A METhOD FOR DETERMINING LOSS OF PAIN SENSATION

FRED E. D’AMOUR AND DONN L. SMITH

From The Biologic Research Laboratory, University of Denver

Received for publication January 27, 1941

In a recent study by Schumacher, Hardy and Wolff (1) the varia-

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Goodell,
tion of the pain threshold in humans was determined under a variety of
conditions. This paper aroused our curiosity as whether the great uniformity
observed in humans also held for animals and also suggested the possibility
of adapting the method to the assay of analgesic drugs. The method used
by the above workers was a simple one, namely the focusing of a beam of
light upon a blackened spot on the forehead for exactly three seconds, with
the intensity controlled by a rheostat; determinations were repeated every
30 seconds until an intensity was reached at which the subject just perceived
pain. An extremely great stability in the pain threshold was found to exist
under a variety of conditions.
In adapting the method to rats the rays from a Mazda 1184, 6 to 8 volt
bulb, with reflector, were focused on the tip of the rat’s tail, which was placed
in a grooved board some six inches below. The set-up includes a voltage
regulator, transformer and rheostat; a stop-watch is operated by the same
switch which makes and breaks the current. In using this device the operator
places the rat’s tail in the groove, switches on the light and stop-watch and
waits for the response, which is a sudden, typical twitch of the tail, when the
animal feels the pain. The switch is then reversed and the time noted.
After a few trials we found that a light intensity which produced a reaction
in about 5 seconds was most convenient.

RESULTS

Normal variation. We were greatly to find

surprised
at last a biologic
reaction subject apparently to very little
individual variation. Up to the
present some 10,000 individual tests have been made on several hundred
rats. It happened that rats were available from another study in which
many states of endocrine dysfunction had been produced. The following is a
list of conditions under which tests were made: Adrenalectomized versus
hyper-cortin, thyroidectomized versus hyper-thyroid, castrate (male and
female) versus hyper-estrin, hyper-progestin, hyper-testosterone, day versus
night, cold room versus hot room, and starvation. Tests were also run twice
per week for two months on the same rats and no conditioning effect was
74
 LOSS OF PAIN SENSATION 75

TABLE 1

REACTION TIME PER CENT RESPONDING

ecn

3-4 0.1
4-S 82.0
5-6 17.6
6-6.5 0.3

TABLE 2
A88ay of cobra venom

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AVERAGE REACTION TIME
NUMBER OP RATS DOSE
After 30 mm. After 2 hours

m.u. per kgm. sec. sec.

12 0 4.8 4.6
12 4 4.7 4.5
12 8 4.8 4.9
12 16 5.0 4.9

TABLE 3
Assay of opi ales

PER CENT COMPLETELY

MATERIAL DOSS
ANALGESIC

mgm. per kgm.

Dilaudid 1 8
Dilaudid 2 75
Dilaudid 4 100
Heroin 1 0
Heroin 2 33
Heroin 4 83
Morphine sulfate 6 0
Morphine sulfate 8 50
Morphine sulfate 10 66
Morphine sulfate 12 92
Codeine sulfate 12 0
Codeine sulfate 18 17
Codeine sulfate 24 75
Codeine sulfate 30 100
Pantopon 12 8
Pantopon 18 66
Pantopon 24 92
All injections were made intraperitoneally, the material being dissolved in approxi-
mately 0.5 cc. of saline.

noted. None of the conditions mentioned affected the reaction time sig-
nificantly, as is shown in table 1, which includes all conditions.
76 FRED E. D’AMOUR AND DONN L. SMITH

TABLE 4
Details of morphine assay

REACTION TIME:
RAT NUMBER WRIGHT ________________________________________________________
Control After 30 minutes After 1 hour After 2.5 hours

Dose: 6 mgm. per kgm. intraperitoneally

gm.

1 240 5.0 5.0 4.9 5.0

2 220 4.5 4.8 4.3 4.8
3 240 4.0 9.0 7.5 4.5

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4 230 4.4 4.7 4.3 4.2
5 270 4.1 4.2 4.2 4.7
6 240 5.0 5.0 5.3 4.6
7 235 4.6 4.2 4.7 4.2
8 265 4.7 5.0 5.5 5.0
9 250 5.0 4.2 4.9 4.8
10 225 4.4 5.8 4.7 4.7
11 240 4.2 4.6 4.6 4.6
12 220 4.7 4.9 4.2 4.5

Dose: 8 mgm. per kgm.

13 240 5.5 6.0 6.0 4.0
14 220 4.5 7.0 7.0 4.5
15 250 4.0 Comp. 9.0 5.5
16 260 5.0 7.0 Comp. 4.0
17 180 4.5 8.0 8.0 5.0
18 200 4.0 Comp. 8.0 5.0
19 200 6.0 Comp. 6.0 4.0
20 230 5.0 7.0 6.0 4.5
21 190 4.5 9.0 8.0 4.0
22 200 4.5 8.0 6.0 5.0
23 230 4.5 Comp. 8.0 5.0
24 250 5.5 Comp. Comp. 5.5

Dose: 10 mgm. per kgm.

25 250 6.0 Comp. 8.0 4.5
26 240 4.0 8.0 8.0 4.0
27 230 5.0 Comp. Comp. 5.5
28 200 5.0 8.0 7.0 4.0
29 180 5.0 7.0 Comp. 4.0
30 200 4.0 Comp. Comp. 6.0
31 220 5.5 8.0 Comp. 4.0
32 250 4.5 Comp. 7.0 7.0
33 200 4.0 Comp. Comp. 5.0
34 240 4.0 7.0 7.0 4.5
35 230 5.0 7.0 4.5 5.0
36 210 4.0 Comp. 7.0 5.0

“Comp.” means complete analgesia.

 LOSS OF PAIN SENSATION 77

TABLE 4-Concluded

REACTION TIME:
EAT NUMBER WEIGHT
Control After 30 minutes After 1 hour After 2.5 hours

Dose: 12 mgm. per kgm.

gm.

37 220 5.0 Comp. Comp. 5.0

38 210 4.5 Comp. Comp. 4.0
39 220 4.0 Comp. Comp. 4.0
40 250 5.0 Comp. Comp. 4.5
41 190 4.5 Comp. Comp. 4.5

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42 210 4.5 Comp. Comp. 5.0
43 210 5.0 Comp. Comp. 4.5
44 260 6.0 Comp. Comp. 4.0
45 250 4.0 Comp. Comp. 6.0
46 200 5.0 8.0 8.0 6.0
47 240 4.0 Comp. Comp. 5.5
48 200 5.0 Comp. Comp. 7.0

The assay of drugs. The method was next applied to the assay of the “anal-
gesic” properties of several drugs. (It is of course realized that the subject
of analgesia is a complex one and we are here using the term analgesia as
synonomous with loss to pain
of reaction merely for convenience sake.) The
following materials showed no analgesic properties: Cobra venom,’ in doses
of 4,8 and 16 m.u. per kilogram; Sodium amytal, in doses of 25 and 50 mgm.
per kilogram; Tarantula venom, in doses of 10 .milkings per rat; Black widow
spider venom, in doses of 4, and 4 lethal doses. Results of the tests on
cobra venom are given in table 2.
Five of the opiates, dilaudid, morphine sulfate, codeine sulfate, heroin and
pantopon, were assayed at either 3 or 4 dosage levels, using 12 animals per
dose. The results are given in table 3, the assay of morphine being repro-
duced in table 4 as an example. By complete analgesia is meant the complete
loss of reaction to pain: the animal makes no movement of the tail whatever
even though it is being burned to a crisp. It is, of course, not necessary to
burn it to that extent, a white blistering appearance being sufficient. In-
cidentally this does the animal no permanent harm, for if the tail tip is badly
burned it merely sloughs off.

DISCUSSION

Since the function of analgesic drugs is to alleviate human pain the human
is the best subject for their study. But when the study concerns new and
untried drugs or an extensive assay of old ones the human subject is obviously
unavailable. As far as animal methods are concerned, the literature is by
1 The cobra venom used in this study was supplied through the kind cooperation of
Dr. Macht, of Hynson, Westcott and Dunning, Inc.
78 FRED E. D’AMOUR AND DONN L. SMITH

no means replete with descriptions of means for measuring analgesia experi-

mentally, and no method seems to have gained widespread acceptance.
Such methods as the piling of weights on a cat’s tail, or heating the skin by
means of hot water passing through tubes, have been described, but these
suffer from two main defects: doubt exists as to the stimulus, i.e., whether
it is pain or merely touch, and there are great individual variations in test
animals. The method most recently reported is that of Macht (2) who used
an electric shock applied to the scrotum. Here again the effective stimulus
in control animals varied from 100 to 800 volts.
of greatest value, of course, would be the development of methods for

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measuring true analgesia, i.e., the loss of conscious sensation of deep seated,
continuous pain, such as that occurring in carcinoma. This is a much more
difficult problem and the method described in this paper makes no pretense of
measuring analgesia of this sort. It is, however, interesting to note that our
rather thorough assay of the opiates gave results in good agreement with
their accepted clinical value. A survey of doses recommended in a number
of treatises on pharmacology indicates that dilaudid and heroin are considered
to be approximately four times as active as morphine, and codein and pan-
topon about half as active as morphine. This is approximately the ratio
of effectiveness found in this study.
We are unable to explain the discrepancy between our findings and those of
Macht (2) concerning cobra venom. On a dosage of 0.5 m.u. (using 7 rats)
he found an increase in the pain threshold, but in no case did the increase
exceed the maximum stimulus (809 volts) found necessary at times in control
rats. Only two rats were used on each of the higher doses and the two on 2.0
m gave inconsistent results in that the pain threshold in one was increased
significantly (from 185 to 1080 volts) but in the other only from 185 to 445
volts. The weight of the rats was not mentioned. In our study, using 12
rats per dose and dosages of 4.0, 8.0 and 16.0 m.u. per kilogram (the rats
weighed approximately 250 grams, therefore the dose was about 1.0, 2.0 and
4.0 m.u. per rat) we found no effect whatever on the reaction time. A dose of
16.0 m.u. per kilogram is approximately one-half the lethal dose.
Although, as stated, no claim is made that this method is capable of de-
termining the analgesic power of drugs when deep-seated, continuous pain
is involved, nevertheless a method as simple and rapid as this should have
value in making it possible to submit to experimental verification many
statements found in books on pharmacology, the evidence for which rests
usually on uncontrolled clinical impressions. Such questions are: the influ-
ence of route of administration upon dosage and duration of analgesia, the
possession of analgesic properties by anti-pyretics, barbiturates and other
drugs, the potentiating effects of one drug upon another, and the testing for
analgesic properties of new drugs. We are at present engaged on certain of
these studies.
 LOSS OF PAIN SENSATION 79

SUMMARY

1. A simple, rapid method for determining the pain threshold in the rat
is described.
2. The individual variation, under a variety of conditions, was found to be
surprisingly small.
3. The method was applied to the determination of analgesic properties
of several substances, including cobra venom. No analgesic property in the
latter could be demonstrated.
4. A comparative assay of five opiates gave results in good agreement with
clinical experience.

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REFERENCES
(1) SCHUMACHER, GOODELL, HARDY AND WOLFF: Science, 92, 110, 1940.
(2) MACHP AND M.&cwr: J. Amer. Pharm’l Ass’n., 29, 193, 1940.