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The effect of hypophysectomy on acupuncture analgesia in the mouse
Brain Research, 202 (1980) 33-39 © Elsevier/North-Holland Biomedical Press
33
T H E E F F E C T OF H Y P O P H Y S E C T O M Y ON A C U P U N C T U R E A N A L G E S I A IN T H E MOUSE
TSU-CHING FU, STEPHEN P. HALENDA and WILLIAM L. DEWEY Department of Pharmacology, Medical College of Virginia, Richmond, Virginia23298 (U.S.A.)
(Accepted May 29th, 1980) Key words: acupuncture - - electroacupuncture - - analgesia - - naloxone - - hypophysectomy
writhing test
SUMMARY Acupuncture analgesia was quantitated in the phenylquinone induced writhing test in mice. Both manual acupuncture and electroacupuncture significantly reduced the number of writhes, i.e. 47 ~ and 51 ~ reduction respectively. Naloxone (2 mg/kg) pretreatment abolished this antinocicpetive effect suggesting that an endogenous opiate-like substance was involved. Hypophysectomy did not alter the electroacupuncture induced inhibition of writhing. These results confirm previous reports that acupuncture causes the release of an endogenous substance(s) with opioid activity, but disagree with previous reports in that our data show that the hypophysis is not involved in the release of this endogenous opiate or in any other mechanism of acupuncture analgesia in the mouse. INTRODUCTION Evidence has accumulated that acupuncture increases experimental pain threshold in humansZ, 17 and in animals2O, 21. Although acupuncture has been studied extensively in recent years its mechanism is still not understood. It takes 15-30 min to reach the peak effect of acupuncture and it dissipates slowly after the end of the stimulation. The duration of analgesia also outlasts the duration of brain area (periaqueductal and periventricular regions) electrical stimulation induced antinociception 12,16,1s. Similarly, both acupuncture and brain area electrical stimulation induced antinociception are reversed by the narcotic antagonist naloxone 1,17. All of these characteristics suggest that a neurohumoral system is involved in the mechanism o f acupuncture antinociception. Recently, we have presented evidence that an endogenous substance was being released by morphine 1°. Convincing evidence exists to
34 indicate that this neurohumoral factor is of central origin and not released at a peripheral site 7. Recent reports suggest that the hypophysis might be the origin of this neurohumoral factor s. The isolation of endorphins from the hypophysis makes this hypothesis quite feasible. The purpose of the present investigation was to determine if the hypophysis is essential for acupuncture. We also demonstrated the use of the writhing test as a model for studying acupuncture in the mouse. MATERIALS AND METHODS A total of 225 male albino mice (24-42 g) were used in these experiments. All animals were unanesthetized and except for controls each mouse was placed in a restrainer (No. 1123 CIO, by Arthur H. Thomas). The rear bottom ends of the restrainers on either side were cut open (20 mm long, 8 mm wide) allowing the two hindlegs to be extended and were immobilized with rubber bands (foot cuffed) for later application of acupuncture needles. The rear end of the restrainer was closed with a tight-fit plate in which a 10 mm diameter half circle was cut. The tail of the mouse was extended through this hole. The other parts of the body including the two forelimbs were free to move in the restrainer. The mice in the restrained group were maintained in the restrainer an equal amount of time as those used in the acupuncture experiments.
Stimulation Acupuncture points for mice were determined by transposing anatomically from the Chinese traditional human acupuncture charts. Only one point, Tsu-San-Li (S-36), located in the lateral upper tibia, was used. This point has been widely used in a number of other animal species and is easily accessible. We used the same point on the leg of each mouse and did not test the effect of stimulation at other sites on the leg or the rest of the body. Since positive results were obtained at this site we did not attempt to define other sites useful for acupuncture analgesia or determine that this is the only site in the leg that would produce these effects. For manual acupuncture, the needle (32-gauge, 1/2 in. handle 1/2 in. long by Trueline Instruments) was carefully inserted into Tsu-San-Li point and manually rotated for 1 min, every 6 rain for the next 30 min. The frequency of the rotation of the needle was 120-130/min. In electro-acupuncture an interdermal needle (32-gauge, 1/4 in. long by Trueline Instruments) soldered to a flexible electrical wire, was inserted into the left hindleg at the Tsu-San-Li point. The second identical electrode, as a positive pole, was inserted into the same leg approximately 5 mm distal to the first electrode. Current was applied from a Grass $88 stimulator through a stimulus isolation unit (SIU5) and constant current until (CCUI), using square wave, 1 msec duration, frequency/2 sec, and current intensity = 6 × T, (T = threshold for causing minimum muscle contractions), it was 1.2-1.5 mA. The total stimulation time was 30 min. In some experiments naloxone (2 mg/kg) was given subcutaneously 10-15 min before acupuncture stimulation.
35
Hypophysectomy The operations were performed under pentobarbital anesthesia and were carried out 1-2 weeks before the acupuncture and writhing tests. The pituitary gland was exposed from the ventral surface by means of a hand drill which was made from stainless steel tubing 2 mm in diameter. A small hole was drilled on the basisphenoid bone in the midline just rostral to the occipito-basisphenoid suture line. The dura mater covering the hypophysis was torn away with a 22-gauge 1.5in. long hypodermic needle (the sharp point of the needle was bent and converted into a fine hook) then a dropper of proper size accomodated by the drilled hole was placed against the hypophysis and the gland was removed by negative pressure. The pituitary gland was carefully examined after removal. Only those mice with complete removal of the hypophysis were used in this study. This observational criteria was confirmed by the determination of serum corticosterone levels. These levels were quantitated by the protein binding assay using human corticosterone binding globulin 14. The average corticosterone level in hypophysectomized mice was 21.9 ng/ml serum (with a range of 10.3-34.7 ng/ml), compared to an average of 240.6 ng/ml (with a range of 107-500 ng/ml) in control or sham-operated mice. Wrffhing test Immediately following the release from the mouse restrainer, each test mouse received phenylquinone (3 mg/kg) i.p. as previously described 9. The number of writhes during the 10th and 15th minute after injection were counted and compared to those observed in non-restrained control mice given phenylquinone. Statistical differences between groups were determined using student's t-test. RESULTS The data presented in Table I show the mean and S. E. of the number of writhes after each treatment and the percentage inhibition for each treated group versus control.
Control series The first 3 treatments did not receive acupuncture stimulation. The mice in treatment 1 were 'non-restrained' controls consisting of 58 mice. They received phenylquinone only, and had a mean of 6.6 writhes per mouse. Treatments 2 and 3 were 'placebo' controls of 2 and 3 groups of 6 mice respectively. The mice in groups 2 and 3 were placed in the mouse restrainers just as the acupuncture mice but acupuncture was not performed. The mice in treatment 2 had an average of 6.17 writhes per mouse. Each mouse in treatment 3 received a random needle insertion in the left hindleg but neither electric current nor needle movement was conducted. The average number of writhes per mouse was 5.27. Acupuncture series The data presented in Table I show that both manual acupuncture and
36 TABLE I Effect of acupuncture on writhing test in mice Treatment
No. of mice (n)
Number of writhes (~ ± S.E.M.)
Percent inhibition
1. 2. 3. 4. 5. 6. 7. 8. 9.
58 12 18 24 24 18 24 24 17
6.60 ± 0.27 6.17 :k 0.61 5.27 i 0.41" 3.50 d- 0.33**,*** 3.25 ± 0.31"*,*** 6.05 4- 0.41 5.67 i 0.38§ 5.25 ± 0.36"*,§§ 3.82 -4- 0.38**
0 6.5 20.1 46.9 50.7 8.3 14.1 20.4 42.1
Control (unrestrained) Restrained Restrained and random needle insertion Acupuncture Electroacupuncture Naloxone and restrained Naloxone and acupuncture Naloxone and electroacupuncture Hypophysectomy and electroacupuncture
* Significantly different from control (P < 0.01) but not significantly different from restrained mice without needle insertion (group 2). ** Significantly different from control at P < 0.001. *** Significantly different from group 3 mice at P < 0.01. § Significantly different from acupuncture (group 4) at P < 0.001. §~ Significantly different from electroacupuncture (group 5) at P < 0.001.
electroacupuncture at the Tsu-San-Li Point significantly decreased the number o f writhes by 47 to 51 ~ respectively. The mean n u m b e r o f writhes for the 24 mice given m a n u a l acupuncture was 3.5. The mice given electroacupuncture showed similar results: the mean n u m b e r o f writhes for these mice was 3.25. Naloxone series
Naloxone alone, as previously reported had no effect on phenylquinone induced writhing. N a l o x o n e given 10-15 min prior to either m a n u a l or electro acupuncture stimulation effectively abolished the effect o f acupuncture on phenylquinone induced writhing. Hypophysectomized mice
The lack o f effects o f h y p o p h y s e c t o m y on electroacupuncutre can be seen by comparing the n u m b e r o f writhes in treatment 9 to those o f treatment 5, in Table I. It is clear f r o m these data that h y p o p h y s e c t o m y did n o t reverse the effects o f electroacupuncture and it is shown that the hypophysis is not critical for this m e t h o d o f increasing pain threshold. DISCUSSION Our data clearly show that both acupuncture and electroacupuncture significantly induce analgesia in mice and this effect can be blocked by naloxone. These results confirm those o f others in suggesting the possibility o f the release o f an endogenous substance(s) which plays a role in acupuncture 17,21. The use o f proper stimulation parameters, especially stimulation intensity and
37 frequency, is essential for obtaining reproducible electroacupuncmre analgesia. Some workers 11 have failed to obtain acupuncture analgesia. We believe one of the main reasons was that they did not choose appropriate stimulation intensity, or frequency, or both. Anderson et al. 3,4 measured the effects of acupuncture on pain threshold in teeth and showed no change at low intensity stimulations. Rather strong stimulations which caused powerful muscle contractions were needed to produce a significant increase in pain threshold. These findings suggest that: (1) activation of the large group I muscle afferents alone is not sufficient to increase pain threshold and; (2) activation of group II or even smaller muscle afferents is necessary to induce acupuncture analgesia. Chiang et al. 7 reported that acupuncture analgesia could still be induced after selective blockade of the transmission of the superficial cutaneous nerves but not following the blockade of muscle nerves. With all these findings in mind, therefore, we used a stimulation intensity of 6 × T, i.e., stimulus intensity at 6 times threshold for causing minimum muscle contraction of the hindlimb. This intensity is believed to be sufficient to activate most group II afferents but only excite a minimum, if any, of the pain fibers (group III) 19. Andersson et al.3,4 showed that stimulation with a low frequency of 2/sec produced a gradual increase in pain threshold which remained at a high level for a long period. However, a frequency of 10/see, or 100/sec, produced an abrupt increase and a rapid decrease of pain threshold. In other words, stimulation with a low frequency (less than 10/sec) is a necessity to induce acupuncture analgesia. A frequency of 2-3/sec is about the upper limit that can be carried out manually. Many workers tend to use higher frequency (more than 10/see) in electroacupuncture. This may be the reason why failure to obtain an analgesic effect has happened more often in electroacupuncture than in acupuncture studies. Possibly the most important finding of this study was that electroacupuncture analgesia was not affected by hypophysectomy in mice. These results suggest that the hypophysis does not play an important role in acupuncture analgesia and clearly differ from those of Pomeranz et al. 2°,21 who showed that the pituitary was necessary for acupuncture analgesia. Goldstein et al. a originally detected the presence of opiatelike peptides in the pituitary gland. However, the role of pituitary fl-endorphin is not clear. It is not thought to be the source of brain opiate peptides since the peptide levels in the brain are not affected by hypophysectomy6,15. Although Met-enkephalin has a wide distribution throughout many brain areas, its rapid inactivation in brain makes it unlikely to play an essential role in such a long-lasting process as acupuncture analgesia, fl-endorphin itself is a potent analgesic and is found in high concentrations in hypothalamusS,2~. Furthermore, Hosobuchi et al. la have reported that electrical stimulation of human periaqueductal gray caused an increase in immuno-reactive flendorphin in CSF. From all of these findings, it is not unreasonable to postulate that acupuncture or electroacupuncture activates group II and possibly some group III afferents which send impulses into the CNS. Two possible mechanisms may be involved. First, these impulses induce short-lasting pre- and post-synaptic inhibitions of pain afferents at the segmental levels. Our previous work 10 which demonstrated the release of endogenous
38 opiates in the CSF suggests that a more probable hypothesis is that some of these impulses are transmitted to the hypothalamus and periaqueductal gray to cause the release of fl-endorphin or some other endogenous opiate into the CSF. This material transported in the CSF to various parts of the brain stem inhibits pain-perceptive neurons and/or the transmission of noxious impulses to the higher centers. ACKNOWLEDGEMENTS
We thank Dr. Suzanne Laychock for the determination of the corticosterone levels. This research was supported in part by USPHS Grant DA 01647, DA 07027 and DA 00490.
REFERENCES 1 Akil, H., Mayer, D. J. and Liebeskind, J. C., Antagonism of stimulation-produced analgesia by naloxone, a narcotic antagonist, Science, 191 (1976) 961-962. 2 Andersson, S. A., Ericson, T., Holmgren, E. and Lindquist, G., Electroacupuncture: effect on pain threshold measured with electrical stimulation of teeth, Brain Research, 63 (1973) 393-396. 3 Andersson, S. A. and Holmgren, E., Analgesic effects of peripheral conditioning stimulation. III. Effect of high frequency stimulation; segmental mechanisms interacting with pain, Acupuncture Electro. Ther. Res. Int. J., 3 (1978) 23-36. 4 Andersson, S. A., Holmgren, E. and Roos, A., Analgesic effects of peripheral conditioningstimulation. II. Importance of certain stimulation parameters, Acupuncture Electro. Ther. Res. Int. J., 2 (1977) 237-246. 5 Bloom, F. E., Rossier, J., Battenberg, E. L., Bayon, A., French, E., Hendriksen, S. J., Siggins, G. R., Segal, D., Browne, R., Ling, N. and Guillemin, R., fi-endorphin: cellular localization, electrophysiological and behavioral effects, Advanc. Biochem. Psychopharmacol., 1 (1978) 89-110. 6 Cheung, A. and Goldstein, A., Failure of hypophysectomy to alter brain content of opioid peptides (endorphins), Life Sci., 19 (1976) 1005-1008. 7 Chiang, C.-Y., Chang, C.-T., Chu, H.-L. and Yang, L.-F., Peripheral afferent pathway for acupuncture analgesia, Scientia Sinca, 16 (1973) 210-217. 8 Cox, B. M., Opheim, K. E., Teschemacher, H. and Goldstein, A., A peptide-like substance from pituitary that acts like morphine, Life Sci., 16 (1975) 1777-1782. 9 Dewey, W. L., Harris, L. S., Howes, J. F. and Nuite, J. A., The effect of various neurohumoral modulators on the activity of morphine and the narcotic antagonists in the tail-flick and phenylquinone tests, J. Pharmacol. exp. Ther., 175 (1970) 435-442. 10 Fu, T. C. and Dewey, W. L., Morphine antinociception: evidence for the release of endogenous substance(s), Life Sci., 25 (1979) 53-60. 11 Galeano, C. and Leung, C. Y., Has acupuncture an analgesic effect in the rabbit?, Pain, 4 (1978) 265-271. 12 Giesler, G. J. and Liebeskind, J. C., Inhibition of visceral pain by electrical stimulation of the periaqueductal gray matter, Pain, 2 (1976) 43 48. 13 Hosobuchi, Y., Rossier, J., Bloom, F. E. and Guillemin, R., Stimulation of human periaqueductal gray for pain relief increases immunoreactive ~-endorphin in ventricular fluid, Science, 203 (1979) 279-281. 14 Jaanus, S. D., Carchman, R. A. and Rubin, R. P., Further studies on the relationships between cyclic AMP levels and adrenocortical activity, Endocrinology, 91 (1972) 887-895. 15 Kobayashi, R. M., Palkovits, M., Miller, R. J., Chang, K. J. and Cuatrecasas, P., Brain enkephalin distribution is unaltered by hypophysectomy, Life Sci., 22 (1978) 527-530. 16 Mayer, D. J. and Liebeskind, J. C., Pain reduction by focal electrical stimulation of the brain: an anatomical behavioral analysis, Brain Research, 68 (1974) 73-93. 17 Mayer, D. J., Price, D. D. and Rafii, A., Antagonismof acupuncture analgesia in man bythe narcotic antagonist naloxone, Brain Research, 121 (1977) 368-372.
39 18 Mayer, D. J., Wolfle, T. L., Akil, H., Carder, B. and Liebeskind, J. C., Analgesia from electrical stimulation in the brain stem of the rat, Science, 174 (1971) 1351-1354. 19 Paintal, A. S., Functional analysis of group III afferent fibers of mammalian muscles, J. PhysioL (Lond.), 152 (1960) 250-270. 20 Pomeranz, B., Cheng, R. and Law, P., Acupuncture reduces electrophysiological and behavioral responses to noxious stimuli: pituitary is implicated, Exp. NeuroL, 54 (1977) 172-178. 21 Pomeranz, B. and Chiu, D., Naloxone blockade of acupuncture analgesia: endorphin implicated, Life Sci., 19 (1976) 1757-1762. 22 Rossier, J., Vargo, T. M., Minick, S., Ling, N., Bloom, F. E. and Guillemin, R., Regional dissociation of fl-endorphin and enkephalin contents in rat brain and pituitary, Proe. nat. Aead. ScL (Wash.), 74 (1977) 5162-5165.
33
T H E E F F E C T OF H Y P O P H Y S E C T O M Y ON A C U P U N C T U R E A N A L G E S I A IN T H E MOUSE
TSU-CHING FU, STEPHEN P. HALENDA and WILLIAM L. DEWEY Department of Pharmacology, Medical College of Virginia, Richmond, Virginia23298 (U.S.A.)
(Accepted May 29th, 1980) Key words: acupuncture - - electroacupuncture - - analgesia - - naloxone - - hypophysectomy
writhing test
SUMMARY Acupuncture analgesia was quantitated in the phenylquinone induced writhing test in mice. Both manual acupuncture and electroacupuncture significantly reduced the number of writhes, i.e. 47 ~ and 51 ~ reduction respectively. Naloxone (2 mg/kg) pretreatment abolished this antinocicpetive effect suggesting that an endogenous opiate-like substance was involved. Hypophysectomy did not alter the electroacupuncture induced inhibition of writhing. These results confirm previous reports that acupuncture causes the release of an endogenous substance(s) with opioid activity, but disagree with previous reports in that our data show that the hypophysis is not involved in the release of this endogenous opiate or in any other mechanism of acupuncture analgesia in the mouse. INTRODUCTION Evidence has accumulated that acupuncture increases experimental pain threshold in humansZ, 17 and in animals2O, 21. Although acupuncture has been studied extensively in recent years its mechanism is still not understood. It takes 15-30 min to reach the peak effect of acupuncture and it dissipates slowly after the end of the stimulation. The duration of analgesia also outlasts the duration of brain area (periaqueductal and periventricular regions) electrical stimulation induced antinociception 12,16,1s. Similarly, both acupuncture and brain area electrical stimulation induced antinociception are reversed by the narcotic antagonist naloxone 1,17. All of these characteristics suggest that a neurohumoral system is involved in the mechanism o f acupuncture antinociception. Recently, we have presented evidence that an endogenous substance was being released by morphine 1°. Convincing evidence exists to
34 indicate that this neurohumoral factor is of central origin and not released at a peripheral site 7. Recent reports suggest that the hypophysis might be the origin of this neurohumoral factor s. The isolation of endorphins from the hypophysis makes this hypothesis quite feasible. The purpose of the present investigation was to determine if the hypophysis is essential for acupuncture. We also demonstrated the use of the writhing test as a model for studying acupuncture in the mouse. MATERIALS AND METHODS A total of 225 male albino mice (24-42 g) were used in these experiments. All animals were unanesthetized and except for controls each mouse was placed in a restrainer (No. 1123 CIO, by Arthur H. Thomas). The rear bottom ends of the restrainers on either side were cut open (20 mm long, 8 mm wide) allowing the two hindlegs to be extended and were immobilized with rubber bands (foot cuffed) for later application of acupuncture needles. The rear end of the restrainer was closed with a tight-fit plate in which a 10 mm diameter half circle was cut. The tail of the mouse was extended through this hole. The other parts of the body including the two forelimbs were free to move in the restrainer. The mice in the restrained group were maintained in the restrainer an equal amount of time as those used in the acupuncture experiments.
Stimulation Acupuncture points for mice were determined by transposing anatomically from the Chinese traditional human acupuncture charts. Only one point, Tsu-San-Li (S-36), located in the lateral upper tibia, was used. This point has been widely used in a number of other animal species and is easily accessible. We used the same point on the leg of each mouse and did not test the effect of stimulation at other sites on the leg or the rest of the body. Since positive results were obtained at this site we did not attempt to define other sites useful for acupuncture analgesia or determine that this is the only site in the leg that would produce these effects. For manual acupuncture, the needle (32-gauge, 1/2 in. handle 1/2 in. long by Trueline Instruments) was carefully inserted into Tsu-San-Li point and manually rotated for 1 min, every 6 rain for the next 30 min. The frequency of the rotation of the needle was 120-130/min. In electro-acupuncture an interdermal needle (32-gauge, 1/4 in. long by Trueline Instruments) soldered to a flexible electrical wire, was inserted into the left hindleg at the Tsu-San-Li point. The second identical electrode, as a positive pole, was inserted into the same leg approximately 5 mm distal to the first electrode. Current was applied from a Grass $88 stimulator through a stimulus isolation unit (SIU5) and constant current until (CCUI), using square wave, 1 msec duration, frequency/2 sec, and current intensity = 6 × T, (T = threshold for causing minimum muscle contractions), it was 1.2-1.5 mA. The total stimulation time was 30 min. In some experiments naloxone (2 mg/kg) was given subcutaneously 10-15 min before acupuncture stimulation.
35
Hypophysectomy The operations were performed under pentobarbital anesthesia and were carried out 1-2 weeks before the acupuncture and writhing tests. The pituitary gland was exposed from the ventral surface by means of a hand drill which was made from stainless steel tubing 2 mm in diameter. A small hole was drilled on the basisphenoid bone in the midline just rostral to the occipito-basisphenoid suture line. The dura mater covering the hypophysis was torn away with a 22-gauge 1.5in. long hypodermic needle (the sharp point of the needle was bent and converted into a fine hook) then a dropper of proper size accomodated by the drilled hole was placed against the hypophysis and the gland was removed by negative pressure. The pituitary gland was carefully examined after removal. Only those mice with complete removal of the hypophysis were used in this study. This observational criteria was confirmed by the determination of serum corticosterone levels. These levels were quantitated by the protein binding assay using human corticosterone binding globulin 14. The average corticosterone level in hypophysectomized mice was 21.9 ng/ml serum (with a range of 10.3-34.7 ng/ml), compared to an average of 240.6 ng/ml (with a range of 107-500 ng/ml) in control or sham-operated mice. Wrffhing test Immediately following the release from the mouse restrainer, each test mouse received phenylquinone (3 mg/kg) i.p. as previously described 9. The number of writhes during the 10th and 15th minute after injection were counted and compared to those observed in non-restrained control mice given phenylquinone. Statistical differences between groups were determined using student's t-test. RESULTS The data presented in Table I show the mean and S. E. of the number of writhes after each treatment and the percentage inhibition for each treated group versus control.
Control series The first 3 treatments did not receive acupuncture stimulation. The mice in treatment 1 were 'non-restrained' controls consisting of 58 mice. They received phenylquinone only, and had a mean of 6.6 writhes per mouse. Treatments 2 and 3 were 'placebo' controls of 2 and 3 groups of 6 mice respectively. The mice in groups 2 and 3 were placed in the mouse restrainers just as the acupuncture mice but acupuncture was not performed. The mice in treatment 2 had an average of 6.17 writhes per mouse. Each mouse in treatment 3 received a random needle insertion in the left hindleg but neither electric current nor needle movement was conducted. The average number of writhes per mouse was 5.27. Acupuncture series The data presented in Table I show that both manual acupuncture and
36 TABLE I Effect of acupuncture on writhing test in mice Treatment
No. of mice (n)
Number of writhes (~ ± S.E.M.)
Percent inhibition
1. 2. 3. 4. 5. 6. 7. 8. 9.
58 12 18 24 24 18 24 24 17
6.60 ± 0.27 6.17 :k 0.61 5.27 i 0.41" 3.50 d- 0.33**,*** 3.25 ± 0.31"*,*** 6.05 4- 0.41 5.67 i 0.38§ 5.25 ± 0.36"*,§§ 3.82 -4- 0.38**
0 6.5 20.1 46.9 50.7 8.3 14.1 20.4 42.1
Control (unrestrained) Restrained Restrained and random needle insertion Acupuncture Electroacupuncture Naloxone and restrained Naloxone and acupuncture Naloxone and electroacupuncture Hypophysectomy and electroacupuncture
* Significantly different from control (P < 0.01) but not significantly different from restrained mice without needle insertion (group 2). ** Significantly different from control at P < 0.001. *** Significantly different from group 3 mice at P < 0.01. § Significantly different from acupuncture (group 4) at P < 0.001. §~ Significantly different from electroacupuncture (group 5) at P < 0.001.
electroacupuncture at the Tsu-San-Li Point significantly decreased the number o f writhes by 47 to 51 ~ respectively. The mean n u m b e r o f writhes for the 24 mice given m a n u a l acupuncture was 3.5. The mice given electroacupuncture showed similar results: the mean n u m b e r o f writhes for these mice was 3.25. Naloxone series
Naloxone alone, as previously reported had no effect on phenylquinone induced writhing. N a l o x o n e given 10-15 min prior to either m a n u a l or electro acupuncture stimulation effectively abolished the effect o f acupuncture on phenylquinone induced writhing. Hypophysectomized mice
The lack o f effects o f h y p o p h y s e c t o m y on electroacupuncutre can be seen by comparing the n u m b e r o f writhes in treatment 9 to those o f treatment 5, in Table I. It is clear f r o m these data that h y p o p h y s e c t o m y did n o t reverse the effects o f electroacupuncture and it is shown that the hypophysis is not critical for this m e t h o d o f increasing pain threshold. DISCUSSION Our data clearly show that both acupuncture and electroacupuncture significantly induce analgesia in mice and this effect can be blocked by naloxone. These results confirm those o f others in suggesting the possibility o f the release o f an endogenous substance(s) which plays a role in acupuncture 17,21. The use o f proper stimulation parameters, especially stimulation intensity and
37 frequency, is essential for obtaining reproducible electroacupuncmre analgesia. Some workers 11 have failed to obtain acupuncture analgesia. We believe one of the main reasons was that they did not choose appropriate stimulation intensity, or frequency, or both. Anderson et al. 3,4 measured the effects of acupuncture on pain threshold in teeth and showed no change at low intensity stimulations. Rather strong stimulations which caused powerful muscle contractions were needed to produce a significant increase in pain threshold. These findings suggest that: (1) activation of the large group I muscle afferents alone is not sufficient to increase pain threshold and; (2) activation of group II or even smaller muscle afferents is necessary to induce acupuncture analgesia. Chiang et al. 7 reported that acupuncture analgesia could still be induced after selective blockade of the transmission of the superficial cutaneous nerves but not following the blockade of muscle nerves. With all these findings in mind, therefore, we used a stimulation intensity of 6 × T, i.e., stimulus intensity at 6 times threshold for causing minimum muscle contraction of the hindlimb. This intensity is believed to be sufficient to activate most group II afferents but only excite a minimum, if any, of the pain fibers (group III) 19. Andersson et al.3,4 showed that stimulation with a low frequency of 2/sec produced a gradual increase in pain threshold which remained at a high level for a long period. However, a frequency of 10/see, or 100/sec, produced an abrupt increase and a rapid decrease of pain threshold. In other words, stimulation with a low frequency (less than 10/sec) is a necessity to induce acupuncture analgesia. A frequency of 2-3/sec is about the upper limit that can be carried out manually. Many workers tend to use higher frequency (more than 10/see) in electroacupuncture. This may be the reason why failure to obtain an analgesic effect has happened more often in electroacupuncture than in acupuncture studies. Possibly the most important finding of this study was that electroacupuncture analgesia was not affected by hypophysectomy in mice. These results suggest that the hypophysis does not play an important role in acupuncture analgesia and clearly differ from those of Pomeranz et al. 2°,21 who showed that the pituitary was necessary for acupuncture analgesia. Goldstein et al. a originally detected the presence of opiatelike peptides in the pituitary gland. However, the role of pituitary fl-endorphin is not clear. It is not thought to be the source of brain opiate peptides since the peptide levels in the brain are not affected by hypophysectomy6,15. Although Met-enkephalin has a wide distribution throughout many brain areas, its rapid inactivation in brain makes it unlikely to play an essential role in such a long-lasting process as acupuncture analgesia, fl-endorphin itself is a potent analgesic and is found in high concentrations in hypothalamusS,2~. Furthermore, Hosobuchi et al. la have reported that electrical stimulation of human periaqueductal gray caused an increase in immuno-reactive flendorphin in CSF. From all of these findings, it is not unreasonable to postulate that acupuncture or electroacupuncture activates group II and possibly some group III afferents which send impulses into the CNS. Two possible mechanisms may be involved. First, these impulses induce short-lasting pre- and post-synaptic inhibitions of pain afferents at the segmental levels. Our previous work 10 which demonstrated the release of endogenous
38 opiates in the CSF suggests that a more probable hypothesis is that some of these impulses are transmitted to the hypothalamus and periaqueductal gray to cause the release of fl-endorphin or some other endogenous opiate into the CSF. This material transported in the CSF to various parts of the brain stem inhibits pain-perceptive neurons and/or the transmission of noxious impulses to the higher centers. ACKNOWLEDGEMENTS
We thank Dr. Suzanne Laychock for the determination of the corticosterone levels. This research was supported in part by USPHS Grant DA 01647, DA 07027 and DA 00490.
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