficacy, controversies such as point specificity and side effects. Our laboratory has been interested in further defining the scientific basis of acupuncture. These studies began as a result of a collaboration between China and the United States, and included investigators who had expertise in peripheral sensory neurophysiology, central autonomic regulation, integrative cardiovascular neurophysiology, exercise physiology and clinical cardiology. The goal of these studies has been to gain a perspective of the mechanisms underlying acupuncture using an experimental animal preparation designed to mimic demand-induced ischemia in patients with symptomatic coronary heart disease.
2. Methods Adult cats anesthetized with ketamine (40 mg/kg), followed by bolus intravenous injection of a-chloralose (50 – 75 mg/kg) were used in these studies. The trachea was
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intubated and respiration maintained with a respirator. The physiological status of the animal was maintained by frequent sampling of arterial blood gases and correction of arterial blood PO2, PCO2 and pH. A femoral vein was cannulated for administration of drugs and fluids. A femoral artery was cannulated for monitoring arterial blood pressure. A ventral midline incision was used to expose the gallbladder (GB) for later chemical stimulation. In some experiments, the median nerves (MN) were isolated for direct stimulation, while in other experiments, acupuncture needles were placed percutaneously through the skin at the Neiguan acupoints. These acupoints are located over the MNs on the forelimbs. Subsequent electrical stimulation of the MNs or acupuncture needles was accomplished with a constant current stimulator and a stimulation isolation unit using pulses of 0.5-ms duration, 5 Hz, 1– 2 V, 1– 2 mA or 2 Hz, 1– 4 V, 1– 10 mA, respectively. In some experiments, the left chest was opened near the midline, two ribs were removed and the pericardium incised to expose the heart and coronary arteries. Subsequently, the proximal left anterior descending coronary artery (LAD) was isolated and partially occluded or a diagonal branch of the LAD was ligated. In some animals, a Doppler pulse flow transducer was positioned over the left anterior descending distal to the partially occluded LAD coronary artery to provide continuous measurement of coronary blood velocity, as an index of cardiac output. In other animals, regional left ventricular (LV) wall thickness was measured with a modified 20-MHz single-transducer sonomicrometer system [6]. This piezoelectric transducer was positioned in the region that would become ischemic as determined during a brief test occlusion of the LAD. In some experiments, a craniotomy was performed either by removing the basal occipital bone to expose the ventral medulla or a partial occipital craniotomy to expose the dorsal medulla. In both circumstances, the animal’s head was stabilized using a stereotaxic apparatus with an attached micropipette assembly [7,8]. Micropipettes were placed in the rostral ventral lateral medulla (rVLM), identified by the pressor response to electrical or chemical stimulations as well as subsequent anatomical localization of an injected dye spot. Thus, at the end of each microinjection experiment the brain stem was removed, fixed in 10% formalin and sectioned (50 m) with a cryostat microtome. After staining the slices with neutral red, the injection site was identified microscopically. Bradykinin (BK) was prepared for application to the GB using a 1-cm2 pledget soaked with BK solution (1 or 10 Ag/ml). The pledget was left on the GB for approximately 30 to 60 s, until the maximum pressor reflex response was attained. Recovery periods of approximately 15 min were provided between applications. In some animals, the area at risk as well as the extent of infarction in the LV were assessed by injecting patent blue violet dye (0.5%) into the left atrium after occlusion of the LAD or its diagonal branch at the end of the experiment [5]. The heart subsequently was removed, sliced in breadloaf fashion into four to six rings from apex to base. The risk area in the LV was defined as the nonblue region. The slices subsequently were weighed and placed in a solution of 1% triphenyltetrazolium chloride in potassium phosphate buffer for 20 min. Regions of viable myocardium stained brick red were distinguished from regions of necrosis within the area at risk. The percent area of infarction was converted to grams of tissue and used for calculation of risk and infarct regions expressed as percentages of total LV weight for each heart.
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3. Protocols 3.1. Effect of MN stimulation on myocardial ischemia [5] Animals were stabilized for 15 min after completion of surgery. Subsequently, BK was administered to the GB approximately every 15 min. After several control stimulations, partial occlusion of the LAD or complete occlusion of the diagonal branch was accomplished. The GB was stimulated twice more with BK while direct electrical stimulation of the MN was accomplished at an intensity sufficient to produce moderate paw twitches for 30 min. During this time BK was applied twice. The stimulus frequency and intensity used in these experiments is similar to that used in clinical EA [9]. After completion of MN stimulation, as a surrogate for EA, BK was applied every 15 min for the next hour. As a time control, five additional animals were studied following exposure of the MN and attachment of the electrodes but without electrical stimulation. Similar to the MN stimulation group, BK was applied at 15-min intervals for a total of 10 applications during the control protocol. 3.2. Afferent fibers activation by median nerve stimulation [5] To identify the fiber types activated by MN stimulation, single-unit afferent recording studies were performed in the desheathed MN isolated in the upper forelimb near the humerus. After splitting of the nerve into fine filaments, a unipolar recording electrode with a high impedance probe was used to record single unit activity. Fibers activated by the stimulus paradigm noted above were recorded. Relating the conduction time between the stimulus and recording electrodes to the conduction distance provided an estimate of conduction velocity. Group IV fibers were identified as axons with conduction velocity of < 2.5 m/s while Group III fibers as those with conduction velocity of z 2.5 m/s. 3.3. Influence of naloxone on EA response [7] In two protocols, the above procedure was repeated with the exception that naloxone was administered 5 –10 min after termination of EA, involving percutaneous needle stimulation of the Neiguan acupoints bilaterally. Naloxone was administered either intravenously (0.4 mg/kg) or by bilateral microinjection (10 nM in 0.1 Al) into the rVLM. Subsequently, the GB was stimulated every 15 min with BK for the next 30 min. 3.4. Effects of specific opioid antagonist and agonists on EA response [8] To identify the opioid receptor subtypes responsible for the EA-induced response, A, y or n receptor antagonists and agonists were administered with a picospritzer (0.1 Al) unilaterally into the rVLM through a micropipette. In this manner, artificial cerebrospinal fluid (CSF) as a control or CTOP (10 – 20 nM), ICI 174,864 (30 nM) or nor-BNI (50 nM), the A, y and n opioid antagonists, respectively, or DAGO (6– 12 nM), DADLE (3 nM), or U50,488 (3 –6 nM), the A, y and n opioid agonists were, administered. The sequence of studies included two to three applications of BK to the GB with a pledget every 15 min
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over a period of 30 –45 min, with percutaneously inserted needles EA at the Neiguan acupoints followed by microinjection of either the CSF control or one of the antagonists after 5 min of acupuncture and subsequent repeat stimulation of the GB with BK every 15 min for the next hour. Alternatively, after basal applications of BK to the GB were completed, one of the three opioid agonists was microinjected into the rVLM in lieu of EA and repeated GB stimulation with BK was conducted every 15 min for 45 min.
4. Data analysis Blood pressure, coronary blood velocity and regional wall motion were recorded on a polygraph. Data also were input to a PC-based computer with an A/D converter data interface card for data acquisition and analysis using a data reduction module (EGAA, R.C. Electronics). The double product was calculated as systolic blood pressure heart rate [10]. LV wall thickness (WTh) was calculated as regional wall motion according to the formula: WTh = 100 [(ESD EDD)/EDD], where ESD = end-systolic diameter, EDD = end-diastolic diameter. Percent wall thickness was calculated as the ratio of [(maximum WTh response to BK pre-BK WTh)/pre-BK WTh] 100 as previously described [11]. Data were presented as mean F SEM. Changes in hemodynamic function produced by administration of BK were analyzed by two-way repeated-measures ANOVA with Bonferroni or Student – Newman– Keuls post hoc test to test for significant differences between preselected comparisons. A statistical software package, SigmaStat (Jandel Scientific) was used for these analyses. The level of statistical significance was P V 0.05.
5. Results 5.1. Feline model of reflex-induced myocardial ischemia response [5] Application of BK to the GB caused a cardiovascular excitatory response including an increase in arterial blood pressure, double product and wall thickening. In the group in which the blood flow was measured, following partial LAD occlusion, the increases in wall thickening and double product were accompanied by an increase in coronary flow velocity. Basal and maximal stimulated coronary blood flows were substantially reduced by arterial occlusion. In contrast to the increase in wall thickening observed before partial coronary occlusion, the increase in coronary flow velocity after occlusion was accompanied by a reduction in regional wall thickening. This response was consistent with the appearance of regional myocardial ischemia during reflex activation of the cardiovascular system following application of BK to the GB. 5.2. Influence of MN stimulation on myocardial ischemia response [5] Direct stimulation of the MN for 30 min, as a surrogate for EA, reduced the reflex pressor response and the resulting increase in double product. In association with a reduced peak double product, we observed that wall thickening again increased (i.e.,
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returned towards normal) after 30 min of MN stimulation. The effect of MN stimulation lasted approximately 1 h. The region at risk for ischemia in the LV was 14% in the partial occlusion group and 2.8% in the complete occlusion group. There was no infarction in animals subjected to partial occlusion and only small infarction in two of the five animals that underwent complete occlusion of the diagonal branch of the LAD. In contrast to the response of the MN stimulation group, we observed no hemodynamic effect in the time control group in which the MN was isolated but not stimulated. 5.3. Influence of naloxone on EA response [7] Intravenous administration of naloxone reversed the blunted cardiovascular reflex pressor response to BK on the GB by percutaneous needle EA within a period of 15 min. Importantly, intravenous naloxone also reversed the beneficial effects that EA had on the myocardial ischemic response as shown by the reversal of wall thickening from a positive to a negative value following the administration of the drug. Microinjection of naloxone into the rVLM, like intravenous administration of the opioid antagonist, reversed the reflex change in blood pressure. This last observation contrasted with the continuing decline in the blood pressure response following EA and microinjection of saline into the rVLM. 5.4. Opioid receptors subtypes in EA response [8] Like naloxone microinjection of CTOP and ICI, the respective A and y opioid receptor antagonists reversed the EA response. This effect lasted for 30 min. Conversely, nor-BNI, the n-receptor antagonist, caused only a transient reversal of the EA-induced response. Microinjection of the A and n opioid agonists into the rVLM, like EA, reversed the reflex pressor response to application of BK on the GB. In contrast, administration of the nopioid agonists into the rVLM did not alter the reflex pressor response to stimulation of chemosensitive receptors on the GB. The effective sites to which the antagonists and agonists were delivered within the boundaries of rVLM in the cat [12 – 14].
6. Discussion Our studies utilized a feline model of demand-induced ischemia to demonstrate that either MN stimulation, as a surrogate for EA, or EA with percutaneous insertion of needles at the Neiguan acupoints bilaterally is capable of reducing the ischemia, as measured by changes in regional wall motion. Regional myocardial thickening has been shown to be a sensitive and accurate index of myocardial function and ischemia in the intact heart. The beneficial effects of the acupuncture began within 15 min and lasted for up to 1 h following its cessation. Potentially, acupuncture could improve cardiac performance by lessening ischemia either through an improvement in blood supply or through a reduction in oxygen demand by the myocardium. Our studies [5] suggest that, in this feline model of demand-induced
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ischemia, acupuncture reduced ischemia through a reduction in oxygen demand rather than an improvement in oxygen supply to the myocardium. This conclusion may be a function of the model in which there is little reflex-induced increase in sympathetic tone to the coronary arterial system [15]. Patients whose angina is precipitated by coronary vasoconstriction, e.g. during static exercise [16], may benefit by a reduction in sympathetic tone to the heart, leading to an increase in coronary flow, which could add to the reduction in oxygen requirements by the myocardium. A number of studies have suggested that acupuncture exerts its influence on the central nervous system through activation of finely myelinated Group III somatic sensory nerves [2]. These studies come primarily from investigations involving recording of multi-unit activity, as well as by sensations reported by patients during treatment. Individuals undergoing EA have reported a set of sensations that the Chinese call DeQi, which represents a heaviness, soreness or deep muscle ache. Our studies demonstrate that although two-thirds of the fibers activated by the stimulus parameters used in our study are Group III afferents, fully one-third of the fibers are Group IV unmyelinated somatic afferents. Thus, it seems reasonable to surmise that both Group III and Group IV fibers participate in the acupuncture-hemodynamic response. The mechanism by which EA or MN stimulation in the cat alters the hemodynamic response to chemosensitive receptor stimulation of the GB involves the opioid system, as revealed by our studies employing either intravenous or rVLM microinjection naloxone. Naloxone substantially reversed the beneficial effects of EA, both with respect to the reflex pressor response and the myocardial ischemic response following partial coronary artery occlusion. These data are consistent with studies by others [2] who have provided at least 10 different lines of experimental evidence implicating the opioid system in acupuncture analgesia. Thus, analgesic and reflex cardiovascular influences of low frequency electroacupuncture appear to operate through similar opioid neuromodulatory mechanisms. Recently, we extended our earlier observations by demonstrating that A and y-opioid receptors play a major role in the beneficial effects of EA on the reflex pressor response during stimulation of chemosensitive receptors by BK. Conversely, n-receptors appear to play little role in the EA-related effects. These data suggest that h-endorphins and enkephalins, but not dynorphins, which serve as the primary ligands for A, y and nreceptors, respectively, are the principle neuromodulators of sympathetic premotor outflow in the rVLM during EA stimulation [17]. We believe that studies like we have conducted are valuable in delineating the central neural physiological mechanisms underlying the autonomic influence of EA during cardiovascular activation. Clinically, EA has been used by traditional Chinese physicians to treat patients with cardiovascular disease. Preliminary reports have suggested that acupuncture may be beneficial in treating hypertension [18,19]. Other studies suggest that acupuncture may be used to treat patients with ischemic heart disease. For instance, patients with angina pectoris may derive benefit when they are treated with a several-week course of acupuncture [20,21]. However, there are a number of concerns relating to these previous clinical investigations. The foremost problem relates to the lack of adequate sham controls used in these early studies. A second concern is the small number of patients included in these investigations as well as the lack of blinding of patients or the
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investigators to the intervention. The issue of sham effects has been reviewed by Ernst and White [22] who noted a number of difficulties in rigorously demonstrating the clinical effectiveness of acupuncture. Furthermore, some studies have used acupuncture points, for instance, in the ear, the so-called auricular acupuncture [23], for which there is little carefully controlled clinical evidence of efficacy. Thus, it is clear that in the future, studies identifying both mechanisms and clinical utility of acupuncture need to be rigorously conducted. Specific questions that need to be addressed include: (1) What is the most appropriate sham control for acupuncture? (2) Which acupuncture point or set of acupuncture points are most efficacious? (3) What are the central neural pathways underlying the effect of acupuncture on sympathetic outflow and on the cardiovascular system? (4) What mechanisms are operative in the central nervous system during EA that reduce the sympathoexcitatory cardiovascular response? These and still other questions will have to be answered before scientists and physicians in all cultures will accept this potentially promising new therapy for the treatment of the cardiovascular disease. Acknowledgements The author acknowledges the collaborators: Peng Li, Stephanie Tjen-A-Looi, Stephen Rendig, Koullis Pitsillides, Hui-Lin Pan, Dong Chao, Lin Shen and the secretarial assistance of Sherry Ong. This work was supported by National Natural Science Foundation of China Grant no. 39610120955, National Heart, Lung, and Blood Institute Grants HL-36527, HL-52165, and HL-07682, Samueli Center/Basic, Samueli Center/ Clinical and DANA Foundation. References [1] J.C. Longhurst, Alternative approaches to the medical management of cardiovascular disease: acupuncture, electrical nerve and spinal cord stimuation, Heart Dis. (2001) 215 – 216. [2] B. Pomeranz, Scientific research into acupuncture for the relief of pain, J. Altern. Complement. Med. 2 (1996) 53 – 60. [3] D.J. Mayer, Acupuncture: an evidence-based review of the clinical literature, Annu. Rev. Med. 51 (2000) 49 – 63. [4] J.C. Longhurst, Acupuncture’s beneficial effects on the cardiovascular system, Prev. Cardiol. 1 (1998) 21 – 33. [5] P. Li, K.F. Pitsillides, S.V. Rendig, H.-L. Pan, J.C. Longhurst, Reversal of reflex-induced myocardial ischemia by median nerve stimulation: a feline model of electroacupuncture, Circulation 97 (1998) 1186 – 1194. [6] K.F. Pitsillides, J.C. Longhurst, An ultrasonic system for measurement of absolute myocardial thickness using a single transducer, Am. J. Physiol. 37 (1995) H1358 – H1367. [7] D.M. Chao, L.L. Shen, S. Tjen-A-Looi, K.F. Pitsillides, P. Li, J.C. Longhurst, Naloxone reverses inhibitory effect of electroacupuncture on sympathetic cardiovascular reflex responses, Am. J. Physiol. 276 (1999) H2127 – H2134. [8] P. Li, S. Tjen-A-Looi, J.C. Longhurst, Rostral ventrolateral medullary opioid receptor subtypes in the inhibitory effect of electroacupuncture on reflex autonomic response in cats, Autonomic Neuroscience: Basic & Clinical 89 (2001) 38 – 47. [9] B. Pomeranz, Scientific basis of acupuncture, in: G. Stux, B. Pomeranz (Eds.), Acupuncture Textbook and Atlas, Springer-Verlag, Berlin, 1987, pp. 1 – 34.
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