Pain, 32 (1988) 77-88 77

Elsevier

PAI 01152

Basic Section
A new and sensitive method for measuring thermal nociception
in cutaneous hyperalgesia

K. Hargreaves, R. Dubner, F. Brown, C. Flores and J. Joris 1

Neurobiology and Anesthesiology Branch, National Institute of Dental Research, National Institutes of Health,
Bethesda, MD 20892 (U.S.A.)

(Received 13 April 1987, revised received 13 August 1987, accepted 17 August 1987)

Summary A method to measure cutaneous hyperalgesia to thermal stimulation in unrestrained animals is described. The testing
paradigm uses an automated detection of the behavioral end-point; repeated testing does not contribute to the development of the
observed hyperalgesia. Carrageenan-induced inflammation resulted in significantly shorter paw withdrawal latencies as compared to
saline-treated paws and these latency changes corresponded to a decreased thermal nociceptive threshold. Both the thermal method
and the Randall-Selitto mechanical method detected dose-related hyperalgesia and its blockade by either morphine or indomethacin.
However, the thermal method showed greater bioassay sensitivity and allowed for the measurement of other behavioral parameters in
addition to the nociceptive threshold.

Key words: Cutaneous hyperalgesia; Thermal nociception

Introduction brous skin (tail flick), and lack of laterality which

removes the possibility of within-subject controls
Despite the advantages of using thermal stimu- (both methods). In addition, neither method has
lation [5], no behavioral method exists for quanti- been extended to investigating behavioral re-
fying thermal nociception in animal models of sponses to hyperalgesia.
hyperalgesia. Determination of acute nociceptive Quantitation of behavioral responses to cuta-
thermal thresholds in animals has primarily relied neous hyperalgesia has relied upon the Randall
upon the tail flick and hot plate methods [4,21]. and Selitto method which uses mechanical force as
Although both methods are used frequently in the nociceptive stimulus [12]. However, this
pharmacological studies, they are not without method also has several limitations. Since mecha-
limitations [3,14,19], including lack of an auto- nical stimuli activate both low and high threshold
mated detection of the end-point (hot plate), mechanoreceptors in cutaneous and non-cuta-
stimulation of a unique, scaly form of non-gla- neous tissue [5,9,16], the relative contribution of
each receptor to the behavioral response detected
by this test is unknown. The Randall and Selitto
J. Joris is a research assistant for the National Fund for method uses a non-automated detection of the
Scientific Research (Belgium).
behavioral end-point which requires substantial
Correspondence to: Dr. Kenneth Hargreaves, NAB, NIDR, observer interaction [6]. In addition, the method
NIH, Building 10, Room lA09, 9000 Rockville Pike, Bethesda, requires the animal to be restrained, providing a
MD 20892, U.S.A. stimulus which activates both the pituitary-adrenal
and sympatho-adrenomedullary axes [l&13]. Last- tion period, the radiant heat source was positioned
ly, it measures only the nociceptive threshold. under the glass floor directly beneath the hind
We describe a new and sensitive method to paw. A trial was commenced by a switch which
measure several behavioral responses to hyperal- activated the radiant heat source and started an
g&a. The method uses radiant heat as a thermal electronic timer. The radiant heat source consisted
stimulus applied to unrestrained animals with an of a high intensity projector lamp bulb (Osram
automated determination of the nociceptive 58-8007, 8 V, SO W) located 40 mm below the
threshold. The sensitivity of this method is com- glass floor and projecting through a 5 mm x 10
pared to the Randall and Selitto mechanical test mm aperture in the top of a movable case. A
for measuring hyperalgesia following administra- photoelectric cell aimed at the aperture detected
tion of carrageenan (CARRA) and for the detec- light reflected from the paw and turned off the
tion of morphine and indomethacin analgesia. lamp and the electronic clock when paw move-
ment interrupted the reflected light. At the same
time, a tone was emitted. The withdrawal latency
to the nearest 0.1 set was determined using the
Methods
electronic clock circuit and a ~crocomputer.
Three other response measures of behavioral
Male Sprague-Dawley rats (Charles River Inc., hyperalgesia to thermal stimuli were taken: (1) the
250-300 g) were maintained in an animal room velocity of the withdrawal reflex (given an ordinal
(lights on 06.00-18.00 h) with water and food ad score of 0 if withdrawal movement was completed
libitum for at least 1 week before testing. All within 1 set, or a score of 1 if the reflex persisted
studies employed randomized, complete block, beyond 1 set); (2) the presence or absence of
vehicle-controlled designs with observers blind as licking; and (3) the duration of the hind paw
to treatment allocation. Animals were tested once withdrawal from the floor. The duration of the
only at each time point. The IASP Ethical Guide- hind paw withdrawal was the time measured with
lines were adhered to in these studies. a stopwatch from the beginning of the withdrawal
Initial studies determined the cutaneous movement, signaled by the tone, to the moment
temperature of the hind paw in 8 rats held on a when the animal returned its paw to the glass
glass floor during exposure to radiant heat. A floor.
thermocouple was placed under the heel of the The time course of these response measure& to
hind paw. The plantar surface of one paw received thermal stimuli was determined in 16 rats who
a subcutaneous (s.c.) injection of 1.0 mg CARRA received an S.C. injection of 1.0 mg CARRA into
in 0.1 ml saline, and the other paw was injected the plantar surface of one hind paw with no
with the same volume of 0.9% saline (SAL). Both injection into the other paw. The behavioral pro-
paws were tested 2 h after injection. The averages files of both the CARRA and untreated, con-
of 3 trials were taken for each paw with 10 min tralateral hind paws were obtained by periodically
between successive tests. The withdrawal latency measuring paw withdrawal latency, paw with-
and the cutaneous temperature at every second drawal velocity, paw licking and the duration of
and at the time of withdrawal were recorded by a paw withdrawal during a 96 h period.
microprocessor. To examine the possible contribution of re-
To assess nociceptive responses to thermal peated thermal stimulation on the development of
stimuli, rats were placed in a clear plastic chamber CARRA-induced hyperalgesia, 5 different groups
(18 cm x 29 cm x 12.5 cm) with a glass floor and of rats (8/group) were tested only once at 1, 2.5.
allowed to acclimate to their environment for 5 4, 8 or 24 h after injection of 3 mg of CARRA.
min before testing. During this time, rats initially Another group of animals (n = 8) were tested at
demonstrated exploratory behavior but subse- baseline and at each of the selected time points.
quently stopped exploring and stood quietly with The 3 mg dose of CARRA was selected to provide
occasional bouts of grooming. After the acclima- a sustained period of hyperaigesia, during which 6
 79

testing sessions with the thermal device were con- TABLE I

ducted. DRUG SCHEDULE FOR THE INDOMETHACIN AND
The thermal device was compared to the MORPHINE STUDY
Randall and Selitto mechanical device for detect-
ing dose-related cutaneous hyperalgesia. Three Group Time zero 60 mill

different doses of CARRA, 0.5 mg, 1.0 mg or 2.0
mg, were used and compared to a SAL control
with 8 rats/group. The drugs were injected S.C.
into one hind paw with no injection into the other
paw. Edema, temperature and hyperalgesia to both
thermal and rn~ha~c~ stimuli were assessed at
baseline, and at 2.5, 4, 7 and 24 h after injection.
Dorsal-plantar paw thickness, measured to 0.1
mm with a vernier caliper, was used as an index of
edema. The temperature of the plantar surface of
the paws was measured by a contact thermocouple
placed under the heel. Paw edema and local hy-
perthermia were used to establish the time course
and magnitude of dose-related CARRA-induced
inflammation.
Nociceptive responses to mechanical stimuli
were determined by the method of Randall and
Selitto [12]. The apparatus employed a pneumatic
system connected to the graphite coated plunger
of a 5 ml glass syringe. The plunger of this syringe
transmitted the mechanical force by a lever to an
acrylic cone (36 slope, rounded tip with a 1.2
mm radius). These dimensions match a common
commercial version of the Randall and Selitto
device (Ugo Basille Analgesy-meter, D. Clark, pers.
commun., Coburn Inst., Lehigh Valley, PA). The
exerted force increased at a constant rate of 25.5
g/see. The nociceptive threshold, expressed in
grams, was that force applied to the dorsal surface
of the rat hind paw which caused each animal to
withdraw the paw. The cut-off was 300 g. The
sequence of testing animals on the thermal and
mechanical devices was counterbalanced such that
half of each group was tested first on the thermal
and second on the mechanical device; the order
was reversed for the remaining rats.
The ability of the thermal and mechanical
methods to detect the blockade of CARRA-in-
duced hyperalgesia by analgesic drugs was as-
sessed and compared in rats pretreated with either
morphine sulfate 3 mg/kg (MS) or indomethacin
2 mg/kg (INDO). Four groups of 10 rats were
used. The groups received 3 injections as seen in
Hind paw * i.p. i.p.
S.C.
1 SAL SAL SAL
2 CARRA SAL SAL
3 CARRA INDO SAL
4 CARRA SAL MS
* The contralateral hind paw was untreated.
Table I. These 4 groups will be referred to as
SAL/SAL, CARRA/SAL, CARRA/INDO and
CARRA/MS. The times of drug injection were
selected to permit concurrent development of peak
analgesic effects of MS and INDO. Behavioral
responses to thermal and mechanical stimuli were
measured for both the CARRA- and contralateral
untreated hind paws at baseline, and at 2 and 3.5
h after the first injection.
Statistical analysis was carried out using the
analysis of variance (ANOVA) for repeated mea-
sured for overall effects, with Duncans new multi-
ple range test for comparison between groups [17].
Students t test was also used when appropriate.
Dose-response relationships at the time of peak
effects were analyzed by linear regression with
ANOVA testing the zero slope hypothesis [17]. A
cl-&square test was used in analyzing the velocity
of withdrawal and paw lick data [17]. Statistical
significance was taken at P < 0.05. Values are
quoted in the text as means k S.E.M.
In all studies, except where noted, 1.0 mg of
lambda CARRA (Sigma Chemical Co., C-3889) in.
0.9% sterile saline was suspended by sonication
and injected S.C.into the plantar surface of a hind
paw with a 25-gauge needle. INDO (Merck, Sharp
and Dohme Co., West Point, PA) was dissolved in
0.1% (w/v) sodium carbonate. Mo~~ne sulfate
was dissolved in 0.9% saline. The S.C. hind paw
injection volume was 0.1 ml while the i.p. injection
volume was 0.1 ml/100 g body weight.
Results tween the CARRA group tested at all time points
and the separate CARRA groups tested at only
The cutaneous temperatures of both CARRA- one time point each.
and saline-treated paws increased during exposure The time course of several behavioral correlates
to radiant heat (Fig. 1). As compared to the of CARRA-induced hyperalgesia was next de-
saline-injected paws, the CARRA-injected paws termined. As seen in Fig. 2A, the paw withdrawal
had significantly greater initial paw temperatures latency decreased significantly (P < 0.01) by 1 h
(30.5 + 0.2C vs. 27.9 & 0.2C; P < 0.001). In ad- after CARRA injection, to reach its minimal value
dition, the CARRA-treated paws exhibited shorter at 2.5 h. At 4 h, the paw withdrawal latency
withdrawal latencies as compared to saline-treated started increasing, and hyperalgesia was not de-
paws (3.5 + 0.6 set vs. 10.9 &-0.6 set; P < 0.001). tected at 8 h. With the onset of hyperalgesia, the
The significantly shorter latencies of CARRA- withdrawal movement of the CARRA-treated paw
treated paws correspond to lower nociceptive became slow (Fig. 2B), the animal did not lick its
threshold temperatures (38.5 _t 0.7 C vs. 45.2 + paw (Fig. 2C) and the paw was held above the
0.2C; P < 0.01). glass floor for a longer period of time (Fig. 2D).
To determine that the time course of hyperalge- Interestingly, these behavioral correlates of hyper-
sia following administration of CARRA was not algesia were detected long after the paw withdrawal
influenced by repeated testing, we compared rats latency had returned to normal (Fig. 2A).
tested at 6 time points to several groups of rats, The thermal device was next evaluated for de-
each tested only once (Table II). While the tecting dose-related inflammation produced by 0.5,
withdrawal latencies of the saline-treated paws 1.0 or 2.0 mg of CARRA. CARRA-treated rats
and the contralateral (untreated) paws of all groups exhibited significantly shorter (F (35, 280) = 6.33;
remained near baseline values throughout the test- P < 0.01) withdrawal latencies to thermal stimula-
ing session, the CARRA-injected paws exhibited tion with an onset by 1 h and a peak at the 2.5 h
significantly shorter latencies from 1 through 24 h time point as compared to saline-treated animals
following injection (Table II). The time course of (Fig. 3). The magnitude of the decrease in
the paw withdrawal latencies did not differ be- withdrawal latency was significantly dose related
(slope = - 3.26; F (1, 30) = 36.58; P < 0.001). The
paw withdrawal exhibited a dose-related peak in
46
its duration at 2.5 h following injection of CARRA
44 I (slope = 7.37; F (1,30) = 13.71; P < 0.01) (Fig.
42
4). Both contralateral, untreated paws and saline-
i treated paws exhibited stable values of paw
g 40 t
withdrawal latency (Fig. 3) and duration of paw
withdrawal (Fig. 4) throughout the testing period.
The time course of edema and local hyperther-
mia after S.C. injection of saline or 0.5, 1.0 and 2.0
mg CARRA is presented in Fig. 5. CARRA-
induced inflammation resulted in significant
edema (F (35. 280) = 39.78; P < 0.001) over time.
There was a significant dose-response rela-
tionship (slope = 1.09; F (1, 30) = 96.83; P c
0.001) between CARRA and edema. The edema
was significantly greater (P < 0.01) for the 3 doses
of CARRA than that observed in the SAL group
Fig. 1. Effects of radiant heat on the cutaneous temperature of
as early as 1 h (Fig. 5A). The peak was reached
rat hind paws. The hind paws of 8 rats were injected with
carrageenan (squares) and saline (circles) 2 h before testing
between 2.5 and 4 h after injection of each dose.
commenced. Edema was still present 24 h after the injection of
 81

1.0 and 2.0 mg CARRA (P -= 0.01). The 2 mg

CARRA group was significantly different from all
the other groups at the 2.5,4 and 7 h time points.
Contralateral edema was not observed. Paw tem-
perature (Fig. 5B) followed a parallel time course
(F (35, 280) = 6.11; P c 0.001). The local hyper-
thermia response was linearly related to the dose
of CARRA {slope = 1.30; P (I, 30) =L= 84.45; P -z
U.001). The onset in local hyperthermia was reach-
ed at f h, while the peak was between 2.5 and 4 h
after injection of CARRA.
Ad~~stration of CARRA also resulted in hy-
peralgesia to mechanical stimuli (F (35, 280) =
2.93; P < 0.01). CARRA-induced hyperalgesia was
not detectable until 2.5 h after injection (Fig. 6).
Dose-related changes in withdrawal pressure were
observed in response to mechanical stimuli (slope
= -1-19; F (1, 30) = 20.08; P < 0.001). Con-
tralateral untreated paws did not exhibit signifi-
cant differences from saline-treated paws.
The ability of the thermal and mechanical de-
vices to detect blockade of CARRA-induced hy-
peralgesia by indomethacin (2 mg,/kg) and
morphine (3 mg/kg) is shown in Figs. 7 and g. c
ANOVA revealed a significant drug effect when lOO-

using radiant heat as the noxious stimulus (F (6,

72) = 8.80; P < 0.01). In the CARRA/SAL group
the CARRA-injected paws exhibited significantly
(P < 0.01) shorter paw withdrawal latencies as
compared to contralateral (untreated) paws at 2
and 3.5 h following injections (Fig. 7). Conversely,
withdrawal latencies of the CARRA-treated paws
of both the indomcthac~ and morphine groups
did not differ from latencies of their contralateral
untreated paws or latencies observed in the TIME Wows Aher CARRAi
SAL/SAL control group (Fig. 7).
When tested with the Randall-Selitto mechani-
D
cal device, the inflamed paws of the CARRA/SAL 20

Fig. 2. The behavioral profite of hyperalgesia. Carrageenan was

injected into a hind paw of 16 rats and the responses of the
inflamed paw were compared to the contralateral untreated
paw following exposure to radiant heat. A: effects of CARRA
on paw withdrawal latency. * * P < 0.01. B: effects of CARRA
on the velocity of paw withdrawd following aposure to radiant
heat. * P ( 0.001. C: effects of CAREU on post-undrawn
paw licking behavior. * P x 0.05, **P < 0.01. D: effects of
CARRA on the duration of paw withdrawal from the glass
floor. * P i 0.05, * * P c 0.01. TIME IHours After CARRAI
TABLE II

THE EFFECT OF REPEATED TESTING ON PAW WITHDRAWAL LATENCY * IN CARRAGEENAN-INDUCED HYPER-

ALGESIA
__- -~-
n Baseline 1h 2.5 11 4h 8h 24 h
Same group tested at each rime poini
(I ) Saline-injected paw 8 9.8 i 0.3 11.4 t 0.4 10.7 10.5 9.6 rt 0.6 9.2 f 0.6 9.8 * (1.2
Contralateral uninjected paw 9.6 IO.4 10.0 * 0.5 9.6 & OS 9.0 & 0.5 9.7 * 0.4 9.4 & 05
(2) Carrageenan-injected paw * * 8 9.lkO.3 3.7i0.6 *** 2.OkO.2 *** 1.81tO.l *** 2.5*0.2 *** 6.5&0.5 ***
Contralateral uninjected paw 10.0 f 0.4 10.0 + 0.3 9.6 + 0.4 9.8 f 0.4 9.0 * 0.4 10.1 + 0.3

Dlfferrnl groups tested only at 1 time point

(3) Carrageenan-injected paw 40 4.6+0.05 *** 1.8kO.3 ***1.8ri:0.01 ***2.8&0.4 *** 58+0.X ***
Contralateral uninjected paw (S/obser- 10.7 _I 0,s x.9 + 0.8 9.9 rt 0.6 9.1 + 0.4 9.1 ?r 0.5
vation)

* Mean & S.E.M. (set).

* * Rat hind paws were injected with either 3 mg carrageenan or saline at time zero.
* * * Significantly different ( P < 0.01) from saline paw and contralateral paw.

group exhibited significantly lower withdrawal Discussion

pressures as compared to their contraiateral un-
treated paws (Fig. 8). ANOVA indicated a signifi-
cant drug effect for mechanical testing (F (6,
72) = 4.01; P < 0.01). The CARRG-induced hy-
peralgesia to mechanical stimuli was not observed
in rats treated with either indomethacin or
morphine sulfate (Fig. 8).
The results of these studies indicate that the
thermal method provides a quantitative measure-
ment of the behavioral correlates of hyperalgesia.
Initial studies demonstrate that the decreased paw
withdrawal latency corresponds to the decreased
nociceptive threshold observed during the course

-
w
SAL
*e5 mg INJECTED
m-4 1.0 mg PAW
- 2.0 mg

5--o CONTRACTUAL
O--O (UNINJECTED)
z=g PAW

IF--+-J
BASELINE 1 2.5 4 7 24
TIME (HOURS AFTER INJECTION)
Fig. 3. Dose-related effects of CARRA on paw withdrawal latency following exposure to radiant heat. Rats (8/group) were injected
with either saline or 0.5. 1.0 or 2.0 mg of CARRA into a hind paw (filled symbols), while the contralateral paw was untreated
(corresponding open symbols). * P < 0.05, * * P i: 0.01.
 83

20 INJECTED PAW
w SAL
18 _ 0.5 mg
w l.Omg
16 - 2.0 mg

CONTRALATERAL PAW
14
e-4
C--4
12
5--if
b-4
10

I I I I 1-4
BASELINE 1 2.5 4 .. 7 24

TIME (Hours After Injection)

Fig. 4. Dose-related effects of CARRA on duration of the paw withdrawal following exposure to radiant heat. The treatment groups
are described in Fig. 3. * P -K0.05,
** P -c0.01.

A .-. SAL o--o

12
B c--2,
t: ::: i-z: D--c!
.. .. &--A
11 u 2.0 mg

5 * pas INJECTED UNINJECTED

f. lK.01 PAW PAW
E 10 - 32
3
8
$! 9- c 31
8
:: 9- 2 30
I
t 7- .. $ 29
w
i%
I- 29
3
2 27
I 1 I 1 I ,

BASELINE 1 2.5 4 174

TIME IHOURS AFTER INJECTlONt TIME iHOURS AFTER INJECTION1

Fig. 5. Dose-related effects of carrageenan on inflammation using the same groups as described in Fig. 3. A: effects of CARRA on
paw edema as measured by dorsal-plantar paw thickness using a vernier caliper. **P -C0.01. B: effects of CARRA on paw
temperature measured with a contact thermocouple. * P < 0.05, * * P i: 0.01.
 INJECTE
)----I l.omg PAW
- 2.0 mg

CONTRALATERAL
(UNtNJECTED)
PAW

L I 1 I t
BASELINE 1 2.5 4 i+-+-+?4
TIME lHOURS AFfER INJECTIONS
Fig. 6. Dose-related effects of CARRA on paw withdrawal CO mechanical pressure using the RandalLSelitto method. The groups are
described in Fig. 3. * P -c 0.05. * * P i 0.01.

in
cl 14 CARRAIINDO: lPSl
B ~AR~/lN~~ CONTRA
2 12 SAL/SAL: CONTRA
u> __.- CARRAIMORPHINE: iPSt
c 10 - - SAL/SAL: IPSI
CARRAIMORPHINE: CONTRA
z
k 8-
4 CARRAISALINE: IPSI
-I 6-

** pa1
$4
is
F2
f f
3 I 1
BL 2 2.5
TiME #IOURS)
Fig. 7. Morphine and indomethacin bkxkade of carrageenan-induced thermal hyperalgesia Carrageenan, ~~d~etha~i~ (2 mg/kg,
i.p i or sabne (i-p.1 were injected at time zero depicted by the first arrow, Morphine (3 mg/kg. i.p.) or saline Q.p.1 were injected 50
min iater (second arrow). * +P i 0.01.
 85

CARRA~MORPHINE: IPSI

CARRA/lNDO: IPSI

CARRA/lNDO: CONTRA
SAL/SAL: CONTRA

CARRAIMS: CONTRA
CARRAISAL: IPSI
CARRAISAL: CONTRA

2
TIME (HOURSI
Fig. 8. Morphine and indomethacin blockade of carrageenan-induced mechanical hyperalgesia. Drug dosage. routes and times of
administration are described in Fig. 7. * * P -z0.01.

z 60 ,
.c I of hyperalgesia. Repeated testing does not alter
z
2 the paw withdrawal latencies of either inflamed or
50
i; untreated paws. Several other response measures
2
determined by this method had a prolonged time
rr 40
2 course to hyperalgesia as compared to changes in
mechanical
I paw withdrawal latency. Both the thermal method
z 30
and the Randall-Selitto method detect dose-
related CARRA hyperalgesia and its blockade by
a prototype non-steroidal anti-inflammatory
(INDO) and opiate (MS) analgesic.
.l 10 Although CARRA-induced inflammation was
Dose of &ra (mg) first described in 1962 (181 and has since been
Fig. 9. Comparison of bioassay sensitivity of thermal and employed widely as an animal model to screen for
mechanical methods. Normalized dose-response data from the clinically useful non-steroidal ~ti-~~a~ato~
thermal and mechanical devices are compared by dividing
observations taken at 2.5 h by baseline values. The slope of the
drugs [ll], most studies have utilized edema as the
thermal device is significantly greater than the slope of the dependent measure. Comparatively little is known
mechanical device (P -z0.01). about CARRA-induced hyperalgesia [15]. Our
findings indicate that administration of 1 mg of nociceptive threshold for a sufficient amount of
CARRA results in the production of a cutaneous time to allow sensitization to occur [1,5]. The
hyperalgesia to thermal stimuli, as indicated by a stability of the control values (i.e., saline and
decreased withdrawal latency, which begins by 1 untreated contralateral paws) in the time course
h. peaks at 2-3 h and subsides by 8 h following experiment (Fig. 2A) constitutes a second argu-
injection. ment against a contribution of repeated testing
The withdrawal latencies of saline-injected paws and indicates that the observed hyperalgesia was
occurred at a nociceptive threshold temperature of due to CARRA.
45.2 + 0.2C. This finding is in agreement with Since the thermal method uses unrestrained
the thermal nociceptive threshold observed in hu- animals, several behavioral responses to cutaneous
mans, monkeys, guinea pigs and rats [5,8,16]. Fol- hyperalgesia can be measured. The normal paw
lowing CARRA inflammation, the paw withdrawal withdrawal occurs after a 9910 set latency and is
occurs at a threshold temperature of 38.5 _t 0.7 o C. characterized as a quick movement, generally fol-
As seen in Fig. 1, the CARRA-induced decrease in lowed by licking the tested paw. The paw is
the paw withdrawal latency corresponds to the returned to the glass floor within a few seconds.
decrease in the thermal nociceptive threshold. For This behavioral profile changes with the onset of
these reasons, we consider the paw withdrawal hyperalgesia. The CARRA-treated paw is with-
latency to be an index of the thermal nociceptive drawn at significantly shorter latencies following
threshold. exposure to radiant heat and the withdrawal is a
Since a CARRA-treated paw exhibits both slow movement, the paw is not licked and it
erythema and hyperthermia, it is possible that the remains elevated from the glass floor for an ex-
inflamed paw absorbs radiant heat more effec- tended duration (Fig. 2). Although changes in all
tively and requires a smaller temperature change response measures peaked at 2.5 h after CARRA
to reach the 38.5C threshold temperature as injection, the velocity of withdrawal, paw lick and
compared to a saline-treated paw. Therefore, we duration of withdrawal can be distinguished from
examined whether these factors contribute to the paw withdrawal latency by their prolonged time
decreased paw withdrawal latency observed after course. Significant alterations in these 3 response
CARRA. Comparison of the time response curves measures can be demonstrated up to 48 h after a
in Fig. 1 indicates that the inflamed paws reach single injection of CARRA, while paw withdrawal
the 38.5 o C nociceptive threshold approximately latency returns to near-baseline values after 8 h
0.6 set sooner than the saline-treated paws. This (Fig. 2). The differences in the time course of
finding indicates that the difference in withdrawal these response measures suggest that they repre-
latencies between the inflamed and saline-treated sent complex behaviors resulting from sensory
paws (i.e., approximately 6-8 set) is predomi- integration from input at different sites in the
nantly due to hyperalgesia and that local erythema CNS. In addition, these findings emphasize the
and hyperthermia contribute very little to the ob- value of a multidimensional measurement of be-
served difference. havioral responses to noxious stimuli.
We ruled out any effect of repeated heating of Increases in the duration of flexion reflexes
the paw on the development of CARRA hyperal- have been noted also in neurophysiologic investi-
gesia. Although heat is known to sensitize C Poly- gations of hyperalgesia. Beitel et al. [l] reported
modal fibers [1,5], we failed to detect any effect of the occurrence of afterdischarge in sensitized poly-
repeated testing on behavioral hyperalgesia (Table modal nociceptors following heat stimuli. Woolf
II). This is probably due to the fact that the rats [20] observed a sustained flexion after injury and
were tested only once at each testing session, and correlated this behavior with an increase of the
that the smallest time interval between tests was 1 afterdischarge in the flexor efferent motor nerve
h. Furthermore, since the animal was allowed to evoked by both thermal and mechanical stimuli.
freely escape from the stimulus, it is likely that the The increased duration of paw withdrawal ob-
cutaneous temperature never exceeded the thermal served in this study could then be due to either a

prolonged afferent discharge or efferent drive.
Comparison of the thermal to the mechanical
method indicates that both devices can measure
dose-related hyperalgesia and the blockade of hy-
peralgesia by either morphine or indomethacin.
However, the two methods differ in their sensitiv-
ity even when testing the same group of animals
on both devices. At 1 h after CARRA injection,
hyperalgesia was not detectable with the mechani-
cal device. In contrast, a robust hyperalgesia was
present in the same groups when testing with
thermal stimuli. Since significant changes in edema
and h~erther~a were also observed at 1 h, the
differences in the mechanical and thermal meth-
ods appear to be due to a difference in bioassay
sensitivity.
Two lines of statistical data provide further
support for a difference in bioassay sensitivity
between the two methods. First, comparison of
the results from the thermal and mechanical de-
vices indicates that the thermal device provides a
greater signal-to-noise ratio. This ratio is indicated
by larger F ratios following ANOVA (i.e., the
ratio of the mean square of the treatment effect to
the mean square of the error). For the compara-
tive studies presented in this paper (CARRA
dose-response and MS/INDO blockade), the
ANOVA F ratios for the thermal device were
more than double those F ratios generated from
data collected with the mechanical device. In more
than 9 months of study, we have consistently
observed F ratios from the thermal device to be
on the average nearly 2-fold greater than F ratios
gathered from the mechanical device. Thus, the
thermal device provides data with greater statisti-
cal power as compared to the Randall-Selitto
mechanical device.
The second line of evidence comes from
comparison of the slopes of the dose-response
curves (Fig. 9). To allow a direct comparison of
the ability of the two methods to measure behav-
ioral hyperalgesia, data from the thermal (Fig. 3)
and mechanical (Fig. 6) devices were normalized
by dividing observations taken at 2.5 h by baseline
values. As described earlier, both methods detect
dose-related hyperalgesia. Further analysis reveals
that the dose-response slope of the thermal
method is more than 2.6-fold steeper than the
87
slope derived from the mechanical method (Fig.
9); these slopes differ significantly (F (2, 43) =
5.38; P < 0.01). These findings indicate that the
thermal method has a greater bioassay sensitivity
for detecting behavioral hyperalgesia, as evidenced
by a given dose of CARRA producing a larger
departure from baseline measures.
The greater bioassay sensitivity of the thermal
device may be due in part to differences in meth-
odology. The thermal device tests an unrestrained
rat exposed to minimal environmental cues which
precede or signal the commencement of a testing
session. In contrast, the Randall-Selitto mechani-
cal device requires a forcibly restrained rat. The
mechanical method may then facilitate a learning
effect [15], in which paw withdrawal occurs at a
force below the nociceptive threshold. An ad-
ditional factor is immobilization, which activates
stress-related systems such as the pituitary-adrenal
and sympatho-adrenomedull~ axes [10,13]. The
amount of i~obilization used in the Randall-
Selitto method is stressful, as indicated both by
behavioral struggling and by higher levels of
blood-borne immunoreactive beta-endorphin [Har-
greaves and Joris, unpublished observations]. Both
a learning effect and stress may diminish the
sensitivity of the Randall-Selitto method.
An additional factor contributing to bioassay
sensitivity may be the development of hyperalge-
sia which differs in responsiveness to thermal and
mechanical stimuli. Supporting this point, Hand-
werker et al. [7] recorded C polymodal nociceptors
in CARRA-inflamed paws after thermal and
mechanical stimulation. Their findings indicate
that CARRA pretreatment alters C-fiber re-
sponsiveness to thermal stimuli more strongly than
to mechanical stimulation. In addition, Benoist et
al. [2] recorded from nociceptive neurons in the
ventrobasal thalamus following injection of
CARRA into the hind paw. They observed that
administration of CARRA decreased the thermal
threshold of these neurons and increased their
responsiveness to thermal stimulation. In contrast,
there were no significant alterations in response to
mechanical stimuli.
The experiments described herein validate the
thermal device as an appropriate and sensitive
method to quantify hyperalgesia. This new method
represents a useful tool in the assessment of he- neural mechanisms of cutaneous hyperalgesia. In: H.L.
havioral hyperalgesia. Together with the Ran- Fields. F. Cervero and R. Dubner (Eds.), Advances in Pain
Research and Therapy, Vol. 9, Raven Press. New York,
dall-Selitto test, these two methods provide be-
1985, pp. 53-71.
havioral correlates for the study of the mecha- 10 Mueller, G., Beta-endorphin immunoreactivity in rat
nisms of hyperalgesia to different noxious stimuli. plasma: variations in response to different physical stimuli,
Life Sci., 29 (1981) 1669-1674.
11 Ottemess, 1.G. and Bliven. M.L., Laboratory models for
testing nonsteroidal antiinflammatory drugs, In: J.
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