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A quantitative study on the tail flick test in the rat
Physiology & Behavior, Vol. 39, pp. 235--240. Copyright © Pergamon Journals Ltd., 1987. Printed in the U.S.A.
0031-9384/87 $3.00 + .00
A Quantitative Study on the Tail Flick Test in the Rat KENJI KAWAKITA 1 AND MASAYA FUNAKOSHI
D e p a r t m e n t o f Oral Physiology, Faculty o f Dentistry, A s a h i University, Gifu 501-02 R e c e i v e d 11 F e b r u a r y 1986 KAWAKITA, K. AND M. FUNAKOSHI. A quantitative study on the tail flick test in the rat. PHYSIOL BEHAV 39(2) 235-240, 1987.--Tail flick latency and response temperature were measured in awake rats. Both of these parameters varied with the change in rate of increase of radiant heat stimulus and the area of skin irradiated. The caloric factor applied to the tail before the response occurred was calculated approximately from the time-temperature curves monitored over the tail and the response latencies. The magnitude of the caloric factor over a tentative threshold temperature was the crucial factor for the induction of tail flick response, because it was similar for the various irradiating conditions. It was proposed that analgesic effects in the tail flick test may be expressed using this caloric factor which allows direct comparison of studies in which different base-line latencies are used. Tail flick test
Pain
Analgesic index
Psychophysics
IN recent studies of pain, measurements have been made using the hot plate test [24], vocalization test [11,23], tail pinch test [25], tail flick test [1,2] and formalin test [10] in awake experimental animals. Of these methods, the tail flick test [8] seems to be the most widely used. The tail flick response, provoked by radiant heat stimulation of the tail, is a stable and stereotyped behavior which can be detected automatically. Although the tail flick response is a spinal reflex [6,16], an increase in latency is induced by the administration of opioid drugs [2, 6, 16], SPA [1] and acupuncture [17], all of which produce analgesia in man [7,15]. Thus, the increase in the tail flick latency correlates with analgesia. The magnitude of the analgesic effect may be expressed as a percentage of the baseline latency: (TL/BL)× 100 [17] or by the formula: ( ( T L - B L ) / ( C T - B L ) ) x 100, where T L = t e s t latency, B L = b a s e l i n e latency and CT=cut-off time. This formula, referred to as the degree of analgesia [1], analgesic index [2], or % MPA (Maximum Possible Analgesia) [28], is commonly used in the study of pain. It is easily seen that the value of % latency of % MPA varies if the baseline latency and/or the cut-off time changes. Routinely the baseline latency is adjusted within the range of 3.0--4.0 sec by regulating the intensity of radiant heat stimulus, and the cut-off time is arbitrarily determined to be approximately 2 times the baseline latency [1, 2, 28]. While these procedures in the tail flick test have shown to be useful in practice, there is little theoretical support for the procedures and for the indexing of analgesic effects by using the baseline latency and test latency. In the present study, the factors that influence the tail flick response are examined and an invariant, quantitative expression of analgesic effect in tail flick test is proposed.
METHOD The present experiments were performed on 43 female Wistar rats (270--350 g). The rats were restrained in a wire mesh holder [22]. Tails were blackened with India ink and placed manually to the plastic plate to measure tail flick latency. To control the size of the irradiated area, reflection tape with slits of 1.0, 2.0, 4.0 and 8.0 mm was wrapped around the tail. The radiant heat of a halogen lamp (100 V, 1000 W) was focused to 10 mm in diameter through a series of optical lenses and the voltage to the lamp was varied at random within the range of 70-110 V in increments of 10 V. The temperature during the radiant heat stimulation was monitored at the center of the focused spot by a needle-type thermo-couple (time constant of 0.1 msec), which was usually set 10 mm over the tail to exclude disturbance of the tail flick response. In some cases, the thermor-couple was inserted into the skin or attached on the skin surface to measure the regional differences of the actual temperature as the tail flick response occurred. The latency and the monitored temperature were indicated on a digital timer and thermometer which were stopped by a signal from the photo cells placed on both sides of the tail. The measurement of the latency was repeated 20 times at intervals of 2 or more min for each rat. The time-temperature curves shown in Fig. 1 are generated by the formula, Y=A×(1-e-~X-C)/B)+P. In the formula, values for A, B and C were determined such that the simulated curves would coincide with the monitored timetemperature curves of the different radiant heat stimulations (see Fig. 2). In Fig. 1, P is the initial temperature. L1, L2 are the tail flick latencies at two radiating conditions, and R1, R2
1Department of Physiology, Meiji College of Oriental Medicine, Hiyoshi, Kyoto 629-03, Japan.
235
236
KAWAKITA AND FUNAKOSHI Y1
C , ~ ~ f ~ =A.{ 1_~(x-C)/B) + p R1 ................................ R2
-
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I C
:
f,.
~.
!:
Xl
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n
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Time FIG. 1. Schematic illustration of the parameters used for calculation in this study. The monitored time-temperature curves simulated by exponential function are shown as Y1 and Y2. L1 and L2 are tail flick latency. R1 and R2 are the response temperatures of tail flick latencies. The magnitude of caloric factor over a tentative threshold temperature (T) is shown as the area of SI or $2. X1 and X2 indicate the time until the monitored temperature reaches to the tentative threshold temperature, m and n are bin numbers (100 ms) from X! to LI and from X2 to L2. P shows the initial temperature of thermoprobe.
are the c o r r e s p o n d i n g r e s p o n s e t e m p e r a t u r e s as the tail flick o c c u r r e d . T h e a r e a s o f S1 and $2 w h i c h are b o u n d e d b y the s i m u l a t e d t i m e - t e m p e r a t u r e c u r v e s (Y1, Y2), a t e n t a t i v e t h r e s h o l d t e m p e r a t u r e (T) a n d l a t e n c i e s (L1, L2) are a p p r o x imate c a l c u l a t i o n s using the t r a p e z o i d a l m e t h o d with a microcomputer. T h e a r e a of S 1 or $2 was c a l c u l a t e d to reflect the magn i t u d e o f the caloric factor. T h e p a r a m e t e r s w h i c h w e r e c o n s i d e r e d to b e r e l a t e d to t h e i n d u c t i o n o f tail flick res p o n s e , s u c h as r e s p o n s e t e m p e r a t u r e , caloric f a c t o r applied to the tail, w e r e m e a s u r e d or calculated. T h e p a r a m e t e r w h i c h r e s u l t e d in the l o w e s t v a l u e for the v a r i a t i o n coeffic i e n t : s t a n d a r d deviation (S.D.)/mean (M), ( n = 5 ) , was considered t h e crucial f a c t o r in the i n d u c t i o n o f tail flick res p o n s e . T h a t is to say, the p a r a m e t e r yielding t h e m o s t similar values for the five radiating c o n d i t i o n s was c o n s i d e r e d the critical factor. T h e effect o f m o r p h i n e a d m i n i s t r a t i o n (2 mg/kg, IP) o n the tail flick l a t e n c y a n d o n t h e c a l c u l a t e d caloric f a c t o r was also e x a m i n e d in s e v e r a l rats.
RESULTS
C l e a r a n d stable tail flick r e s p o n s e s w e r e i n d u c e d b y r a d i a n t h e a t s t i m u l a t i o n applied to t h e tail. T h e l a t e n c y a n d the r e s p o n s e t e m p e r a t u r e o f tail flick v a r i e d w i t h the differe n t radiating c o n d i t i o n s . A s s h o w n in T a b l e 1, t h e tail flick i a t e n c i e s d e c r e a s e d f r o m 5.9 to 1.9 sec as the s u p p l y voltage to the radiating l a m p i n c r e a s e d f r o m 70 to 110 V. T h e res p o n s e t e m p e r a t u r e s m o n i t o r e d o v e r , u n d e r a n d o n the tail
TABLE
1
T H E T A I L F L I C K L A T E N C I E S (MEAN -+ S.D.) AND T H E RESPONSE T E M P E R A T U R E S MONITORED OVER, U N D E R AND ON T H E T A I L SKIN IN T H E 5 D I F F E R E N T R A D I A T I N G CONDITIONS
Voltage (V) 70 80 90 100 110
Monitored Response Temperatures (°C) Latency (sec) (n=42) over (n=20) under (n=8) on (n=12) 5.9 3.7 2.7 2.2 1.9
_+ 1.0 _+ 0.6 + 0.3 _+ 0.2 _+ 0.2
36.2 39.0 41.0 43.5 44.1
_+ 1.8 _+ 2.2 _+ 2.1 _+ 2.6 _+ 1.8
44.3 45.1 47.6 48.3 48.6
_+ 1.3 _+ 0.9 _+ 1.4 _+ 1.8 _+ 1.6
49.1 56.1 61.8 65.3 66.5
_+ 1.4 _ 1.8 _+ 2.3 -+ 4.2 _+ 3.5
v a r i e d with the c h a n g e in the a r e a o f the tail skin w h i c h was e x p o s e d . T h e r e was a t e n d e n c y in t h e r e s p o n s e t e m p e r a t u r e to i n c r e a s e with a n i n c r e a s e of s u p p l y voltage for r a d i a n t h e a t stimulus. T h e r e s p o n s e t e m p e r a t u r e s m o n i t o r e d u n d e r t h e skin s h o w e d the l e a s t v a r i a t i o n s ; h o w e v e r , the differe n c e s b e t w e e n the r e s p o n s e t e m p e r a t u r e at the s u p p l y voltage o f 70 a n d t h a t o f 90, 100 a n d 110 V w e r e statistically significant ( p < 0 . 0 1 , t-test). D e c r e a s i n g t h e size o f t h e r a d i a t e d a r e a o n the tail also s t r o n g l y i n f l u e n c e d t h e tail flick l a t e n c y a n d t h e r e s p o n s e t e m p e r a t u r e . T a b l e 2 s h o w s t h a t t h e l a t e n c y a n d the res p o n s e t e m p e r a t u r e i n c r e a s e d as the r a d i a t e d a r e a de-
A Q U A N T I T A T I V E EXPRESSION IN TAIL F L I C K TEST
237
TABLE 2 EFFECTS OF T H E D I M I N I S H M E N T OF T H E R A D I A T E D SIZE ON T H E T A I L F L I C K L A T E N C Y (TFL) A N D T H E RESPONSE T E M P E R A T U R E (RT) M O N I T O R E D OVER T H E T A I L IN T H E 5 D I F F E R E N T R A D I A T I N G CONDITIONS
Radiating Size of the Tail (Slit Size) 8 mm (n=28) Voltage (V)
TFL (sec),
70 80 90 100 I10
5.9 3.7 2.7 2.1 1.9
4 mm (n=14)
RT (°C)
- 1.0, --- 0.7, -+ 0.4, ± 0.2, ± 0.2,
TFL (sec),
36.2 38.8 40.8 42.7 43.9
7.6 4.2 3.7 2.6 2.3
2 mm (n=14)
RT (°C)
+_ 1.8, _+ 0.7, ± 0.7, ± 0.5, ± 0.1,
TFL (sec),
37.1 39.9 44.7 45.9 47.9
6.4 4.8 3.3 3.0
1 mm (n=14)
RT (°C)
. --- 1.3, ± 1.4, ± 0.8, ± 1.0,
.
TFL (sec),
.
. 9.5 5.5 4.7 3.3
42.7 46.7 49.2 52.1
RT (°C)
-+ 1,1, ± 2.1, ± 1.5, ± 1.3,
44.1 47.6 52.9 53.6
C 40
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FIG. 2. The monitored and the simulated time-temperature curves in 5 different radiating conditions. Open circles and vertical short bars are the mean values and standard deviations of monitored temperatures at intervals of 0.5 sec. The dotted lines show the simulated time-temperature curves used for the calculation in this study.
FIG. 3. Magnitude of caloric factor over various tentative threshold temperature and their coefficient of variation (S.D./Mean) in 5 different radiating conditions. The small numbers show the supplying voltages for the radiation of lamp. The solid lines are the calculated caloric factor (left ordinate) and the dotted line with solid circles is the coefficient of variation (right ordinate).
creased. These results indicate that the temperature at which a tail flick response occurs is not absolute but depends on the radiating condition. Therefore, another factor responsible for the provocation of tall flick must be considered. Hardy et al. [13,14] previously demonstrated the threshold of pain as a caloric parameter (mcal/sec/cm2). We calculated an approximation of the heat applied to the tall until the provocation of the tall flick response. Figure 2 shows the monitored and the simulated time-temperature curves of the five radiating conditions. The time-temperature curves based on monitoring above the focused spot were simulated with the formula: Y=Ax(1-e-(X-C)~B)+P. The values of A, B and C used in this calculation were as follows: 15.0, 25.5 and 8.0 for 70 V of supplied voltage; 22.0, 24.0 and 7.0 for 80 V; 27.0, 18.0 and 7.0 for 90 V; 34.0, 17.5 and 5.5 for
100 V; 39.0, 17.5 and 5.5 for 110 V, respectively. P was kept at 24.0°C. The correlation coefficients between the paired temperatures (monitored and simulated) at intervals of 0.5 sec for all five of the radiating conditions were high enough to be used for further analysis (r>0.999). The magnitude of the caloric factor calculated from the simulated temperature curve varied with the radiating conditions. When a more rapid increase of radiant heat was applied to the tail, a smaller caloric factor was required to provoke the tail flick response. Figure 3 shows the influence of a tentative threshold temperature on the magnitude of the caloric factor (open circles, solid line, left ordinate) and the variation coefficient (S.D./M) (solid circles and dotted line, right ordinate) for the 5 different radiating conditions. The small numbers indicate
238
K A W A K I T A AND F U N A K O S H I TABLE
3
9-
2.8s
I N F L U E N C E OF RADIATED A R E A ON T H E RCTFs C A L C U L A T E D FROM T H E T A I L F L I C K L A T E N C I E S IN T H E 5 D I F F E R E N T R A D I A T I N G CONDITIONS "
Radiated Size of the Tail (Slit) Voltage (V)
8 mm
4 mm
2 mm
1 mm
110
70 68 67 62 62
150 106 180 120 120
-322 339 243 251
-696 450 528 317
Mean S.D.
65.8 3.6
136.6 29.0
288.8 48.8
497.8 158.3
70 80 90 100
Percentages (%)
100
208
439
757
RCTF × Slit (mm)
800
832
878
757
O
7"
._
6
/o3.2s
36s
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5/ /
32 1 0 75
100 Tail
125 150 175 Flick Latency
200
225 (%)
FIG. 4. Relationship between analgesic index and % latency with various baseline latencies. Analgesic index is indicated as the ratio of RCTF (RCTF of test latency/RCTF of baseline latency). The radiating condition in this calculation was 80 V and 8.0 mm slit. The average tail flick latency in this condition was 3.8 sec.
the supply voltages for the lamp. The magnitude of the caloric factor over a tentative threshold temperature decreases almost linearly as the tentative threshold temperature increases. When lower supply voltages were used, the magnitude of the caloric factor decreases more steeply. On the other hand, the variation coefficient (S.D./M) of the calculated caloric factor for the 5 radiating conditions decreases to a minimum value (0.055) at the tentative threshold temperature of P+8.8 (P=24.0°C), and then rapidly increases. The tentative threshold temperature of P+8.8°C monitored on and under the tail skin were 46.3-+1.8 and 37.7-+1.7°C (mean-+S.D., n=5), respectively. Since the magnitude of the caloric factor over a tentative threshold temperature of P+8.8 showed a similar value for the 5 different radiating conditions, it was considered to be the crucial factor in provoking a tail flick response. This factor, the Required Calories for Tail Flick (RCTF) is used for subsequent calculations in this paper. The quantitative relationship between the radiated area and the tail flick latency or the response temperature of tail flick is not apparent in Table 2. The relationship between the RCTF and radiated area was examined. As shown in Table 3, at different radiant heat conditions, the calculated RCTFs show a tendency to increase as the size of slit decreases. The ratios of the mean RCTF of the 5 radiating conditions at 4, 2 and 1 mm slit are 2.08, 4.39 and 7.57, respectively. These results are statistically significant (chi square test, p<0.05). That is, the total magnitudes of caloric factor (RCTF × radiated area (in slit) applied to the tail until the occurrence of tail flick response are very similar for the different radiating conditions. These facts suggest that the RCTF is a useful quantitative index of pain in the tail flick test. Table 4 shows that the latencies clearly increase to 155173% of baseline latency at 20-30 min after the administra-
tion of morphine (2 mg/kg, IP). When the lower voltage was supplied to the lamp, a greater increase in % latency was observed. On the other hand, the ratios of RCTF (RCTF of test latency/RCTF of baseline latency) after the injection of morphine were 3.64-4.25. Each value was similar and independent of the radiating conditions. The variation coefficient in % latency was smaller than that for the ratios of RCTF; however, it indicates only the utility of the expression of % latency. On the other hand, the similar values of the ratios of RCTF indicate not only its utility but also its theoretical efficacy. Therefore, we proposed the ratio of RCTF as a new analgesic index. Relationship between % latency and analgesic index (ratio of RCTF) is shown in Fig. 4. The radiating condition was 80 V with a 8 mm slit; the average latency of normal rats in this radiating condition was 3.8 sec (solid circles), as presented in Tables 1 and 4. The analgesic index increases with the increase of % latency in a gradual manner. A steeper increase of analgesic index is observed with a shorter baseline latency. Similar tendencies are observed in the different radiating conditions. The low supply voltage causes a mild increase of analgesic index while the high voltage induces a steep increase in analgesic index. It is clear that the same value of% latency does not indicate the same analgesic index when the radiating condition and/or baseline latency is different. DISCUSSION
The present study demonstrates that the response temperature of tail flick varies with the size of radiated area and voltage supplied to the radiating lamp. No absolute temperature consistently elicits a tail flick response. These observations suggest that a constant temperature of pain threshold reported in previous investigations [14,21] might depend on
A Q U A N T I T A T I V E E X P R E S S I O N IN T A I L F L I C K TEST
239
TABLE 4 EFFECTS OF MORPHINE ADMINISTRATIONON THE TAIL FLICK LATENCY, RCTF, % LATENCY AND THE ANALGESIC INDEX IN THE DIFFERENT RADIATINGCONDITIONS Baseline
After Morphine
Voltage (V)
TFL (sec),
RCTF
TFL (sec),
RCTF
70 80 90 100 110
6.2 3.9 2.8 2.2 2.0
82 82 76 74 75
10.7 ± 2.8, 6.6 --- 1.3, 4.7 ± 1.0, 3.5 ± 0.4, 3.1 ± 0.4,
323 345 323 280 273
173 169 168 159 155
0.101
0.046
S.D./Mean
-+ 0.5, ± 0.7, ± 0.2, ± 0.2, ± 0.1,
0.050
their method of pain measurement when exposed to constant radiant heat stimulation. Hardy et al. [13] characterized pain threshold by a caloric unit (mcal/sec/cm 2) instead of considering a threshold temperature. In their study, the calories applied to the skin were not estimated but the intensity of radiant heat lamp itself was measured. In contrast, we calculated the approximate caloric factor, which when applied to the tail resulted in a tail flick response. This factor was estimated from the simulated time-temperature curve and tail flick latency. The magnitude of the caloric factor above a tentative threshold temperature (P+8.8°C) is considered the most crucial factor for the provocation of tail flick response. This caloric factor (here referred to as the Required Calories for Tail Flick or RCTF), quantitatively changes with the size of the area heated and it similarly increases after the administration of morphine under the different radiating conditions. However, the physiological basis of the RCTF is not yet understood. F r o m a physiological point of view, the R C T F applied to the skin should be converted into the afferent impulses by heat sensitive receptors. The existence of skin receptors which respond to radiant heat has been reported in rats [19,20], cats [2,5], monkeys [4, 9, 18] and man [12, 26, 27]. The participation of these heat sensitive neurons in the provocation of pain sensation [18, 27, 28] and avoidance responses [9,19] has been strongly suggested. Dubner et al. [9] demonstrated that the total impulse number from A-delta heat receptors during radiant heat stimulation applied to the monkey's face is closely related to the provocation of the avoidance response. On the other hand, the mean impulse rate of C polymodal receptors is shown to be related to pain sensation [18]. These studies indicate the close relation between the afferent impulse volley from the receptor and pain sensation or avoidance responses. The present problem is determining the physiological basis of RCTF. Therefore, we calculated the hypothetical afferent impulse rate and total impulse number elicited by the radiant heat stimulation of the tail skin. The assumptions were as follows. The impulses are elicited in accordance with Steven's power function: N = k x ( T - T o ) n, where N is im-
% Latency
Analgesic Index 3.94 4.20 4.25 3.78 3.64 0.066
pulse number, k is a proportional constant, T is monitored temperature of heat stimulus, To is threshold of the heat sensitive receptor and n is an exponent respectively. The tentative threshold temperature used in the calculation of RCTF is substituted as To. The final impulse rate is obtained as the impulse number as the tail flick response occurred. Total impulse number is calculated as the sum of N in each bin (re,n) from X to L (see Fig. 1). The calculated final impulse rate varies with the radiating condition for the 5 radiating conditions when the value of exponent varied from 0.1 to 2.0 in increments of 0.1. On the other hand, calculated total impulse numbers for the 5 radiating conditions resulted in similar values when the exponent was 1.2; that is, the value of the coefficient of variation was minimum 0.069, with an exponent of 1.2. According to Necker and Hellon [20], there exist heat sensitive receptors in the rat tail skin; their response characteristics are in agreement with the power function, and their exponent is about 1.2 (roughly estimated from their data). The existence of a very similar receptor predicted from a hypothetical calculation based on the R C T F and the tentative threshold temperature indicates that the RCTF and the tentative threshold used in the present study are likely parameters for the expression of tail flick response. The purpose of the present study was to establish a reasonable expression of analgesic effect in the tail flick test. The analgesic index (ratio of RCTF) proposed in this study is shown to be reasonable and useful; however, it is somewhat difficult to use routinely. On the other hand, % latency is proportionally changed by a change in the analgesic index with a limited radiating condition and baseline latency (Fig. 4). Therefore, the present study demonstrates that the expressions of analgesic effect by % latency and analgesic index (ratio of RCTF) are both useful, quantitative expression in the tail flick test. However, it should be noted that the expression of analgesic effects by % latency must not be used when the magnitudes of analgesia were compared between the animals in the different baseline latencies. In these cases, only the analgesic index (ratio of RCTF) could indicate the precise comparison of the analgesic effects.
240
KAWAKITA AND FUNAKOSHI REFERENCES
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16. Irwin, S., R. W. Houde, D. R. Beneatt, L. C. Hendershot and M. H. Seevers. The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. J Pharmacol Exp Ther 101: 132-143, 1951. 17. Kawakita, K. and M. Funakoshi. Role of the polymodal receptors in acupuncture analgesia of the rat. Comp Med East West 6: 313-321, 1978. 18. Lamotte, R. H., J. G. Thalhammer and C. J. Robinson. Peripheral neural correlates of magnitude of cutaneous pain and hyperalgesia: A comparison of neural events in monkey with sensory judgements in human. J Neurophysiol 50: 1-26, 1983. 19. Mitchell, D. and R. F. Hellon. Neuronal and behavioural responses in rats during noxious stimulation of the tail. Proc R Soc Lond [Biol] 197: 16%197, 1977. 20. Necker, R. and R. F. Hellon. Noxious thermal input from the rat tail: Modulation by descending inhibitory influences. Pain 4: 231-242, 1978. 21. Neisser, U. Temperature thresholds for cutaneous pain. J Appl Physiol 14: 368-372, 1959. 22. Ohara, K., H. Sato, N. Okuda, Y. Makino and Y. Isobe. Responses in rectal and skin temperatures to centrifugal forces in rats of different ambient temperatures. Int J Biometeorol 26: 61-72, 1982. 23. Pomeranz, B. and D. Chiu. Naloxone blockade of acupuncture anlagesia: Endorphin implicated. Life Sci 19: 1757-1762, 1976. 24. Segal, M. and D. Sandberg. Analgesia produced by electrical stimulation of catecholamine nuclei in the rat brain. Brain Res 123: 362-372, 1977. 25. Takagi, H., M. Sato, A. Araike, T. Shibata, H. Yajima and H. Ogawa. Analgesia by enkephalins injected into the nucleus reticularis gigantocellularis of rat medulla oblongata. Eur J Pharmacol 49: ll3-116, 1978. 26. Torebjork, H. E. Afferent C units responding to mechanical, thermal and chemical stimuli in human non-glabrous skin. Acta Physiol Scand 92: 374-390, 1974. 27. Van Hees, J. and J. Gybels. C nociceptor activity in human nerve during painful and non-painful skin stimulation. J Neurosurg Psychiatry 44: 600-607, 1981. 28. Watkins, L. R. and D. J. Mayer. Involvement of spinal opioid systems in footshock-induced analgesia: Antagonism by naloxone is possible only before induction of analgesia. Brain Res 242: 30%316, 1982.
0031-9384/87 $3.00 + .00
A Quantitative Study on the Tail Flick Test in the Rat KENJI KAWAKITA 1 AND MASAYA FUNAKOSHI
D e p a r t m e n t o f Oral Physiology, Faculty o f Dentistry, A s a h i University, Gifu 501-02 R e c e i v e d 11 F e b r u a r y 1986 KAWAKITA, K. AND M. FUNAKOSHI. A quantitative study on the tail flick test in the rat. PHYSIOL BEHAV 39(2) 235-240, 1987.--Tail flick latency and response temperature were measured in awake rats. Both of these parameters varied with the change in rate of increase of radiant heat stimulus and the area of skin irradiated. The caloric factor applied to the tail before the response occurred was calculated approximately from the time-temperature curves monitored over the tail and the response latencies. The magnitude of the caloric factor over a tentative threshold temperature was the crucial factor for the induction of tail flick response, because it was similar for the various irradiating conditions. It was proposed that analgesic effects in the tail flick test may be expressed using this caloric factor which allows direct comparison of studies in which different base-line latencies are used. Tail flick test
Pain
Analgesic index
Psychophysics
IN recent studies of pain, measurements have been made using the hot plate test [24], vocalization test [11,23], tail pinch test [25], tail flick test [1,2] and formalin test [10] in awake experimental animals. Of these methods, the tail flick test [8] seems to be the most widely used. The tail flick response, provoked by radiant heat stimulation of the tail, is a stable and stereotyped behavior which can be detected automatically. Although the tail flick response is a spinal reflex [6,16], an increase in latency is induced by the administration of opioid drugs [2, 6, 16], SPA [1] and acupuncture [17], all of which produce analgesia in man [7,15]. Thus, the increase in the tail flick latency correlates with analgesia. The magnitude of the analgesic effect may be expressed as a percentage of the baseline latency: (TL/BL)× 100 [17] or by the formula: ( ( T L - B L ) / ( C T - B L ) ) x 100, where T L = t e s t latency, B L = b a s e l i n e latency and CT=cut-off time. This formula, referred to as the degree of analgesia [1], analgesic index [2], or % MPA (Maximum Possible Analgesia) [28], is commonly used in the study of pain. It is easily seen that the value of % latency of % MPA varies if the baseline latency and/or the cut-off time changes. Routinely the baseline latency is adjusted within the range of 3.0--4.0 sec by regulating the intensity of radiant heat stimulus, and the cut-off time is arbitrarily determined to be approximately 2 times the baseline latency [1, 2, 28]. While these procedures in the tail flick test have shown to be useful in practice, there is little theoretical support for the procedures and for the indexing of analgesic effects by using the baseline latency and test latency. In the present study, the factors that influence the tail flick response are examined and an invariant, quantitative expression of analgesic effect in tail flick test is proposed.
METHOD The present experiments were performed on 43 female Wistar rats (270--350 g). The rats were restrained in a wire mesh holder [22]. Tails were blackened with India ink and placed manually to the plastic plate to measure tail flick latency. To control the size of the irradiated area, reflection tape with slits of 1.0, 2.0, 4.0 and 8.0 mm was wrapped around the tail. The radiant heat of a halogen lamp (100 V, 1000 W) was focused to 10 mm in diameter through a series of optical lenses and the voltage to the lamp was varied at random within the range of 70-110 V in increments of 10 V. The temperature during the radiant heat stimulation was monitored at the center of the focused spot by a needle-type thermo-couple (time constant of 0.1 msec), which was usually set 10 mm over the tail to exclude disturbance of the tail flick response. In some cases, the thermor-couple was inserted into the skin or attached on the skin surface to measure the regional differences of the actual temperature as the tail flick response occurred. The latency and the monitored temperature were indicated on a digital timer and thermometer which were stopped by a signal from the photo cells placed on both sides of the tail. The measurement of the latency was repeated 20 times at intervals of 2 or more min for each rat. The time-temperature curves shown in Fig. 1 are generated by the formula, Y=A×(1-e-~X-C)/B)+P. In the formula, values for A, B and C were determined such that the simulated curves would coincide with the monitored timetemperature curves of the different radiant heat stimulations (see Fig. 2). In Fig. 1, P is the initial temperature. L1, L2 are the tail flick latencies at two radiating conditions, and R1, R2
1Department of Physiology, Meiji College of Oriental Medicine, Hiyoshi, Kyoto 629-03, Japan.
235
236
KAWAKITA AND FUNAKOSHI Y1
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:
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~.
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Time FIG. 1. Schematic illustration of the parameters used for calculation in this study. The monitored time-temperature curves simulated by exponential function are shown as Y1 and Y2. L1 and L2 are tail flick latency. R1 and R2 are the response temperatures of tail flick latencies. The magnitude of caloric factor over a tentative threshold temperature (T) is shown as the area of SI or $2. X1 and X2 indicate the time until the monitored temperature reaches to the tentative threshold temperature, m and n are bin numbers (100 ms) from X! to LI and from X2 to L2. P shows the initial temperature of thermoprobe.
are the c o r r e s p o n d i n g r e s p o n s e t e m p e r a t u r e s as the tail flick o c c u r r e d . T h e a r e a s o f S1 and $2 w h i c h are b o u n d e d b y the s i m u l a t e d t i m e - t e m p e r a t u r e c u r v e s (Y1, Y2), a t e n t a t i v e t h r e s h o l d t e m p e r a t u r e (T) a n d l a t e n c i e s (L1, L2) are a p p r o x imate c a l c u l a t i o n s using the t r a p e z o i d a l m e t h o d with a microcomputer. T h e a r e a of S 1 or $2 was c a l c u l a t e d to reflect the magn i t u d e o f the caloric factor. T h e p a r a m e t e r s w h i c h w e r e c o n s i d e r e d to b e r e l a t e d to t h e i n d u c t i o n o f tail flick res p o n s e , s u c h as r e s p o n s e t e m p e r a t u r e , caloric f a c t o r applied to the tail, w e r e m e a s u r e d or calculated. T h e p a r a m e t e r w h i c h r e s u l t e d in the l o w e s t v a l u e for the v a r i a t i o n coeffic i e n t : s t a n d a r d deviation (S.D.)/mean (M), ( n = 5 ) , was considered t h e crucial f a c t o r in the i n d u c t i o n o f tail flick res p o n s e . T h a t is to say, the p a r a m e t e r yielding t h e m o s t similar values for the five radiating c o n d i t i o n s was c o n s i d e r e d the critical factor. T h e effect o f m o r p h i n e a d m i n i s t r a t i o n (2 mg/kg, IP) o n the tail flick l a t e n c y a n d o n t h e c a l c u l a t e d caloric f a c t o r was also e x a m i n e d in s e v e r a l rats.
RESULTS
C l e a r a n d stable tail flick r e s p o n s e s w e r e i n d u c e d b y r a d i a n t h e a t s t i m u l a t i o n applied to t h e tail. T h e l a t e n c y a n d the r e s p o n s e t e m p e r a t u r e o f tail flick v a r i e d w i t h the differe n t radiating c o n d i t i o n s . A s s h o w n in T a b l e 1, t h e tail flick i a t e n c i e s d e c r e a s e d f r o m 5.9 to 1.9 sec as the s u p p l y voltage to the radiating l a m p i n c r e a s e d f r o m 70 to 110 V. T h e res p o n s e t e m p e r a t u r e s m o n i t o r e d o v e r , u n d e r a n d o n the tail
TABLE
1
T H E T A I L F L I C K L A T E N C I E S (MEAN -+ S.D.) AND T H E RESPONSE T E M P E R A T U R E S MONITORED OVER, U N D E R AND ON T H E T A I L SKIN IN T H E 5 D I F F E R E N T R A D I A T I N G CONDITIONS
Voltage (V) 70 80 90 100 110
Monitored Response Temperatures (°C) Latency (sec) (n=42) over (n=20) under (n=8) on (n=12) 5.9 3.7 2.7 2.2 1.9
_+ 1.0 _+ 0.6 + 0.3 _+ 0.2 _+ 0.2
36.2 39.0 41.0 43.5 44.1
_+ 1.8 _+ 2.2 _+ 2.1 _+ 2.6 _+ 1.8
44.3 45.1 47.6 48.3 48.6
_+ 1.3 _+ 0.9 _+ 1.4 _+ 1.8 _+ 1.6
49.1 56.1 61.8 65.3 66.5
_+ 1.4 _ 1.8 _+ 2.3 -+ 4.2 _+ 3.5
v a r i e d with the c h a n g e in the a r e a o f the tail skin w h i c h was e x p o s e d . T h e r e was a t e n d e n c y in t h e r e s p o n s e t e m p e r a t u r e to i n c r e a s e with a n i n c r e a s e of s u p p l y voltage for r a d i a n t h e a t stimulus. T h e r e s p o n s e t e m p e r a t u r e s m o n i t o r e d u n d e r t h e skin s h o w e d the l e a s t v a r i a t i o n s ; h o w e v e r , the differe n c e s b e t w e e n the r e s p o n s e t e m p e r a t u r e at the s u p p l y voltage o f 70 a n d t h a t o f 90, 100 a n d 110 V w e r e statistically significant ( p < 0 . 0 1 , t-test). D e c r e a s i n g t h e size o f t h e r a d i a t e d a r e a o n the tail also s t r o n g l y i n f l u e n c e d t h e tail flick l a t e n c y a n d t h e r e s p o n s e t e m p e r a t u r e . T a b l e 2 s h o w s t h a t t h e l a t e n c y a n d the res p o n s e t e m p e r a t u r e i n c r e a s e d as the r a d i a t e d a r e a de-
A Q U A N T I T A T I V E EXPRESSION IN TAIL F L I C K TEST
237
TABLE 2 EFFECTS OF T H E D I M I N I S H M E N T OF T H E R A D I A T E D SIZE ON T H E T A I L F L I C K L A T E N C Y (TFL) A N D T H E RESPONSE T E M P E R A T U R E (RT) M O N I T O R E D OVER T H E T A I L IN T H E 5 D I F F E R E N T R A D I A T I N G CONDITIONS
Radiating Size of the Tail (Slit Size) 8 mm (n=28) Voltage (V)
TFL (sec),
70 80 90 100 I10
5.9 3.7 2.7 2.1 1.9
4 mm (n=14)
RT (°C)
- 1.0, --- 0.7, -+ 0.4, ± 0.2, ± 0.2,
TFL (sec),
36.2 38.8 40.8 42.7 43.9
7.6 4.2 3.7 2.6 2.3
2 mm (n=14)
RT (°C)
+_ 1.8, _+ 0.7, ± 0.7, ± 0.5, ± 0.1,
TFL (sec),
37.1 39.9 44.7 45.9 47.9
6.4 4.8 3.3 3.0
1 mm (n=14)
RT (°C)
. --- 1.3, ± 1.4, ± 0.8, ± 1.0,
.
TFL (sec),
.
. 9.5 5.5 4.7 3.3
42.7 46.7 49.2 52.1
RT (°C)
-+ 1,1, ± 2.1, ± 1.5, ± 1.3,
44.1 47.6 52.9 53.6
C 40
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FIG. 2. The monitored and the simulated time-temperature curves in 5 different radiating conditions. Open circles and vertical short bars are the mean values and standard deviations of monitored temperatures at intervals of 0.5 sec. The dotted lines show the simulated time-temperature curves used for the calculation in this study.
FIG. 3. Magnitude of caloric factor over various tentative threshold temperature and their coefficient of variation (S.D./Mean) in 5 different radiating conditions. The small numbers show the supplying voltages for the radiation of lamp. The solid lines are the calculated caloric factor (left ordinate) and the dotted line with solid circles is the coefficient of variation (right ordinate).
creased. These results indicate that the temperature at which a tail flick response occurs is not absolute but depends on the radiating condition. Therefore, another factor responsible for the provocation of tall flick must be considered. Hardy et al. [13,14] previously demonstrated the threshold of pain as a caloric parameter (mcal/sec/cm2). We calculated an approximation of the heat applied to the tall until the provocation of the tall flick response. Figure 2 shows the monitored and the simulated time-temperature curves of the five radiating conditions. The time-temperature curves based on monitoring above the focused spot were simulated with the formula: Y=Ax(1-e-(X-C)~B)+P. The values of A, B and C used in this calculation were as follows: 15.0, 25.5 and 8.0 for 70 V of supplied voltage; 22.0, 24.0 and 7.0 for 80 V; 27.0, 18.0 and 7.0 for 90 V; 34.0, 17.5 and 5.5 for
100 V; 39.0, 17.5 and 5.5 for 110 V, respectively. P was kept at 24.0°C. The correlation coefficients between the paired temperatures (monitored and simulated) at intervals of 0.5 sec for all five of the radiating conditions were high enough to be used for further analysis (r>0.999). The magnitude of the caloric factor calculated from the simulated temperature curve varied with the radiating conditions. When a more rapid increase of radiant heat was applied to the tail, a smaller caloric factor was required to provoke the tail flick response. Figure 3 shows the influence of a tentative threshold temperature on the magnitude of the caloric factor (open circles, solid line, left ordinate) and the variation coefficient (S.D./M) (solid circles and dotted line, right ordinate) for the 5 different radiating conditions. The small numbers indicate
238
K A W A K I T A AND F U N A K O S H I TABLE
3
9-
2.8s
I N F L U E N C E OF RADIATED A R E A ON T H E RCTFs C A L C U L A T E D FROM T H E T A I L F L I C K L A T E N C I E S IN T H E 5 D I F F E R E N T R A D I A T I N G CONDITIONS "
Radiated Size of the Tail (Slit) Voltage (V)
8 mm
4 mm
2 mm
1 mm
110
70 68 67 62 62
150 106 180 120 120
-322 339 243 251
-696 450 528 317
Mean S.D.
65.8 3.6
136.6 29.0
288.8 48.8
497.8 158.3
70 80 90 100
Percentages (%)
100
208
439
757
RCTF × Slit (mm)
800
832
878
757
O
7"
._
6
/o3.2s
36s
' /
5/ /
32 1 0 75
100 Tail
125 150 175 Flick Latency
200
225 (%)
FIG. 4. Relationship between analgesic index and % latency with various baseline latencies. Analgesic index is indicated as the ratio of RCTF (RCTF of test latency/RCTF of baseline latency). The radiating condition in this calculation was 80 V and 8.0 mm slit. The average tail flick latency in this condition was 3.8 sec.
the supply voltages for the lamp. The magnitude of the caloric factor over a tentative threshold temperature decreases almost linearly as the tentative threshold temperature increases. When lower supply voltages were used, the magnitude of the caloric factor decreases more steeply. On the other hand, the variation coefficient (S.D./M) of the calculated caloric factor for the 5 radiating conditions decreases to a minimum value (0.055) at the tentative threshold temperature of P+8.8 (P=24.0°C), and then rapidly increases. The tentative threshold temperature of P+8.8°C monitored on and under the tail skin were 46.3-+1.8 and 37.7-+1.7°C (mean-+S.D., n=5), respectively. Since the magnitude of the caloric factor over a tentative threshold temperature of P+8.8 showed a similar value for the 5 different radiating conditions, it was considered to be the crucial factor in provoking a tail flick response. This factor, the Required Calories for Tail Flick (RCTF) is used for subsequent calculations in this paper. The quantitative relationship between the radiated area and the tail flick latency or the response temperature of tail flick is not apparent in Table 2. The relationship between the RCTF and radiated area was examined. As shown in Table 3, at different radiant heat conditions, the calculated RCTFs show a tendency to increase as the size of slit decreases. The ratios of the mean RCTF of the 5 radiating conditions at 4, 2 and 1 mm slit are 2.08, 4.39 and 7.57, respectively. These results are statistically significant (chi square test, p<0.05). That is, the total magnitudes of caloric factor (RCTF × radiated area (in slit) applied to the tail until the occurrence of tail flick response are very similar for the different radiating conditions. These facts suggest that the RCTF is a useful quantitative index of pain in the tail flick test. Table 4 shows that the latencies clearly increase to 155173% of baseline latency at 20-30 min after the administra-
tion of morphine (2 mg/kg, IP). When the lower voltage was supplied to the lamp, a greater increase in % latency was observed. On the other hand, the ratios of RCTF (RCTF of test latency/RCTF of baseline latency) after the injection of morphine were 3.64-4.25. Each value was similar and independent of the radiating conditions. The variation coefficient in % latency was smaller than that for the ratios of RCTF; however, it indicates only the utility of the expression of % latency. On the other hand, the similar values of the ratios of RCTF indicate not only its utility but also its theoretical efficacy. Therefore, we proposed the ratio of RCTF as a new analgesic index. Relationship between % latency and analgesic index (ratio of RCTF) is shown in Fig. 4. The radiating condition was 80 V with a 8 mm slit; the average latency of normal rats in this radiating condition was 3.8 sec (solid circles), as presented in Tables 1 and 4. The analgesic index increases with the increase of % latency in a gradual manner. A steeper increase of analgesic index is observed with a shorter baseline latency. Similar tendencies are observed in the different radiating conditions. The low supply voltage causes a mild increase of analgesic index while the high voltage induces a steep increase in analgesic index. It is clear that the same value of% latency does not indicate the same analgesic index when the radiating condition and/or baseline latency is different. DISCUSSION
The present study demonstrates that the response temperature of tail flick varies with the size of radiated area and voltage supplied to the radiating lamp. No absolute temperature consistently elicits a tail flick response. These observations suggest that a constant temperature of pain threshold reported in previous investigations [14,21] might depend on
A Q U A N T I T A T I V E E X P R E S S I O N IN T A I L F L I C K TEST
239
TABLE 4 EFFECTS OF MORPHINE ADMINISTRATIONON THE TAIL FLICK LATENCY, RCTF, % LATENCY AND THE ANALGESIC INDEX IN THE DIFFERENT RADIATINGCONDITIONS Baseline
After Morphine
Voltage (V)
TFL (sec),
RCTF
TFL (sec),
RCTF
70 80 90 100 110
6.2 3.9 2.8 2.2 2.0
82 82 76 74 75
10.7 ± 2.8, 6.6 --- 1.3, 4.7 ± 1.0, 3.5 ± 0.4, 3.1 ± 0.4,
323 345 323 280 273
173 169 168 159 155
0.101
0.046
S.D./Mean
-+ 0.5, ± 0.7, ± 0.2, ± 0.2, ± 0.1,
0.050
their method of pain measurement when exposed to constant radiant heat stimulation. Hardy et al. [13] characterized pain threshold by a caloric unit (mcal/sec/cm 2) instead of considering a threshold temperature. In their study, the calories applied to the skin were not estimated but the intensity of radiant heat lamp itself was measured. In contrast, we calculated the approximate caloric factor, which when applied to the tail resulted in a tail flick response. This factor was estimated from the simulated time-temperature curve and tail flick latency. The magnitude of the caloric factor above a tentative threshold temperature (P+8.8°C) is considered the most crucial factor for the provocation of tail flick response. This caloric factor (here referred to as the Required Calories for Tail Flick or RCTF), quantitatively changes with the size of the area heated and it similarly increases after the administration of morphine under the different radiating conditions. However, the physiological basis of the RCTF is not yet understood. F r o m a physiological point of view, the R C T F applied to the skin should be converted into the afferent impulses by heat sensitive receptors. The existence of skin receptors which respond to radiant heat has been reported in rats [19,20], cats [2,5], monkeys [4, 9, 18] and man [12, 26, 27]. The participation of these heat sensitive neurons in the provocation of pain sensation [18, 27, 28] and avoidance responses [9,19] has been strongly suggested. Dubner et al. [9] demonstrated that the total impulse number from A-delta heat receptors during radiant heat stimulation applied to the monkey's face is closely related to the provocation of the avoidance response. On the other hand, the mean impulse rate of C polymodal receptors is shown to be related to pain sensation [18]. These studies indicate the close relation between the afferent impulse volley from the receptor and pain sensation or avoidance responses. The present problem is determining the physiological basis of RCTF. Therefore, we calculated the hypothetical afferent impulse rate and total impulse number elicited by the radiant heat stimulation of the tail skin. The assumptions were as follows. The impulses are elicited in accordance with Steven's power function: N = k x ( T - T o ) n, where N is im-
% Latency
Analgesic Index 3.94 4.20 4.25 3.78 3.64 0.066
pulse number, k is a proportional constant, T is monitored temperature of heat stimulus, To is threshold of the heat sensitive receptor and n is an exponent respectively. The tentative threshold temperature used in the calculation of RCTF is substituted as To. The final impulse rate is obtained as the impulse number as the tail flick response occurred. Total impulse number is calculated as the sum of N in each bin (re,n) from X to L (see Fig. 1). The calculated final impulse rate varies with the radiating condition for the 5 radiating conditions when the value of exponent varied from 0.1 to 2.0 in increments of 0.1. On the other hand, calculated total impulse numbers for the 5 radiating conditions resulted in similar values when the exponent was 1.2; that is, the value of the coefficient of variation was minimum 0.069, with an exponent of 1.2. According to Necker and Hellon [20], there exist heat sensitive receptors in the rat tail skin; their response characteristics are in agreement with the power function, and their exponent is about 1.2 (roughly estimated from their data). The existence of a very similar receptor predicted from a hypothetical calculation based on the R C T F and the tentative threshold temperature indicates that the RCTF and the tentative threshold used in the present study are likely parameters for the expression of tail flick response. The purpose of the present study was to establish a reasonable expression of analgesic effect in the tail flick test. The analgesic index (ratio of RCTF) proposed in this study is shown to be reasonable and useful; however, it is somewhat difficult to use routinely. On the other hand, % latency is proportionally changed by a change in the analgesic index with a limited radiating condition and baseline latency (Fig. 4). Therefore, the present study demonstrates that the expressions of analgesic effect by % latency and analgesic index (ratio of RCTF) are both useful, quantitative expression in the tail flick test. However, it should be noted that the expression of analgesic effects by % latency must not be used when the magnitudes of analgesia were compared between the animals in the different baseline latencies. In these cases, only the analgesic index (ratio of RCTF) could indicate the precise comparison of the analgesic effects.
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