Pain, 46 (1991) 89-95 0 1991 Elsevier Science Publishers ADONIS 030439599100158M
89 B.V. 0304-3959/91/$03.50
PAIN 01807
Stress analgesia: the opioid analgesia of long swims suppresses the non-opioid analgesia induced by short swims in mice Garth Tierney, John Carmody and Dana Jamieson School of Physiology and Pharmacology, Unkersity of New South Wales, Kensington, Sydney (Austmlial (Received
18 June 1990, revision
received
26 November
1990, accepted
17 December
1990)
In mice, room temperature swimming for as short a period as 15 see has been found to induce a Summary non-opioid analgesia with a time course of lo-12 min. As the duration of the swim is increased, an opioid analgesia develops with a longer persistence (25-30 min); the development of the opioid analgesia appears to suppress the expression of the non-opioid analgesia so that none of the latter is evident after 3 min swims. The characteristics of the tail-flick nociceptive test are also described. Key words: Swim stress; Nociception; Tail-flick test; Stress analgesia; Autoanalgesia; analgesia; Non-opioid anaIgesia; (Albino mouse, MUS~~~c~Z~S)
Introduction Over the centuries a great deal of effort has gone into devising methods for suppressing pain and over the last decade or so, concomitant with the growth of knowledge of the endogenous opioids and their receptors, “stress-induced analgesia” has been of enduring interest. A variety of “stressors” has been shown to induce this analgesia [for typical reviews see 15,19,23], though it must be conceded that “stress” is often a term of convenience rather than a strictly defined scientific term. The authenticity of the phenomenon is now undisputed but there has been some controversy over whether it is based on opioid or non-opioid mechanisms. In this laboratory swimming has been the preferred (but not exclusive) stressor, usually in water at 20°C [9]. In such circumstances, a powerful analgesia has been observed in mice which persists for 0.5 h and is whohy opioid in nature; furthermore, the magnitude of the analgesia is a function of swim duration. We have also found that exposure to a 10 min period of intermittent electric footshock produces a purely opi-
Co~~es~~~e~ee fo: Dr. J.J. Carmody, Pharmacology, University of New South Australia.
School of Physiology and Wales, Kensington 2033,
Cross-tolerance;
Opioid
oid analgesia [l]. It has been argued, however, that the duration of the stress is important in determining whether the h~oalgesia is opioid or non-opioid in nature. Lewis et al. have reported [16] that, whereas prolonged periods of exposure to electric footshock induce an opioid-based analgesia, the analgesia which follows shorter periods of exposure is non-opioid in nature. On the other hand, Terman et al. state that shorter periods of electrical stress induce opioid analgesia and the longer periods produce non-opioid analgesia [l&191. A more thorough examination of our previously published results has shown that, although the analgesia increases as swimming is prolonged (up to about 8 min) and a distinct analgesia is present with swims as short as 15 set, the time dependence is apparent only when the swims are longer than 30 sec. It seemed likely, therefore, that with swim stress also, two mechanisms might be at work [4], so experiments were designed to test this idea more thoroughly and to evaluate the basis of the associated analgesia more comprehensively. We have employed the reproducible “tailflick” test of nociception together with the opioid antagonist naloxone and the technique of inducing (or attempting to induce1 morphine cross-tolerance. The special advantage of swimming as a form of stress is particularly -apparent in an experiment of this type: although it cannot be asserted that each animal truly
experiences an identical stress. at least the applicution of the stress can be controlled in terms of its duration and the temperature of the water. Some of these results have previously been communicated in brief form [3.20].
Methods Animal., Naive female albino mice of QS strain, 20-25 g in weight, were used (bred by the Animal Breeding and Holding Unit, University of NSW). They had never previously been used for any experimentation and had ad libitum access to dry food (Allied Mills, Sydney) and water until used for the experiment. As handling can itself be stressful, they were handled to a minimal but uniform extent and because of circadian variations in nociceptive sensitivity [ 111 the experiments were always done at the same time of day. The “stress” was an individual swim in water at room temperature (2022°C) with the details essentially as have been described previously [Y]. All injections were given intraperitoneally in 0.9%’ saline in a volume of 10 ml/kg. Pain measurement Nociception was assessed by a modification of D‘Amour and Smith’s tail-flick method [lO] involving the latency for a flick response when the animal was held comfortably with its tail wholly immersed in hot water (47°C being routinely used). The choice of any nociceptive measure is to some extent arbitrary and a matter of experimental convenience, but this tail-flick test has the advantages that it requires simple and inexpensive apparatus and that it is highly reproducible (see Results). It therefore has the additional statistical advantage that every animal may validly be used as its own control and the ethical advantage that it allows the number of mice to be minimised. This test is also convenient because it can be performed by a single observer while holding a mouse in one hand and a stop-watch in the other. The hot-plate test 1251, on the other hand, requires two observers for greatest accuracy and cannot be used repeatedly on the same animal because the mice rapidly learn and quickly attempt to jump off the hot-plate on subsequent exposures, failing to display the relevant behaviour. Even so, this test was used in one experiment as part of a corroboration of the range of results obtained previously with the hot-plate test, the results of Cooper and Carmody [91 having been obtained entirely with the hot-plate test. Tolerunce studies Animals were made stress tolerant and morphine tolerant. (i) Stress tolerance. This was done in 2 groups, one subjected to 3 min swims (“long swim animals”) and
the other to I5 SW swims (“short swim animals”). On day 1. each mouse was pre-tested and then \WLIIII ;14 described (above). Nociception was then mcasurcd I. 3. 5. 7. 0 and I I min post swim. The animals wcrc subsequently swum once daily. between IO.00 a.m. and 12.00 noon. Nociception was tested (as above) on day 14. the only other day on which it was measured. fii) Morphine cr-o.vs-to1emnc.r. There wcrc 2 cxpcrimental groups in which each animal was trcatcd for 5 days: the mice in the control group received suhcutancous 0.0% NaCl solution once daily while the othci animals were given morphine daily. lnjcctions wcrc given in the morning, subcutaneously (to slow the action of the drug) at a dose of IO mg/kg. On day 7. the groups were subdivided (with 8 animals per sub-group): group I: saline-treated, short swim; group 2: morphinc-treated, short swim; group 3: saline-treated, long swim; group 4: morphine-treated, long swim. Nociception was tested before the swim and then the nociceptive time-course was determined after the swim (as above). Stutistical analysis Unpaired “Student’s” t tests were done to assess the statistical significance of the data, with a I-’ value of 5%’ or less being taken (as is conventional) to indicate a “significant” difference. No pre-determined difference magnitude was set as the criterion for analgesia as has been done by some authors; for instance, Chesher et al. [7] determined what they called a “critical reaction time” which was 3 S.D.s greater than the mean tail-flick latency in the control animals and this had to be exceeded before analgesia was held to exist.
Results C’haracteristics of the tail-flick test A great advantage of the tail-flick test is the reproducibility of the results, as Fig. 1 shows. The latency showed no variation with repeated exposure: all of the results (at 47°C) lie between 9 and 10 set and the co-efficient of variation was always less than 18%. As Fig. 2 shows, this latency was highly dependent upon the water temperature. A temperature of 47°C was chosen and routinely used because it produced a response time which was long enough for the observer’s reaction time to be an insignificant proportion of the overall latency, but not so long that the mouse had to be held for an unduly prolonged time. Opioid and non-opioid analgesia: growth and decuy As reported by Cooper and Carmody [9], following a swim as short as 15 set, a marked analgesia was apparent which increased progressively as swim duration was
91
1”
44
t
”
46
TEMPERATURE
I,, 0
, , , , , , , , 2
4
6
8
c IO
TIME fminsf Fig. 1. Tail-flick latencies with repeated measurements. At a water temperature of 47°C each of 8 mice was repeatedly tested at 2 min intervals over 10 min. The curve plots mean values with the error bars showing s~~n~~~~cirt&ztiorzs in order to demonstrate the ru~rge of the measurements. It can be seen that over these 6 repeated exposures flick latencies remain very constant.
extended up to 5 min. Fig. 3 shows this result: the latency of the tail-flick (measured 1 min post swim) increased by 13.1 set from a control value of 9.5 set, i.e., by 138% after a I5 set swim, and by 47 set (N 500%) after a 3 min swim. If the reciprocal of this latency is taken as an index of pain severity, these results indicate that the short swim reduced nociception by 58% (measured 1 min after the stress) while the 3 min swim reduced nociception by 83% (saline-treated animals). In fact, although it is not shown in Fig. 3, swims as short as 5 set induced a significant analgesia, increasing latency by 58% and reducing the “pain index” by 37%. The short-swim (15 set) analgesia was marked, too, when the hot-plate test was used: foot-flick latency increased from 10.5 see (n = 10; S.E,M. = 0.8) to 22.1 see (S.E.M. = 1.51, i.e., pain index was reduced by 52%. In animals which were pre-treated with naloxone at a dosage considered sufficient to block endogenous opioids (5 mg/kg, determined from the knowledge [4] that 100 pg/kg is sufficient to block 15 mg/kg morphine in mice, and extrapolating from the data of Lord et al. f17]), analgesia after the 15 set swim was identical to that in the saline-treated control animals (latency increase: 12 set; pain index reduced by 65%). Fig. 3 alsa shows that as the duration of the swim was ex-
’ “I’ 50
48
52
”
54
f=C)
Fig. 2. Tait-flick latency as a function of temperature of the test water. There were 11 separate groups of animals in these experiments, with 15 mice per group. Mean latency values and standard errors of the means are plotted. Response latency is inversely related to the stimulus magnjtude in a complex fashion.
tended, this non-opioid analgesia became less and less significant, so that after 3 min swims it was non-existent (confirming the report [9] that swims of this duration induce a purely opioid analgesia). The anaIysis has been extended in Fig. 3 to determine the growth curve of this opioid analgesia by subtracting the latency changes of the naloxone-treated mice from the latency values obtained with control mice swum for the 6 durations depicted in Fig. 3. It can be seen that there was a progressive growth in latency (from there being rro opioid analgesia at 15 set> as the swims were extended to 5 min duration. Naloxone was also without influence on the short-swim analgesia when the hotplate test was used (data not shown). The magnitude of this non-opioid analgesia (following 15 set swims) showed a distinct temperature dependence quite different from the pattern of the opioid analgesia induced by 3 min swims [93. As water temperature was reduced in those experiments [9], opioid analgesia increased in magnitude only very gradually until the vicinity of 2O”C, whereupon the analgesia increased steeply. The norm-opioid analgesia, on the other hand, increased uniformly and briskly with decline in water temperature (Fig. 4) and, further, as water temperature was increased, the analgesia increased from a minimum at approximately 35°C which is close to Herrington’s [12] thermoneutral temperature in these animals (31.5”C). Time-course of he nomopioid analgesia Two groups of mice were swum for 15 set and serial measurements made of the tail-flick latency fsee Meth-
SWIM TIME Pig. 3. Effect post-swim marked
of swim duration
tail-flick increase
lengthened. when
latencies. in response
This
plot
the experiments
non-opioid between
and character show the results
latency
I
delineates were
analgesia.
on the magnitude The open squares (measured
total
done
analgesia,
these two sets of values. swims. The error
analgesia.
with
10 saline
The vertical
the end of the swim) and the intensity the opioid
group
IO animals
indicate
bar shows the S.E.M.
of
declines
and the non-opioid which
were
with prolongation
the time course
ads). One group had been pre-treated with saline and the other with naloxone (5 mg/kg). The time-course of the decline in the magnitude of the analgesia (Fig. 5)
animals:
with
The
opioid
analgesia
increased
control
show
the latency
i.e.. delineating
are the arithmetic
increases
corrected
and
is seen a
as the swimc were
squares
(5 mg/kg),
squares
which
from the ANOVAR
between
a IS XC swim there
hatched
naloxone
of the swims. The fjlled
calculated
with
of the analgesia
components.
pre-treated
of the putatively
of a single observation
axis ir the difference
pre-treated
min after
of the analgesia
i.e.. they
obtained
i.e., both
in a different
This component
of the evoked
(min)
with
the
differences
lengthening
of the
sum-of-squares.
was identical in the two groups and, as can be seen from the figure, this analgesia persisted for 10-12 min. The naloxone pre-treatment itself induced some hyperalgesia, increasing the pain index by 28%. as has been reported previously with the hot-plate test [4,13].
25 ,
-B-
NALOXONE SALINE
-) 8-
o!
.
I
I
10
0
SWIM Fig. 4. lntluence
on analgesia
which
the mice
made
on X separate
measured
results
represent
indicated ohtained
swum groups
immediately
on the graph Iatcnciea
were
prior
for
temperature
15 sec. These
of X-10
mice
mean values
the swum animals
plot is from unswum
Nociception with
w,hile the lower
control
animal\.
shows
*
I 4
’
1 6
Time-course
were was
the
(horizontal)
.
/ 8
TIME
in
the measured plot
I 2
0
a IS set swim. Points upper
om
(“C)
measurements
and S.E.M.s axis. The
50
of the water
each.
to and I min after
on the vertical
from
TEMPERATURE
of the
1
1
40
30
20
The vertical increasing from
of the short
axis shows increased intervals
after
10 saline-treated
the filled
squares depict
the swim.
animals
’
I 1:
.
1 14
(min)
swim-induced latency
I IO
non-opioid
of the tail-flick
i.e., increased
are shown
the results obtained
by open with
analgesia. measured
at
analgesia.
Results
squares
whereas
10 naloxone-treated
mice (5 mg/kg. i.p., 15 min prior to the swim). The half-life naloxone is approximately 30 min in mice.
of i.p.
93
Cross-tolerance of swim-stress analgesia
22 -
It has been reported [7] that the stress of a 3 min swim shows a partial cross-tolerance with morphine, which is a further indication of an opioid component. We have used this experimental approach in the investigation of the short-swim, putatively non-opioid analgesia. Fig. 6A shows that a distinct tolerance developed to the effects of the long swim. The animals which had been subjected to a daily 3 min swim over a fortnight had substantially shorter tail-flick latencies compared with the non-tolerant animals, i.e., they had an attenuated analgesic response. One minute after the swim
A 20 -
18 -
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PRE POST
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B 20 -
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SALINE
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-
SALINE
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- TREATED -TREATED
25 -
20 -
15 -
Wn)
Fig. 7. Lack of tolerance or cross-tolerance with short swims. A: the curves show the magnitude and the time-course of the analgesia induced by short swims, before and after a 5 day period of conditioning during which (see Methods) each of 8 animals was subjected to a daily swim of I5 sec. The filled symbols indicate the results obtained prior to the conditioning regime while the open symbols show the results obtained after the 5 days of swimming; the curves are essentially identical, indicating that no tolerance has developed. B: likewise, there is no difference in the analgesic responses in the mice subjected to the morphine or saline conditioning (see Methods), indicating, from the lack of any cross-tolerance, that there is no opioid component in the analgesia induced by the short swims.
10 -
5-
o+ 0
.
, 2
.
,
.
4
, 6
.
, 8
.
, 10
,
, 12
TIME (min) Fig. 6. Development of tolerance to the effects of long swims and cross-tolerance with morphine. A: this panel shows the time-course of the nociceptive responses in 8 mice before the period of conditioning (see Methods) and in the same animals at the end of the conditioning programme using long (3 mitt) swims. There is a clear disparity in the results, indicating an attenuation of the swim-induced analgesia; in other words, tolerance has developed to this stressor. B: this panel shows that, likewise, the 2 week period of morphine treatment induces a clear tolerance to the analgesic effect of these long swims. Such cross-tolerance indicates that this analgesia is opioid-based.
the latency in the naive animals was 40.7 set (control value: 9.1 set; Fig. 6A), whereas in the “tolerant” animals the 1 min latency was 25.5 set (control value: 9.6 set). In terms of the “pain index” this result shows that the naive animals had their pain sensitivity reduced to 23% of control values, whereas the reduction was only to 38% in the “tolerant” animals. The latency values are different to a highly significant degree over the first 10 min of this time-course. Part B of Fig. 6 demonstrates that these animals also showed a striking cross-tolerance between the long swim and morphine. One minute post swim the naive animals had a tail-flick latency of 39.9 set whereas the latency was 28 set in the morphine-tolerant animals.
Fig. 7 shows completely contrasted results with the stress of the 15 set swims. In Part A it is clear that a 14 day programme of daily short swims induces IIO tolcrante at aII and, further, thcrc was absolutely no crosstolerance demonstrable in the rnorphinc-trcatcd~/ tolerant mice (Fig. 7H).
Discussion The plethora of stress “recipes” which have been published as a means of evoking autoanalgesia can seem, at times, like alchemy. This paper, while adding to that variety of techniques. confirms that there are at least two systems involved in this analgesia and examines some of their functional characteristics. One of them has a crucial site of opiate action (whatever other neurotransmitters or modulators might he involved) while the second has no opioid involvement at all. On the other hand. our results are in disagreement with some previous investigators about the severity and extent of the stress that is required to activate these two systems rrtzll about the time-course of their efficacy. We have used two criteria to distinguish between these two autoanalgesic systems. We have argued. first, that if the opiate antagonist naloxone abolishes part or all of this analgesia then such a result indicates an opioid basis and second. that if tolerance develops which parallels a tolerance to morphine then that result, too. indicates an opioid basis. While our previous work has shown that a dose of naloxone as low as 100 pg/kg (i.p.) is sufficient to block the analgesia evoked by a 3 min swim in water at 20°C [24], the higher dose of 5 mg/kg was chosen to ensure that opioid receptors of lower affinity for naloxone were also unequivocally blocked: Lord et al. [ 171 show a differential affinity up to 12-fold, so a SO-fold incrcment in dosage should be sufficient to block p and K receptors without inducing naloxone agonist action [14]. The fact that no tolerance develops to the 15 set swim and the results of the morphine cross-tolerance experimcnts reinforce this view since morphine acts at ;I range of opioid receptors [21,22]. Even if these two experimental procedures do not absolutely exclude opioid involvement, they do indicate that there are two distinct forms of analgesia and that their neurochemical bases are quite different. They also indicate that activation of these systems follows quite different time-courses. Previous work [Fig. 1 in ref. 91 showed that the opioid analgesia resulting from the 3 min swim stress persisted for 25-30 min whereas the current experiments show that the non-opioid analgesia from the IS set swimming endures for only 10-12 min (Fig. 5). These results arc in agreement with those of Lewis ct al. [16] who reported that increasing the stress
;ictivatcs 1hc opioid system. ‘I‘hcy used clc%ctric f0(1l
89 B.V. 0304-3959/91/$03.50
PAIN 01807
Stress analgesia: the opioid analgesia of long swims suppresses the non-opioid analgesia induced by short swims in mice Garth Tierney, John Carmody and Dana Jamieson School of Physiology and Pharmacology, Unkersity of New South Wales, Kensington, Sydney (Austmlial (Received
18 June 1990, revision
received
26 November
1990, accepted
17 December
1990)
In mice, room temperature swimming for as short a period as 15 see has been found to induce a Summary non-opioid analgesia with a time course of lo-12 min. As the duration of the swim is increased, an opioid analgesia develops with a longer persistence (25-30 min); the development of the opioid analgesia appears to suppress the expression of the non-opioid analgesia so that none of the latter is evident after 3 min swims. The characteristics of the tail-flick nociceptive test are also described. Key words: Swim stress; Nociception; Tail-flick test; Stress analgesia; Autoanalgesia; analgesia; Non-opioid anaIgesia; (Albino mouse, MUS~~~c~Z~S)
Introduction Over the centuries a great deal of effort has gone into devising methods for suppressing pain and over the last decade or so, concomitant with the growth of knowledge of the endogenous opioids and their receptors, “stress-induced analgesia” has been of enduring interest. A variety of “stressors” has been shown to induce this analgesia [for typical reviews see 15,19,23], though it must be conceded that “stress” is often a term of convenience rather than a strictly defined scientific term. The authenticity of the phenomenon is now undisputed but there has been some controversy over whether it is based on opioid or non-opioid mechanisms. In this laboratory swimming has been the preferred (but not exclusive) stressor, usually in water at 20°C [9]. In such circumstances, a powerful analgesia has been observed in mice which persists for 0.5 h and is whohy opioid in nature; furthermore, the magnitude of the analgesia is a function of swim duration. We have also found that exposure to a 10 min period of intermittent electric footshock produces a purely opi-
Co~~es~~~e~ee fo: Dr. J.J. Carmody, Pharmacology, University of New South Australia.
School of Physiology and Wales, Kensington 2033,
Cross-tolerance;
Opioid
oid analgesia [l]. It has been argued, however, that the duration of the stress is important in determining whether the h~oalgesia is opioid or non-opioid in nature. Lewis et al. have reported [16] that, whereas prolonged periods of exposure to electric footshock induce an opioid-based analgesia, the analgesia which follows shorter periods of exposure is non-opioid in nature. On the other hand, Terman et al. state that shorter periods of electrical stress induce opioid analgesia and the longer periods produce non-opioid analgesia [l&191. A more thorough examination of our previously published results has shown that, although the analgesia increases as swimming is prolonged (up to about 8 min) and a distinct analgesia is present with swims as short as 15 set, the time dependence is apparent only when the swims are longer than 30 sec. It seemed likely, therefore, that with swim stress also, two mechanisms might be at work [4], so experiments were designed to test this idea more thoroughly and to evaluate the basis of the associated analgesia more comprehensively. We have employed the reproducible “tailflick” test of nociception together with the opioid antagonist naloxone and the technique of inducing (or attempting to induce1 morphine cross-tolerance. The special advantage of swimming as a form of stress is particularly -apparent in an experiment of this type: although it cannot be asserted that each animal truly
experiences an identical stress. at least the applicution of the stress can be controlled in terms of its duration and the temperature of the water. Some of these results have previously been communicated in brief form [3.20].
Methods Animal., Naive female albino mice of QS strain, 20-25 g in weight, were used (bred by the Animal Breeding and Holding Unit, University of NSW). They had never previously been used for any experimentation and had ad libitum access to dry food (Allied Mills, Sydney) and water until used for the experiment. As handling can itself be stressful, they were handled to a minimal but uniform extent and because of circadian variations in nociceptive sensitivity [ 111 the experiments were always done at the same time of day. The “stress” was an individual swim in water at room temperature (2022°C) with the details essentially as have been described previously [Y]. All injections were given intraperitoneally in 0.9%’ saline in a volume of 10 ml/kg. Pain measurement Nociception was assessed by a modification of D‘Amour and Smith’s tail-flick method [lO] involving the latency for a flick response when the animal was held comfortably with its tail wholly immersed in hot water (47°C being routinely used). The choice of any nociceptive measure is to some extent arbitrary and a matter of experimental convenience, but this tail-flick test has the advantages that it requires simple and inexpensive apparatus and that it is highly reproducible (see Results). It therefore has the additional statistical advantage that every animal may validly be used as its own control and the ethical advantage that it allows the number of mice to be minimised. This test is also convenient because it can be performed by a single observer while holding a mouse in one hand and a stop-watch in the other. The hot-plate test 1251, on the other hand, requires two observers for greatest accuracy and cannot be used repeatedly on the same animal because the mice rapidly learn and quickly attempt to jump off the hot-plate on subsequent exposures, failing to display the relevant behaviour. Even so, this test was used in one experiment as part of a corroboration of the range of results obtained previously with the hot-plate test, the results of Cooper and Carmody [91 having been obtained entirely with the hot-plate test. Tolerunce studies Animals were made stress tolerant and morphine tolerant. (i) Stress tolerance. This was done in 2 groups, one subjected to 3 min swims (“long swim animals”) and
the other to I5 SW swims (“short swim animals”). On day 1. each mouse was pre-tested and then \WLIIII ;14 described (above). Nociception was then mcasurcd I. 3. 5. 7. 0 and I I min post swim. The animals wcrc subsequently swum once daily. between IO.00 a.m. and 12.00 noon. Nociception was tested (as above) on day 14. the only other day on which it was measured. fii) Morphine cr-o.vs-to1emnc.r. There wcrc 2 cxpcrimental groups in which each animal was trcatcd for 5 days: the mice in the control group received suhcutancous 0.0% NaCl solution once daily while the othci animals were given morphine daily. lnjcctions wcrc given in the morning, subcutaneously (to slow the action of the drug) at a dose of IO mg/kg. On day 7. the groups were subdivided (with 8 animals per sub-group): group I: saline-treated, short swim; group 2: morphinc-treated, short swim; group 3: saline-treated, long swim; group 4: morphine-treated, long swim. Nociception was tested before the swim and then the nociceptive time-course was determined after the swim (as above). Stutistical analysis Unpaired “Student’s” t tests were done to assess the statistical significance of the data, with a I-’ value of 5%’ or less being taken (as is conventional) to indicate a “significant” difference. No pre-determined difference magnitude was set as the criterion for analgesia as has been done by some authors; for instance, Chesher et al. [7] determined what they called a “critical reaction time” which was 3 S.D.s greater than the mean tail-flick latency in the control animals and this had to be exceeded before analgesia was held to exist.
Results C’haracteristics of the tail-flick test A great advantage of the tail-flick test is the reproducibility of the results, as Fig. 1 shows. The latency showed no variation with repeated exposure: all of the results (at 47°C) lie between 9 and 10 set and the co-efficient of variation was always less than 18%. As Fig. 2 shows, this latency was highly dependent upon the water temperature. A temperature of 47°C was chosen and routinely used because it produced a response time which was long enough for the observer’s reaction time to be an insignificant proportion of the overall latency, but not so long that the mouse had to be held for an unduly prolonged time. Opioid and non-opioid analgesia: growth and decuy As reported by Cooper and Carmody [9], following a swim as short as 15 set, a marked analgesia was apparent which increased progressively as swim duration was
91
1”
44
t
”
46
TEMPERATURE
I,, 0
, , , , , , , , 2
4
6
8
c IO
TIME fminsf Fig. 1. Tail-flick latencies with repeated measurements. At a water temperature of 47°C each of 8 mice was repeatedly tested at 2 min intervals over 10 min. The curve plots mean values with the error bars showing s~~n~~~~cirt&ztiorzs in order to demonstrate the ru~rge of the measurements. It can be seen that over these 6 repeated exposures flick latencies remain very constant.
extended up to 5 min. Fig. 3 shows this result: the latency of the tail-flick (measured 1 min post swim) increased by 13.1 set from a control value of 9.5 set, i.e., by 138% after a I5 set swim, and by 47 set (N 500%) after a 3 min swim. If the reciprocal of this latency is taken as an index of pain severity, these results indicate that the short swim reduced nociception by 58% (measured 1 min after the stress) while the 3 min swim reduced nociception by 83% (saline-treated animals). In fact, although it is not shown in Fig. 3, swims as short as 5 set induced a significant analgesia, increasing latency by 58% and reducing the “pain index” by 37%. The short-swim (15 set) analgesia was marked, too, when the hot-plate test was used: foot-flick latency increased from 10.5 see (n = 10; S.E,M. = 0.8) to 22.1 see (S.E.M. = 1.51, i.e., pain index was reduced by 52%. In animals which were pre-treated with naloxone at a dosage considered sufficient to block endogenous opioids (5 mg/kg, determined from the knowledge [4] that 100 pg/kg is sufficient to block 15 mg/kg morphine in mice, and extrapolating from the data of Lord et al. f17]), analgesia after the 15 set swim was identical to that in the saline-treated control animals (latency increase: 12 set; pain index reduced by 65%). Fig. 3 alsa shows that as the duration of the swim was ex-
’ “I’ 50
48
52
”
54
f=C)
Fig. 2. Tait-flick latency as a function of temperature of the test water. There were 11 separate groups of animals in these experiments, with 15 mice per group. Mean latency values and standard errors of the means are plotted. Response latency is inversely related to the stimulus magnjtude in a complex fashion.
tended, this non-opioid analgesia became less and less significant, so that after 3 min swims it was non-existent (confirming the report [9] that swims of this duration induce a purely opioid analgesia). The anaIysis has been extended in Fig. 3 to determine the growth curve of this opioid analgesia by subtracting the latency changes of the naloxone-treated mice from the latency values obtained with control mice swum for the 6 durations depicted in Fig. 3. It can be seen that there was a progressive growth in latency (from there being rro opioid analgesia at 15 set> as the swims were extended to 5 min duration. Naloxone was also without influence on the short-swim analgesia when the hotplate test was used (data not shown). The magnitude of this non-opioid analgesia (following 15 set swims) showed a distinct temperature dependence quite different from the pattern of the opioid analgesia induced by 3 min swims [93. As water temperature was reduced in those experiments [9], opioid analgesia increased in magnitude only very gradually until the vicinity of 2O”C, whereupon the analgesia increased steeply. The norm-opioid analgesia, on the other hand, increased uniformly and briskly with decline in water temperature (Fig. 4) and, further, as water temperature was increased, the analgesia increased from a minimum at approximately 35°C which is close to Herrington’s [12] thermoneutral temperature in these animals (31.5”C). Time-course of he nomopioid analgesia Two groups of mice were swum for 15 set and serial measurements made of the tail-flick latency fsee Meth-
SWIM TIME Pig. 3. Effect post-swim marked
of swim duration
tail-flick increase
lengthened. when
latencies. in response
This
plot
the experiments
non-opioid between
and character show the results
latency
I
delineates were
analgesia.
on the magnitude The open squares (measured
total
done
analgesia,
these two sets of values. swims. The error
analgesia.
with
10 saline
The vertical
the end of the swim) and the intensity the opioid
group
IO animals
indicate
bar shows the S.E.M.
of
declines
and the non-opioid which
were
with prolongation
the time course
ads). One group had been pre-treated with saline and the other with naloxone (5 mg/kg). The time-course of the decline in the magnitude of the analgesia (Fig. 5)
animals:
with
The
opioid
analgesia
increased
control
show
the latency
i.e.. delineating
are the arithmetic
increases
corrected
and
is seen a
as the swimc were
squares
(5 mg/kg),
squares
which
from the ANOVAR
between
a IS XC swim there
hatched
naloxone
of the swims. The fjlled
calculated
with
of the analgesia
components.
pre-treated
of the putatively
of a single observation
axis ir the difference
pre-treated
min after
of the analgesia
i.e.. they
obtained
i.e., both
in a different
This component
of the evoked
(min)
with
the
differences
lengthening
of the
sum-of-squares.
was identical in the two groups and, as can be seen from the figure, this analgesia persisted for 10-12 min. The naloxone pre-treatment itself induced some hyperalgesia, increasing the pain index by 28%. as has been reported previously with the hot-plate test [4,13].
25 ,
-B-
NALOXONE SALINE
-) 8-
o!
.
I
I
10
0
SWIM Fig. 4. lntluence
on analgesia
which
the mice
made
on X separate
measured
results
represent
indicated ohtained
swum groups
immediately
on the graph Iatcnciea
were
prior
for
temperature
15 sec. These
of X-10
mice
mean values
the swum animals
plot is from unswum
Nociception with
w,hile the lower
control
animal\.
shows
*
I 4
’
1 6
Time-course
were was
the
(horizontal)
.
/ 8
TIME
in
the measured plot
I 2
0
a IS set swim. Points upper
om
(“C)
measurements
and S.E.M.s axis. The
50
of the water
each.
to and I min after
on the vertical
from
TEMPERATURE
of the
1
1
40
30
20
The vertical increasing from
of the short
axis shows increased intervals
after
10 saline-treated
the filled
squares depict
the swim.
animals
’
I 1:
.
1 14
(min)
swim-induced latency
I IO
non-opioid
of the tail-flick
i.e., increased
are shown
the results obtained
by open with
analgesia. measured
at
analgesia.
Results
squares
whereas
10 naloxone-treated
mice (5 mg/kg. i.p., 15 min prior to the swim). The half-life naloxone is approximately 30 min in mice.
of i.p.
93
Cross-tolerance of swim-stress analgesia
22 -
It has been reported [7] that the stress of a 3 min swim shows a partial cross-tolerance with morphine, which is a further indication of an opioid component. We have used this experimental approach in the investigation of the short-swim, putatively non-opioid analgesia. Fig. 6A shows that a distinct tolerance developed to the effects of the long swim. The animals which had been subjected to a daily 3 min swim over a fortnight had substantially shorter tail-flick latencies compared with the non-tolerant animals, i.e., they had an attenuated analgesic response. One minute after the swim
A 20 -
18 -
16 -
PRE POST
14 -
12 -
lo-
8: 0
!
d
I
*
I
2
-
4 TIME
45 -
9
I.
I.
6
8
I
1
-
10
12
(min)
A 22 -
40 -
B 20 -
35 -
30 -
*
0 FRE POST
18 -
SALINE
TREATED
tvQ3RPHINE -TREATED
16 25 14 20 12 1510 1 5
10 -
c!
.
,
,
,
,
2
4
6
8
TIME
.
,
,
10
12
0:.
i
0
, 2
.
, 4
,
.
6
I.,
8
.
10
(
12
(min) TIME
40 -!
B
35 -
30 -
.
T
-
SALINE
+
WHINE
- TREATED -TREATED
25 -
20 -
15 -
Wn)
Fig. 7. Lack of tolerance or cross-tolerance with short swims. A: the curves show the magnitude and the time-course of the analgesia induced by short swims, before and after a 5 day period of conditioning during which (see Methods) each of 8 animals was subjected to a daily swim of I5 sec. The filled symbols indicate the results obtained prior to the conditioning regime while the open symbols show the results obtained after the 5 days of swimming; the curves are essentially identical, indicating that no tolerance has developed. B: likewise, there is no difference in the analgesic responses in the mice subjected to the morphine or saline conditioning (see Methods), indicating, from the lack of any cross-tolerance, that there is no opioid component in the analgesia induced by the short swims.
10 -
5-
o+ 0
.
, 2
.
,
.
4
, 6
.
, 8
.
, 10
,
, 12
TIME (min) Fig. 6. Development of tolerance to the effects of long swims and cross-tolerance with morphine. A: this panel shows the time-course of the nociceptive responses in 8 mice before the period of conditioning (see Methods) and in the same animals at the end of the conditioning programme using long (3 mitt) swims. There is a clear disparity in the results, indicating an attenuation of the swim-induced analgesia; in other words, tolerance has developed to this stressor. B: this panel shows that, likewise, the 2 week period of morphine treatment induces a clear tolerance to the analgesic effect of these long swims. Such cross-tolerance indicates that this analgesia is opioid-based.
the latency in the naive animals was 40.7 set (control value: 9.1 set; Fig. 6A), whereas in the “tolerant” animals the 1 min latency was 25.5 set (control value: 9.6 set). In terms of the “pain index” this result shows that the naive animals had their pain sensitivity reduced to 23% of control values, whereas the reduction was only to 38% in the “tolerant” animals. The latency values are different to a highly significant degree over the first 10 min of this time-course. Part B of Fig. 6 demonstrates that these animals also showed a striking cross-tolerance between the long swim and morphine. One minute post swim the naive animals had a tail-flick latency of 39.9 set whereas the latency was 28 set in the morphine-tolerant animals.
Fig. 7 shows completely contrasted results with the stress of the 15 set swims. In Part A it is clear that a 14 day programme of daily short swims induces IIO tolcrante at aII and, further, thcrc was absolutely no crosstolerance demonstrable in the rnorphinc-trcatcd~/ tolerant mice (Fig. 7H).
Discussion The plethora of stress “recipes” which have been published as a means of evoking autoanalgesia can seem, at times, like alchemy. This paper, while adding to that variety of techniques. confirms that there are at least two systems involved in this analgesia and examines some of their functional characteristics. One of them has a crucial site of opiate action (whatever other neurotransmitters or modulators might he involved) while the second has no opioid involvement at all. On the other hand. our results are in disagreement with some previous investigators about the severity and extent of the stress that is required to activate these two systems rrtzll about the time-course of their efficacy. We have used two criteria to distinguish between these two autoanalgesic systems. We have argued. first, that if the opiate antagonist naloxone abolishes part or all of this analgesia then such a result indicates an opioid basis and second. that if tolerance develops which parallels a tolerance to morphine then that result, too. indicates an opioid basis. While our previous work has shown that a dose of naloxone as low as 100 pg/kg (i.p.) is sufficient to block the analgesia evoked by a 3 min swim in water at 20°C [24], the higher dose of 5 mg/kg was chosen to ensure that opioid receptors of lower affinity for naloxone were also unequivocally blocked: Lord et al. [ 171 show a differential affinity up to 12-fold, so a SO-fold incrcment in dosage should be sufficient to block p and K receptors without inducing naloxone agonist action [14]. The fact that no tolerance develops to the 15 set swim and the results of the morphine cross-tolerance experimcnts reinforce this view since morphine acts at ;I range of opioid receptors [21,22]. Even if these two experimental procedures do not absolutely exclude opioid involvement, they do indicate that there are two distinct forms of analgesia and that their neurochemical bases are quite different. They also indicate that activation of these systems follows quite different time-courses. Previous work [Fig. 1 in ref. 91 showed that the opioid analgesia resulting from the 3 min swim stress persisted for 25-30 min whereas the current experiments show that the non-opioid analgesia from the IS set swimming endures for only 10-12 min (Fig. 5). These results arc in agreement with those of Lewis ct al. [16] who reported that increasing the stress
;ictivatcs 1hc opioid system. ‘I‘hcy used clc%ctric f0(1l
