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Life Sciences Vol. 19, pp . 1757-1762, 1976 .

Pergamon Press
Printed in the U.S .A .

NALORONE BLOCKADE OF

ACUPUNCTURE ANALGESIA :

ENDORPHIN IMPLICATED

Bruce Pomeranz and Daryl Chiu

Department of Zoology

University of Toronto

Toronto, Ontario, Canada, M5S lAl

(Received in final form October 25, 1976)

Summary

Electroacupuncture in awake mice produced analgesia to noxious heat


stimuli causing a 54X increase in latency to squeak . Subcutaneous
naloxone completely abolished this acupuncture analgesia implica-
ting endorphin . Naloxone injections in control mice caused a 17X
hyperalgeaia suggesting that "normal" mice also release endorphin .
These results imply that endorphin is released at a low basal rate
in "normal" mice, and at a much higher rate during acupuncture .

The mechanism of acupuncture analgesia is poorly understood . There are


three favoured hypotheses . First, that acupuncture causes a hypnotic, auto-
suggeative or distracting effect (1,2) . This is supported by the observations
that patients in China are carefully selected for acupuncture anesthesia, and
then are psychologically prepared for one week prior to the surgery . Secondly,
some authors suggest that acupuncture causes preaynaptic inhibition of sensory
fibres by activating large diameter afferents in a manner similar to electro-
analgeaia (3) . It is consistent with this second hypothesis that many acu-
puncture points have a segmental dermatomal relationship to the site of pain
being treated. A third hypothesis proposes that acupuncture activates a
central biasing mechanism in the brainstem which then sends out inhibitory
influences to block synaptic transmission in pain pathways (4) . This pro-
posal is supported by the finding that electrical stimulation of the brainstem
in awake animals produces profound analgesia (5) .

In this paper we support a fourth hypothesis ; that acupuncture releases


endorphin which causes analgesia (6) . In another paper (7) we showed that the
pituitary may release this endorphin to produce acupuncture analgesia .

Methods

Latency to squeak procedure: In order to obtain a good animal model for


acupuncture analgesia we screened numerous strains of mice for the presence of
a reproducible squeak response to noxious heat stimuli . We selected the females
of an F1 cross between C57BL/6J and A/J obtained from Jackson Laboratories, aged
7 to 8 weeks . Each mouse was placed in a soft cardboard restrainer in the shape
of a funnel . The nose protruded from the narrow end of the funnel ; the wide
end was compressed and stapled shut . The forelegs were pulled out with forceps

1757
1758 Naloxone Blocks Acupuncture Analgesia Vol . 19, No . 11

through holes in the aide of the funnel and were immobilized with masking tape
for later application of acupuncture needles (Control mice not receiving acu-
puncture were immobilized in the same manner) . Squeaks were elicited using
noxious heat from a elide projector lamp (GE-ELH) pointed at the protruding
nose at a distance of 10 cm . The restrained mouse and lamp were in a sound
proof chamber as ambient noises disturbed the animal . The squeak was monitored
through a microphone, headphones, and a storage oscilloscope (Tektronix 5103N)
externally triggered by the lamp switch . The latency of the squeak was measur
ed accurately on the oscilloscope and averaged 3 .0 seconds (S .E . _+ 0 .05) . The
oscilloscope traces were used to exclude small amplitude squeaks which occas-
ionally occurred within 2 seconds of turning on the lamp . These were discarded
from the data as they resembled startle reaponaea with small intensities quite
different from the stressful sounds occurring at 3 .0 seconds . The criteria for
rejecting small early squeaks were any sounds with amplitudes less than one
half that of the true stressful response and occurring within 2 seconds of the
onset of the heat stimulus . Before starting the various treatments, each
mouse was given four control tests, two minutes apart, and the mean latency was
used for zero time control values . We selected those mice which gave 4 reprod-
ucible control reaponaea with latencies between 2 to 4 seconds ; we rejected 5
out of 75 mice . Next, we tested each mouse 6 times over 140 mina .

Treatments : There were 7 matched groups of mice with 10 mice in each


group : I acupuncture, II sham acupuncture, III acupuncture plus naloxone, IV
acupuncture plus saline, V naloxone, VI saline, VII no treatment . For groups
I, II and IV, electroacupuncture was applied just after the control readings
at zero time . Needles were inserted to as acupuncture point called Large
Intestine 4 or Hoku situated between the first and second metacarpal bones of
the foreleg in a position proportional to the human charts . This point, one
of the most successful for acupuncture analgesia, is used extensively in China
for face surgery on humans (8) . One needle, 1 cm long by 0 .2 mm diameter,
soldered to flexible electrical wire, was inserted into each foreleg at the
Hoku points, at zero time, and was removed at 20 minutes . Current was applied
from a Grass SD9 stimulator using 4 volts, 6Hz, 0 .1 maec duration, 1 mamp,
biphasic square pulses throughout the 20 minute treatment . The voltage was
adjusted around the above values sufficient to produce small vibrations in the
forepaw . The electroacupuncture was not painful or stressful as judged by the
lack of withdrawal, struggling or squeaking (also voltages were below usual A
delta fiber thresholds) . The vibrations were important indications that A
alpha fiber thresholds for muscle nerves were exceeded . Group II, received
sham electroacupuncture in the deltoid region of the foreleg, subcutaneously at
non-acupuncture sites . At the above voltage, sham acupuncture did not produce
any muscle vibrations ae the needles were in a subcutaneous location . Groups
III and V received injections of naloxone, 0 .9 mg/kg in one half ml saline (9),
aubcutaneously just after the zero time control readings and again at 20 min-
utes . Groups IV and VI were given isotonic saline as control for naloxone .
These injections were given in a blind protocol, so that the experimenter was
not aware of which animals received naloxone and which received saline .

Reeulta

Figures 1 and 2 and table 1 summarize the averaged data for each group,
giving percentage change in latency to squeak . Figure 1 shows the values
obtained from groups I to IV and figure 2 the data from groups V to VII . The P
values indicated by letters were obtained by comparing the means at a given
time with zero time control values . (All P values in this paper were calcula-
ted from two-tailed student t tests .)

The first result to observe ie the time course of acupuncture analgesia in


groups I and IV, which occurs at 40 minutes, with a 54x increase in latency
Vol . 19, No . 11 Naloxone Blocks Acupuncture Analgesia 1759

-20

-ao ~+ ~ ±
O 6 20 "0 BO BO 100 120

TIME (MIN .1

Effect öf aaloxone on acupuncture analgesia. Percentage change in


latency to squeak as compared to zero time pretreatment control
values . Closed circle solid line for group I - acupuncture ; open
circle dashed line for group II - sham acupuncture ; closed triangles
dashed line for group III - acupuncture plus naloxone ; open triangles
dotted line for group IV - acupuncture plan saline . Each point is
average for 10 mice . Barn show S .E . Arrows indicate treatments : for
groups I, III and IV acupuncture started after zero time and stopped
at 20 minutes; group II received sham needling ; groups III and IV
received injections at the arrows for zero time and 20 minute boosters .

os 20 ~o eo so lo0 120 lno

TIME (MIN .)

Same se figure 1 for additional control groups . Open circles


dashed line for group V - naloxone ; closed triangles dashed line
for group VI - saline ; closed circles solid line for group VII -
ao treatment . At the arrows groups V and VI received injections .
1760 Naloxone Blocks Acupuncture Analgesia Vol . 19, No . 11

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Vol . 19, No . 11 Naloxone Blocks Acupuncture Analgesia 1761

for acupunctured mice (P« ,001) and a 44% increase for acupuncture plus saline
(P« ,001) . In addition to these within group statistics, we also did t tests
to compare the percentage changes between pairs of groups at 40 minutes. These
results show the following : First, that sham acupuncture in group II produced
no analgesia, showing that group I had a true acupuncture effect (group I ve
group II P« .001) . Next, the results show that in group III naloxone blocks
acupuncture analgesia (group I vs group III P « ,001), while the saline control
group IV has no such effect (group I vs group IV P = 0 .25) . Consequently group
IV showed far more analgesia than group III (group III versus group IV P «,001) .

All the three control experiments in figure 2 showed no analgesia (no in-
creased latency) . The small decrease of latency in group VII at 60 minutes
(P<0 .05) may reflect sensitization of the nose with repeated noxious stimuli.
The larger decrease in latency observed at 40 minutes with âaloxone group V
was significantly greater than saline group VI (group V va group VI P«,001) or
no treatment group VII (group V va group VII P<,005) . This will be discussed
below in relation to hyperalgesia . Finally it should be noted that naloxone
plus acupuncture was not different to the plain naloxone treatment (group III
versus group V P>0 .7) .

DISCUSSION

These experiments clearly demonstrate that naloxone blocks acupuncture


analgesia . These data are similar to preliminary results obtained with acupun-
cture analgesia in human subjects (6) .

Our results suggest two main conclusions . First, acupuncture may have
released endorphin which was blocked by naloxone (group III) . We are now test-
ing this conclusion by extracting endorphin during acupuncture and assaying for
endorphin on guinea pig ilium (10) . Secondly, we may conclude that the brain
releases small amounts of endorphin even without acupuncture since naloxone in-
creased pain sensitivity in groups III and V; this is consistent with findings
of endorphin in brains of normal animals (10, 11) . Naloxone hyperalgesia in
mice was previously reported (9) and dose response curves reached plateau at
about 0 .9 mg/kg .

Further support for the endorphin hypothesis of acupuncture analgesia


comes from the time course of the acupuncture effect in groups I and IV . The
peak effect took 40 minutes and recovery occurred after 2 hours . These delays
indicate that a humoral factor might be involved such as endorphin . A recent
Chinese paper reported cerebrospinal fluid transfer studies in mice where the
recipients experienced analgesia from acupuncture of donor animals (12) .
Finally, clinical reports usually find prolonged time courses consistent with
the endorphin hypothesis (8) .

The site of release of this endorphin during acupuncture is unclear . One


paper reported the hormone to be concentrated in the pituitary (13) . In another
paper from our laboratory (7) we showed that hypophysectomy abolished acupunc
ture analgesia in mice ; we also reported that decerebration or spinalization in
cats abolished acupuncture blockade of spinal cord single neuron responses to
noxious stimuli . The simplest explanation for all these results would be that
acupuncture releases endorphin from the pituitary to produce analgesia . This
mechanism is consistent with the generalized analgesia reported clinically (g) .
However it does not explain the specific localized effects so often seen with
acupuncture analgesia (8) . For this we can postulate that the central biasing
mechanism in the brain stem sends axons with endorphin to specific spinal cord
segments . Several experiments support this hypothesis . First, morphine ion-
tophoresis blocks transmission in spinal cord pain pathways (14) . Secondly,
brainstem stimulation produces analgesia in specific regions (15), and blocks
1762 Naloaone Blocks Acupuncture Analgesia Vol . 19, No . 11

transmission in spinal cord pain pathways (16) . Third, this brainstem analge-
sic effect is abolished by naloxone (5) .

In conclusion, therefore, we propose the following hypothesis for acupun-


cture analgesia: needling in appropriate points stimulates sensory nerves
which activate the pituitary or brainstem (central biasing mechanism) to rele
ase endorphin which reduces transmission in pain pathways .

Acknowledgements

The authors are grateful to Mary Adams, Richard Cheng and Elliot Goldner
for their assistance . This research was supported by a grant from Ontario
Ministry of Health ACB.

References

1. P.D . Wall, New Scientist 3, 31-34 (1974) .


2. W .S . Kroger, Amer . J . Psych . _130, 855-860 (1973) .
3. C.L . Li, Amer . J . Chin Med. _1, 61-74 (1973) .
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1973, p . 89 .
5. H . Akil, D.J . Mayer, J .C . Liebeskind, Science _191, 961-962 (1976) .
6. D.J . Mayer, Neurosciences Res . ProR . _13, 98 (1975) .
7. B . Pomeranz, R . Cheng, and P . Law, Exp . Neurol . (in press) .
8. Acupuncture Anesthesia , Shanghai Institute, Shan-Wo, Hong Kong 1973 .
9. J.J . Jacob, E .C . Tremblay, and M.C . Colombel, Psychopharmacologia _37,
217-223 (1974) .
10 . J . Hughes, T . Smith, H.W . Kosterlitz, L.A . Fothergill, B. Morgen and H.R .
Morris Nature 258, 577-579 (1975) .
11 . R. Simaatov, and S .H . Snyder, Life Sciences _18, 781-788 (1976) .
12 . Peking Research Group of Acupuncture Anesthesia, Scieniia Sinica _17,
112-130 (1974) .
13 . B .M . Cox, K.E . Ophiem, H. Techemacher, and A . Goldstein, Life Sciences _16
1777-1785 (1975) .
14 . J . Dostrovsky and B . Pomeranz, Nature (New Biology) _246, 222-224 (1973) .
15 . D.J . Mayer and J .C . Liebeskind, Brain Res . _68 73-93 (1974) .
16 . J.L . Obverse, J .M . Besson, G. Guilbaud and J .C . Liebeskind, Eap . Brain
Rea . 20, 32-44 (1974) .

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