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The heterotopic effects of visceral pain: Behavioural and electrophysiological approaches in the rat
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Pain, 20 (1984) 261-271 Elsevier
PA1 00695
The Heterotopic Effects of Visceral Pain: Behavioural and Electrophysiological Approaches in the Rat
B. Calvin0 ‘, L. Villanueva
and D. Le Bars
Unitk de Recherches de Neurophysiologie Pharmacologique de I’INSERM (U. 161), 2 rue d’Albia, 75014 Paris (France) (Received 6 April 1984, accepted 8 June 1984)
Summary
The heterotopic effects of peritoneo-visceral pain were investigated in behavioural and electrophysiological experiments performed on rats. The intraperitoneal administration of acetic acid (i.p. AA), an algesic agent commonly used to induce writhing behaviour in rodents, was used as a conditioning stimulus in two parallel series of experiments involving 3 behavioural tests and recordings of dorsal horn convergent neurones. The responses to a nociceptive stimulus applied to the tail or paws were lowered by i.p. AA but these effects depended on the behavioural test used: in the tail-flick test, AA produced a transient low magnitude increase in latencies; the threshold for vocalization induced by electrical stimulation of the tail was clearly (25%) and sustainedly (full recovery taking up to 1 h) increased; the jump latency in hot plate was markedly increased (100% at 15 min). Intraperitoneal AA strongly depressed the C fibre evoked responses of coccygeal convergent neurones to suprathreshold transcutaneous electrical stimulation applied on their tail excitatory receptive fields. The time course of these inhibitory effects roughly paralleled the behavioural hypoalgesic effect observed in the vocalization test. These results are discussed with reference to diffuse noxious inhibitory controls (DNIC). Analogies with counter-irritation phenomena are emphasized.
’ Correspondence
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to B. Calvino (same address). Maitre-Assistant
0 1984 Elsevier Science Publishers B.V.
UniversitC Paris-Nord.
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Introduction Convergent neurones are those cells of the dorsal horn which receive inputs from both low threshold afferents and nociceptors and are probably essential in the integrative mechanisms which process nociceptive information at the spinal level. Previous studies [19] have shown that a wide variety of noxious stimuli applied on widespread areas of the body by mechanical, thermal or chemical agents, induce specific and powerful depressions of the activities of this type of neurone. To be effective, conditioning stimuli must be applied on an area distant to the receptive field of the cell under study and this phenomenon, which has been called diffuse noxious inhibitory controls (DNIC), is characterized by its potency and the presence of long lasting post-effects. On the basis of DNIC we have proposed [20] that pain is associated with a double processing on convergent neurones: an activation of a segmental pool, surrounded by the inhibition of the remaining population. However our previous studies envisaged ‘epicritic’ stimuli and, therefore, it was essential to investigate the way in which such systems respond to situations more relevant to clinical pain; the first aim of the present work was to study the effect of a long lasting visceral nociceptive stimulus upon the responsiveness of convergent neurones with receptive fields located in areas remote from viscera (e.g., on the tail). The second aim was an attempt to correlate electrophysiological and behavioural investigations of this phenomenon. We have reported [17,18] that a noxious peritoneo-visceral stimulus (i.p. injection of phenylbenzoquinone (PBQ)) is able to produce a significant increase in the threshold for vocalization provoked by transcutaneous electrical stimulation of the tail, and this effect was interpreted as ‘a behavioural model of DNIC.’ In the present paper the effects of an analogous conditioning stimulus, i.p. acetic acid, has been tested using both behavioural (i.e., classical tail-flick, hot plate and vocalization tests) and electrophysiological models. In the latter case, the activity of convergent neurones in the dorsal horn was produced by transcutaneous electrical stimulation of their excitatory receptive fields located on the tail; this part of the body was chosen not only because of its distance from viscera but also to provide an element of comparison with two of the behavioural tests we used. Acetic acid was chosen as the conditioning stimulus because it is classically used for the screening of putative analgesic drugs (writhing test) and, in this case, writhing responses are less variable than in the equivalent PBQ test, particularly in terms of seasonal changes ]321.
Methods One hundred and sixty-one male Sprague-Dawley rats weighing 250-300 g were used. They were allowed to habituate to the laboratory for at least 96 h before testing. They were housed 5 per cage under diurnal lighting conditions with light on from 08.00 to 20.00 h and were given food and water ad libitum.
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(I) Behavioural experiments Experiments were carried out in a quiet and indirectly lit room, and were performed between 09.00 and 13.00 h. Tail-flick test [S]. A needle fixed on a length of plastic tubing was inserted intraperitoneally and held in place with elastoplast, thus allowing subsequent injection to be made without disturbing the animals. Thereafter, the rat was kept for 30 min before the beginning of the experiment in an individual plexiglass restraining chamber with a hole to allow access to the tail. Tail-flick latency was measured following exposure of the blackened tip of the tail to radiant heat from a 150 W lamp condensed with a convergent lens to a 1.5 mm diameter spot. Baseline latencies were in around 2.5 set and the automatic cut-off time was 10 sec. A baseline was established over at least 25 min (5 consecutive trials at 5 min intervals) before the injection; results were expressed as percentages with reference to the last three of these control tail-flick latencies. Vocalization test [2/. The rat was kept in an individual cylindric plexiglass cage (20 cm diameter) with a hole at the base to allow the access of wires to two needles previously inserted subcutaneously on either side of the tail. Following a 30 min resting period, tests were performed every 5 min throughout the experiment. To produce vocalization, electric shocks were applied to the tail, through the two needles and these consisted of 25 monophasic pulses (2 msec width, 50 Hz, 500 msec duration). The current was increased in successive 0.2 mA steps until the threshold for vocalization was reached. Three successive control thresholds were used as the baseline reference before i.p. injection of acetic acid or vehicle. Results were expressed as percentages with reference to this baseline. Hot plate test [8]. The rat was placed in a plexiglass cage (20 cm diameter) 30 min before and 15 or 30 min after i.p. injection of acetic acid or vehicle. For testing, the rat was placed on the hot plate and hind-paw-lick and jump latencies were measured with a 45 set cut-off time. The device consisted of a metal surface (25 cm x 25 cm) maintained at 60 f 0.5 ‘C. A glass cylinder (16 cm diameter: 30 cm high) also maintained at 60 f l°C, was placed on the metal surface. In order to avoid sensitization or desensitization phenomena induced by the stimulus, only one test was performed for each rat; the test was applied 15 or 30 min after the i.p. injection. Results were therefore expressed as latencies. (2) Electrophysiological experimetns The methods used were essentially those described in detail previously [19] and so only a brief account will be given here. Rats were deeply anaesthetized with halothane (2%) in a nitrous oxide/oxygen mixture (2/3, l/3) and, following tracheal and jugular cannulation, a laminectomy was performed, the spinal cord exposed between vertebrae L3 and L4 - corresponding to S4-Co3 segments [33] - and the area of cord giving the maximal dorsum potential to electrical stimulation of the tail was determined; this area was found at Co2 level. Anaesthesia was then lowered to 0.5% halothane and the animal was immobilized with gallamine triethiotide. This procedure appeared to achieve an adequate level of anaesthesia from ethical consideration whilst not excessively
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depressing neuronal responses to noxious stimuli [I]. Extracellular unitary recordings were made with glass micropipettes filled with pontamine sky blue and NaCl, and the cord was stabilized with agar. Convergent neurones were characterized by their responses to innocuous (touch, pressure . . .) and noxious (heating, strong pinch) stimuli applied to their peripheral receptive fields located on the tail. Suitable units were then activated by transcutaneous electrical stimulation (2 msec pulses, 0.6 Hz) of their receptive fields, which produced clear C fibre evoked responses as gauged by latency measurements. Tests were made using currents which were 2-5-fold threshold, and which resulted in reproducible responses to C fibre activation. Such transcutaneous electrical stimulation has been shown to induce responses very similar to those evoked by direct nerve stimulation [27]. Following a suitable number of control stimuli, strong pinches were applied to a hind paw in order to verify the ability of the C fibre evoked responses to be modulated by a heterotopic noxious stimulus using a well established paradigm 1191: one hundred single transcutaneous electrical shocks were applied to the tai1; the pinch to the hind paw was applied from trials 46 to 70; poststimulus histograms (PSHs) were built from trials 31-45 for control, from trials 56-70 for the conditioning period and from trials 71-85 and 86-100 to evaluate post-effects. The effects of i.p. acetic acid injection were investigated as follows: sequences of 50 suprathreshold stimuli were applied every 5 min. After two reproducible response sequences, acetic acid was injected via an intraperitoneal cannula previousIy inserted and held in place. Only one cell was studied per animal. For each sequence, PSHs were built for the analysis of C fibre responses. Results were expressed as percentages of the mean control responses. At the conclusion of each experiment, the recording site was marked by electrophoresis of pontamine sky blue for further histological identification. (3) Conditioning stimuli In all behavioural and electrophysiological experiments, identical acetic acid (AA) or vehicle (V) solutions were used. AA was prepared by dissolving 1 g arabic gum in 9 ml of a 1% aqueous solution of acetic acid (pH = 3.20 at 22OC), and V by dissolving 1 g arabic gum in 9 ml of distilled water (pH = 4.31 at 22’C). Each animal received 5 mg/kg of one of these solutions.
Results Both behavioural and electrophysiolo~cal responses plied to the tail were conditioned by i.p. administration
to nociceptive stimuli apof the algogenic agent AA.
(I) Behavioural experiments The responses to a nociceptive stimulus applied to the tail or paws were lowered by the i.p. AA injection but these effects varied depending upon the beharioural test used. (a) Tail-flick test (Fig. IA). Predrug tail flick latencies were not statistically different for the 2 groups of rats: 2.57 _t 0.33 and 2.43 & 0.19 set for control and
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experimental groups respectively. In individual experiments, the i.p. AA induced variable changes in tail-flick latencies, which, in any case, were of low magnitude. As shown in Fig. lA, a very transient increase (10%) was observed 5 min after the i.p. injection; during the following 55 min, differences between control and experimental groups were not significant. (6) Vocalization test (Fig. IB). Predrug vocalization thresholds were not statistically different for the 2 groups of rats: 1.96 k 0.12 and 1.71 + 0.10 mA for control and experimental groups respectively. AA injection induced a very significant increase in the threshold for vocalization, reaching a maximum at 10 min with a progressive recovery to baseline level within 1 h. (c) Hot plate test (Fig. 1 C). In this case, rats were tested only once, and therefore comparison has been made between rats receiving vehicle or acetic acid at fixed times (15 and 30 min) after i.p. injection. As shown in Fig. 1C (left), jump latency was significantly increased (98%) 15 min after i.p. AA, whereas the increase (27%)
[TAIL-FLICK TEST]
[ HOT - PLATE TEST ]
15min.
30min. JJMP
1 Bmin.
PAW
30min.
LuxuiG
Fig. 1. Time courses of the paradoxical hypoalgesic effects of i.p. AA (see text). A: tail flick test. B: vocalization test, In both cases, results are expressed as percentages of the mean pre-injection baseline values (A: latencies; B: current intensities). Dotted line: group receiving i.p. vehicle; solid line: group receiving i.p. AA; n = number of animals in each group. C: hot plate test (6OOC). Jump (left) and paw-licking (right) latencies (ordinate in set) were measured in different groups of animals 15 and 30 min after i.p. vehicle (open columns) or AA (cross-hatched columns). * P < 0.02; ** P -C0.01; *** P < 0.001; unpaired 1 test.
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observed 30 min after i.p. AA was not significant. On the other hand, hind-paw licking (Fig. lC, right) was significantly affected both 15 and 30 min after i.p. AA (37% and 34% increase respectively). (2) Electrophysiologicul experiments (a) General characteristics of cells. A total number of 16 neurones were recorded. They all exhibited the characteristics common to convergent units, responding to both noxious and innocuous stimuli applied to their excitatory receptive fields. They were located in the medial part of the dorsal horn of the second coccygeal segment, in an area corresponding to laminae III-V, as defined by the approximate laminar organization delineated by Grossman et al. [lo]. All units were activated by noxious stimuli applied to a clearly defined area, which in every case included both sides of the tail. The extent of these excitatory receptive fields was variable (rostro-caudal extension ranging from 2 to 14.5 cm) but most were large (mean rostro-caudal extension: 7.8 f 1.2 cm). In most cases (14/16). the receptive field was organized in two concentric areas with the centre responding to both noxious and innocuous stimuli and the periphery being activated only by noxious heat and noxious pinch; the mean extent of the centre was 37.1 t_ 4.0% of the total receptive field. A typical example is shown in Fig. 2 (right). In the two remaining cases the receptive field was homogenous but very small and responded to both noxious and innocuous stimuli. By applying transcutaneous electrical stimulation to the centre of the excitatory receptive field on either side of the tail (2 msec pulses; 0.6 Hz), all the neurones could be shown to respond to both Aa and C fibre inputs; some (7/16) also responded to A6 inputs. Only the C fibre evoked responses were considered in the present study. The mean threshold for obtaining a C fibre response was 6.2 f 1.6 A.A. IP-lnlectlon
Control
I
5min.
,,
15min.
25min. 1
LL
II&L
1 ! !
1 \
> \ ,‘, \),,:s il 0 ;lya
Fig. 2. Example of i.p. AA effects on C fibre evoked responses of a convergent neurone to tramcutaneous suprathreshold electrical stimulation of the tail (peripheral excitatory field depicted on the right). Poststimulus histograms (bin width: 5 msec) were built with 50 consecutive trials (see text).
261 mA,
and the current was systematically increased to a suprathreshold value (mean: 3.6 x threshold) which was sufficient to give a maximal, stable C fibre response. In these conditions, the latencies of the maximum C fibre responses (259 k 20 msec), as gauged by poststimulus histogram analysis, were found to correspond to peripheral fibres with a mean conduction velocity of 0.73 -t- 0.12 m/set. We examined the possibility that these cells were subject to diffuse noxious inhibitory controls. As previously described in the lumbar cord [19], the activity of convergent neurones with excitatory receptive fields located on the extremity of one hind paw, can be powerfully inhibited by various noxious stimuli applied to various parts of the body; for example, it has been reported that the responses of such neurones to C fibre activation are strongly inhibited by a pinch applied to the tail. In the present study, we have investigated the converse situation and found that the C fibre responses of sacro-coccygeal cells produced by transcutaneous electrical stimulation of the tail were strongly depressed by a strong pinch applied to the left hind paw; the mean inhibition was 81.2 k 6.5% and was followed by marked post-effects (62.0 k 11% inhibition during the 22.5 set period following the conditioning stimulus). All neurones recorded were therefore under the influence of DNIC. (b) Effect of i.p. AA on C fibre evoked activities. For 14/16 cells, the i.p. AA injection induced a clear inhibition of the C fibre evoked responses to supramaximal electrical stimulation of the tail: 5 min after i.p. AA, a mean inhibition of 57.3 k 8.0% was observed. Fig. 2 shows a typical individual example: note that the inhibition was very marked 5 min after the injection and that following a transient recovery at 15 min, the inhibition was strong again at 25 and 35 min, with a progressive recovery to the baseline level occurring within an hour. Eleven units were recorded for enough time to investigate the time course of the i.p. AA effects. One of these was completely inhibited for 1 h and has been excluded from the mean data shown in Fig. 3: again note the biphasic effect induced by i.p. AA. b % inhibition n=lO 50-
15
20
25
30
35
40
45
50
55
60 min.
I.P. Injection Fig. 3. Time course of the inhibitory effect induced by i.p. AA on C fibre evoked activities. Ordinate: mean percentage of inhibition with reference to the pre-injection baseline: abscissa: time. * P < 0.02; ** P < 0.01; *** P < 0.001; paired t test.
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Finally, 2/16 cells showed an atypical effect of i.p. AA: the C fibre evoked responses of one cell were transiently facilitated 5 min after the injection while, in the remaining case, they were permanently, albeit variably, facilitated. Discussion These results demonstrate that i.p. injection of acetic acid, an algogenic agent commonly used to induce writhing behaviour in rats [29] and mice [16], when used as a conditioning stimulus, is able to modify both behavioural and electrophysiological responses to nociceptive stimuli. Our experiments confirm that dull pain of visceral origin can block some behavioural responses to a heterotopic nociceptive stimulus. Winter and Flataker [36] and Kraus et al. [17,18] reported that in the rat, there was a long lasting ‘antinociceptive effect’ induced by i.p. PBQ or stearic acid when pinches or electric shocks applied to the tail were used as the conditioned stimuli. Other i.p. chemical irritants, such as croton oil, formalin or kaolin, induced similar effects on the pressure pain test in rats or hot plate test in mice [14]. In the rat, hypertonic saline, a strong painful stimulant of the viscera [4] produced an elevation in the threshold for vocalization induced by electrical stimulation of the tail and a transient (< 4 min) increase in the tail flick latency [12,15]. On the other hand, Chapman and Way [3] reported an increase of tail-flick latency in mice, which lasted up to 1 h after i.p. acetic acid. In our study in rats, the tail-flick test was less sensitive to i.p. AA than the hot plate and vocalization tests. The small (10%) short duration (5 min) increase of tail-flick latency which we observed is in good agreement with the observations by Hayes et al. [12] of a short acting effect of i.p. hypertonic saline, but is at variance with the magnitude (35%) and duration (1 h) of the effects reported by Chapman and Way [3] in mice after i.p. AA. It is most probable that the discrepancies in these observations result from species differences: indeed, for a given algogenic agent, writhing is easier to induce in mice than in rats. In addition, writhing behaviour in rats is easy to produce in light-weight animals [4,29] and such a factor could contribute to discrepancies in the results. On the other hand, there is general agreement that stressful factors. including strong nociceptive stimuli [12] are able to increase tail-flick latency and this phenomenon has been described under the term ‘stress-induced analgesia.’ In these experiments the hypoalgesic effects lasted from 10 to 30 min, depending on the type and duration of the conditioning nociceptive stimulus. Variations in hypoalgesic effects may also result from experimental conditions such as the way in which rats are restrained for testing. There are no data available concerning the effect of visceral pain on spinal reflex activities in man. However some experimental situations may well be relevant: for instance, the polysynaptic reflex response, RIII, recorded from the biceps femoris when the sural nerve is stimulated, can be depressed by the application of nociceptive stimuli to the contralateral hand [22,35]. With regard to animal studies, when tests involve processes more integrated than
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a spinal reflex, there is general agreement in the descriptions of the paradoxical hypoalgesic effects of heterotopic nociceptive stimuli. In our experiments, these effects concern both a threshold behavioural reaction (vocalization test) and responses to a suprathreshold stimulus (hot plate test). These observations are in keeping with psychophysiological data obtained in man reporting that both the pain threshold [7,9,11,21,31] and the threshold for intolerable pain [35] are elevated by heterotopic nociceptive stimuli. These experimental observations confirm earlier reports of the pain-relieving effects of counter-irritation [24,30,34; and see refs. in 221. Our present electrophysiological experiments strongly suggest that the neuronal processes involved in the above described effects originate, at least partly, from the spinal cord. Indeed we found that i.p. AA induced a long lasting inhibition of C fibre evoked responses of convergent dorsal horn neurones which most probably play an important role in the transmission of nociceptive information [see refs. in 191. In the parallel situations, i.e., the comparable conditioning visceral stimulation and conditioned tail stimulation, the time courses of effects were roughly parallel in the behavioural (vocalization test) and electrophysiological experiments. However, the time course observed for the depression of neuronal activities was biphasic with a powerful (50% inhibition) transient effect followed by a long lasting but smaller (25-30%) effect. The short lasting inhibition characterizing the first period (5-15 min) may be interpreted as a primary acute irritant action of acetic acid, i.e., a local effect upon peritoneal and visceral tissues; this may be the result of stimulation of chemoreceptors associated with free terminals of C-sympathetical afferent fibres [23,26]. The long lasting inhibition characterizing the second period (20-60 min) may be interpreted as a secondary action resulting from inflammatory processes. The mechanisms leading to abdominal contractions induced by algogenic substances are not known; Niemegeers et al. [29] have postulated a relationship of acetic acid induced writhing with prostaglandin biosynthesis, a hypothesis which appeared in accordance with the relatively long period of writhing. Such an interpretation is also supported by the latency of writhing occurring after i.p. AA, at least in rats: in a previous experiment, in rats weighing 280-320 g [22], we found that writhing began 15 min after the administration of the algogenic substance. It seems important that another factor be taken into consideration. Our study was an attempt to parallel behavioural and electrophysiological investigations. However, the experimental situations differ markedly since in the former case, rats were freely moving whereas in the latter they were anaesthetized and paralysed. It is well known that halothane induces relaxation of the gastrointestinal musculature, inhibiting its motility [25]. In a control study, we have injected the AA solution dyed with pontamine sky blue (5%) in the two experimental situations and sacrified the animals 5, 15 or 45 min after the injection. In paralysed anaesthetized animals the dye was concentrated in peritoneum and intestinal loops near the injection site, even 45 min after its administration. In freely moving animals, we observed an increasing spread of dye in the viscera with time: intestinal loops surrounding the injection site were involved at 5 min; most of the intestine was dyed at 15 min, whereas all viscera, including the liver, were marked at 45 min. These observations underline the obvious
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difficulties in comparing behavioural and electrophysiological experiments and could explain the slight dissociation in the time courses observed in the two experimental situations. In any case, our present results confirm earlier reports indicating that the short-acting algogenic agent bradykinin administered intraperitoneally induced strong inhibitions (80-90%) of C fibre evoked responses of convergent neurones recorded in the lumbar dorsal horn [19] or trigeminal nucleus caudalis [6]; in this case effects were of short duration ( -c 5 min) and easily reproducible. In our present experiments DNIC triggered by noxious pinches of the hind paw were also powerful (mean inhibition about 80%) and reproducible. By contrast, the dull pain which probably follows an i.p. AA injection, is associated with smaller inhibitory effects which, in addition, show a much greater variability. We believe that the present experimental situation is closer to clinical pain than those envisaged in our earlier reports. Our data clearly indicate that a long duration dull pain of visceral origin is associated with sustained inhibitory processes occurring at the spinal level and strong enough to induce paradoxical heterotopic hypoalgesic effects in a correlative fashion. The clinical observation that pain elevates experimental thresholds tested on body areas distant from the painful focus, strongly supports this proposition [13.28].
Acknowledgements We wish to thank Dr. S.W. Cadden for English corrections, Mr. E. Dehausse for drawing and photography and Miss M. Hoch for secretarial help. This work was supported by I’INSERM (CRL No. 82 60 29). Luis Villanueva was supported by a scholarship from the French Government.
References 1 Benoist, J.M., Kayser, V., Gautron, M. and Guilbaud, G., Letter to the Editor, Pain, 18 (1984) 410-411. 2 Carrol, M.N. and Lim, R.K.S., Observationson the neuropharmacology of morphine and morphine-like analgesia, Arch. int. Pharmacodyn., 167 (1960) 383-403. 3 Chapman, D.B. and Way, E.L., Modification of endorphin/enkephalin analgesia and stress-induced analgesia by divalent cations, a cation chelator, and an ionophore, Brit. J. Pharmacol., 75 (1982) 389-396. 4 Collier, H.O. and Schneider, C., Profiles of activity in rodents of some narcotic and narcotic antagonist drugs, Nature (Lond.), 224 (1969) 610-612. 5 D’Amour, F.E. and Smith, D.L., A method for determining loss of pain sensation, J. Pharmacol. exp. Ther., 72 (1941) 74-79. 6 Dickenson, A.H., Le Bars, D. and Besson, J.M., Diffuse noxious inhibitory controls (DNIC). Effects on trigeminal nucleus caudalis neurones in the rat, Brain Res., 200 (1980) 293-305. 7 Duncker, K., Some preliminary experiments on the mutual influence of pains, Psychol. Forsch., 21 (1937) 311-326. 8 Eddy, N.B. and Leimbach, D., Synthetic analgesics. II. Dithienylbutenyl and dithienylbutylamines, J. Pharmacol. exp. Ther., 107 (1953) 385-393. 9 Gammon, G.D. and Starr, I., Studies on the relief of pain by counter-irritation, J. clin. Invest., 20 (1941) 13-20.
271
10 Grossman, M.L., Basbaum, AI. and Fields, H.L., Afferent and efferent connections of the rat tail flick reflex (a model used to analyze pain control mechanisms), J. camp. Neurol., 206 (1982) 9-16. 11 Hardy, J.D., Wolff, H.G. and Goodell, H., Studies on pain. A new method for measuring pain threshold: observations on spatial summation of pain, J. clin. Invest., 19 (1940) 649-657. 12 Hayes, R.L., Bennett, G.J., Newlon, P.G. and Mayer, D.J., Behavioral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli, Brain Res., 155 (1978) 69-90. 13 Hazouri, L.A. and Mueller, A.D., Pain threshold studies on paraplegic patients, Arch. Neurol. Psychiat. (Chic.), 64 (1950) 607-613. 14 Hitchens, J.T., Goldstein, S., Shemano, 1. and Beiler, J.M., Analgesic effects of irritants in three models of experimentally-induced pain, Arch. int. Pharmacodyn., 169 (1967) 384-393. 15 Komisaruk, B.R. and Wallman, J., Antinociceptive effects of vaginal stimulation in rats: neurophysiological and behavioral studies, Brain Res., 137 (1977) 85-107. 16 Koster, R., Anderson, M. and De Beer, E.J., Acetic acid for analgesic screening, Fed. Proc., 18 (1959) 412. 17 Kraus, E., Le Bars, D. and Besson, J.M., Behavioral confirmation of ‘diffuse noxious inhibitory controls’ (DNIC) and evidence for a role of endogenouos opiates, Brain Res., 266 (1981) 495-499. 18 Kraus, E., Besson, J.M. and Le Bars, D., Behavioral model for diffuse noxious inhibitory controls (DNIC): potentiation by 5-hydroxytryptophan, Brain Res., 231 (1982) 461-465. 19 Le Bars, D., Dickenson, A.H. and Besson, J.M., Diffuse noxious inhibitory controls (DNIC). I. Effects on dorsal horn convergent neurones in the rat, Pain, 6 (1979) 283-304. 20 Le Bars, D., Dickenson, A.H. and Besson, J.M., Diffuse noxious inhibitory controls (DNIC). II. Lack of effect on non-convergent neurones, supraspinal involvement and theoretical implications, Pain, 6 (1979) 305-327. 21 Le Bars, D., Roby, A. and Willer, J.C., Effects of heterotopic thermal stimuli on a nociceptive flexion reflex and the related pain sensation in man, J. Physiol. (Lond.), 336 (1983) 31P-32P. 22 Le Bars, D., Calvino, B., Villanueva, L. and Cadden, S., Physiological approaches to counter-irritation phenomena. In: G. Curzon and M.D. Tricklebank (Eds.), Stress Induced Analgesia, Wiley, London, 1984, in press. 23 Lim, R.K.S., Pain, Ann. Rev. Physiol., 32 (1970) 269-288. J.G. and Alstead, S., Counter-irritants. A method of assessing their effect, Lancet, ii 24 MacArthur, (1953) 1060-1062. F.N., Pittinger, C.B. and Long, J.P., Effects of halothane on gastrointestinal motility, 25 Marshall, Anesthesiology, 22 (1961) 363-366. 26 Mei, N., La sensibilite viscerale, J. Physiol. (Paris), 77 (1981) 597-612. 27 Menetrey, D., Giesler, G.J. and Besson, J.M., An analysis of response properties of spinal cord dorsal horn neurones to non-noxious and noxious stimuli in the spinal rat, Brain Res., 27 (1977) 15-33. and neurological 28 Merskey, H. and Evans, P.R., Variation in pain complaint threshold in psychiatric patients with pain, Pain, 1 (1975) 73-79. C.J.E., Van Bruggen, J.A.A. and Janssen, P.A.J., Suprofen, a potent antagonist of 29 Niemegeers, acetic-acid induced writhing in rats, Arzneimittel-Forsch., 25 (1975) 1505-1509. 30 Parsons, C.M. and Goetzl, F.R., Effect of induced pain on pain threshold, Proc. Sot. exp. Biol. (N.Y.), 60 (1945) 327-329. 31 Pertovaara, A., Kemppainen, P., Johansson, G. and Karonen, S.L., Ischemic pain non-segmentally produces a predominant reduction of pain and thermal sensitivity in man: a selective role for endogenous opioids, Brain Res., 251 (1982) 83-92. 32 Taber, R.I., Greenhouse, D.D., Rendell, J.K. and Irwin, S., Agonist and antagonist interactions of opioids on acetic acid-induced abdominal stretching in mice, J. Pharmacol. exp. Ther., 169 (1969) 29-38. 33 Waibl, H., Zur Topographie der Medulla spinalis der Albinoratte (Rarrm noruegicus), Adv. Anat. Embryol. Cell Biol., 47 (1973) 7-42. 34 Wand-Tetley, J.I., Historical methods of counter-irritation, Ann. phys. Med., 3 (1956) 90-98. 35 Willer, J.C., Roby, A. and Le Bars, D., Psychophysical and electrophysiological approaches to the pain relieving effects of heterotopic nociceptive stimuli, Brain, (1984) in press. 36 Winter, C.A. and Flataker, L., Nociceptive thresholds as affected by parenteral administration of irritants and of various antinociceptive drugs, J. Pharmacol. exp. Ther., 148 (1965) 373-379.
Pain, 20 (1984) 261-271 Elsevier
PA1 00695
The Heterotopic Effects of Visceral Pain: Behavioural and Electrophysiological Approaches in the Rat
B. Calvin0 ‘, L. Villanueva
and D. Le Bars
Unitk de Recherches de Neurophysiologie Pharmacologique de I’INSERM (U. 161), 2 rue d’Albia, 75014 Paris (France) (Received 6 April 1984, accepted 8 June 1984)
Summary
The heterotopic effects of peritoneo-visceral pain were investigated in behavioural and electrophysiological experiments performed on rats. The intraperitoneal administration of acetic acid (i.p. AA), an algesic agent commonly used to induce writhing behaviour in rodents, was used as a conditioning stimulus in two parallel series of experiments involving 3 behavioural tests and recordings of dorsal horn convergent neurones. The responses to a nociceptive stimulus applied to the tail or paws were lowered by i.p. AA but these effects depended on the behavioural test used: in the tail-flick test, AA produced a transient low magnitude increase in latencies; the threshold for vocalization induced by electrical stimulation of the tail was clearly (25%) and sustainedly (full recovery taking up to 1 h) increased; the jump latency in hot plate was markedly increased (100% at 15 min). Intraperitoneal AA strongly depressed the C fibre evoked responses of coccygeal convergent neurones to suprathreshold transcutaneous electrical stimulation applied on their tail excitatory receptive fields. The time course of these inhibitory effects roughly paralleled the behavioural hypoalgesic effect observed in the vocalization test. These results are discussed with reference to diffuse noxious inhibitory controls (DNIC). Analogies with counter-irritation phenomena are emphasized.
’ Correspondence
0304-3959/84/$03.00
to B. Calvino (same address). Maitre-Assistant
0 1984 Elsevier Science Publishers B.V.
UniversitC Paris-Nord.
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Introduction Convergent neurones are those cells of the dorsal horn which receive inputs from both low threshold afferents and nociceptors and are probably essential in the integrative mechanisms which process nociceptive information at the spinal level. Previous studies [19] have shown that a wide variety of noxious stimuli applied on widespread areas of the body by mechanical, thermal or chemical agents, induce specific and powerful depressions of the activities of this type of neurone. To be effective, conditioning stimuli must be applied on an area distant to the receptive field of the cell under study and this phenomenon, which has been called diffuse noxious inhibitory controls (DNIC), is characterized by its potency and the presence of long lasting post-effects. On the basis of DNIC we have proposed [20] that pain is associated with a double processing on convergent neurones: an activation of a segmental pool, surrounded by the inhibition of the remaining population. However our previous studies envisaged ‘epicritic’ stimuli and, therefore, it was essential to investigate the way in which such systems respond to situations more relevant to clinical pain; the first aim of the present work was to study the effect of a long lasting visceral nociceptive stimulus upon the responsiveness of convergent neurones with receptive fields located in areas remote from viscera (e.g., on the tail). The second aim was an attempt to correlate electrophysiological and behavioural investigations of this phenomenon. We have reported [17,18] that a noxious peritoneo-visceral stimulus (i.p. injection of phenylbenzoquinone (PBQ)) is able to produce a significant increase in the threshold for vocalization provoked by transcutaneous electrical stimulation of the tail, and this effect was interpreted as ‘a behavioural model of DNIC.’ In the present paper the effects of an analogous conditioning stimulus, i.p. acetic acid, has been tested using both behavioural (i.e., classical tail-flick, hot plate and vocalization tests) and electrophysiological models. In the latter case, the activity of convergent neurones in the dorsal horn was produced by transcutaneous electrical stimulation of their excitatory receptive fields located on the tail; this part of the body was chosen not only because of its distance from viscera but also to provide an element of comparison with two of the behavioural tests we used. Acetic acid was chosen as the conditioning stimulus because it is classically used for the screening of putative analgesic drugs (writhing test) and, in this case, writhing responses are less variable than in the equivalent PBQ test, particularly in terms of seasonal changes ]321.
Methods One hundred and sixty-one male Sprague-Dawley rats weighing 250-300 g were used. They were allowed to habituate to the laboratory for at least 96 h before testing. They were housed 5 per cage under diurnal lighting conditions with light on from 08.00 to 20.00 h and were given food and water ad libitum.
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(I) Behavioural experiments Experiments were carried out in a quiet and indirectly lit room, and were performed between 09.00 and 13.00 h. Tail-flick test [S]. A needle fixed on a length of plastic tubing was inserted intraperitoneally and held in place with elastoplast, thus allowing subsequent injection to be made without disturbing the animals. Thereafter, the rat was kept for 30 min before the beginning of the experiment in an individual plexiglass restraining chamber with a hole to allow access to the tail. Tail-flick latency was measured following exposure of the blackened tip of the tail to radiant heat from a 150 W lamp condensed with a convergent lens to a 1.5 mm diameter spot. Baseline latencies were in around 2.5 set and the automatic cut-off time was 10 sec. A baseline was established over at least 25 min (5 consecutive trials at 5 min intervals) before the injection; results were expressed as percentages with reference to the last three of these control tail-flick latencies. Vocalization test [2/. The rat was kept in an individual cylindric plexiglass cage (20 cm diameter) with a hole at the base to allow the access of wires to two needles previously inserted subcutaneously on either side of the tail. Following a 30 min resting period, tests were performed every 5 min throughout the experiment. To produce vocalization, electric shocks were applied to the tail, through the two needles and these consisted of 25 monophasic pulses (2 msec width, 50 Hz, 500 msec duration). The current was increased in successive 0.2 mA steps until the threshold for vocalization was reached. Three successive control thresholds were used as the baseline reference before i.p. injection of acetic acid or vehicle. Results were expressed as percentages with reference to this baseline. Hot plate test [8]. The rat was placed in a plexiglass cage (20 cm diameter) 30 min before and 15 or 30 min after i.p. injection of acetic acid or vehicle. For testing, the rat was placed on the hot plate and hind-paw-lick and jump latencies were measured with a 45 set cut-off time. The device consisted of a metal surface (25 cm x 25 cm) maintained at 60 f 0.5 ‘C. A glass cylinder (16 cm diameter: 30 cm high) also maintained at 60 f l°C, was placed on the metal surface. In order to avoid sensitization or desensitization phenomena induced by the stimulus, only one test was performed for each rat; the test was applied 15 or 30 min after the i.p. injection. Results were therefore expressed as latencies. (2) Electrophysiological experimetns The methods used were essentially those described in detail previously [19] and so only a brief account will be given here. Rats were deeply anaesthetized with halothane (2%) in a nitrous oxide/oxygen mixture (2/3, l/3) and, following tracheal and jugular cannulation, a laminectomy was performed, the spinal cord exposed between vertebrae L3 and L4 - corresponding to S4-Co3 segments [33] - and the area of cord giving the maximal dorsum potential to electrical stimulation of the tail was determined; this area was found at Co2 level. Anaesthesia was then lowered to 0.5% halothane and the animal was immobilized with gallamine triethiotide. This procedure appeared to achieve an adequate level of anaesthesia from ethical consideration whilst not excessively
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depressing neuronal responses to noxious stimuli [I]. Extracellular unitary recordings were made with glass micropipettes filled with pontamine sky blue and NaCl, and the cord was stabilized with agar. Convergent neurones were characterized by their responses to innocuous (touch, pressure . . .) and noxious (heating, strong pinch) stimuli applied to their peripheral receptive fields located on the tail. Suitable units were then activated by transcutaneous electrical stimulation (2 msec pulses, 0.6 Hz) of their receptive fields, which produced clear C fibre evoked responses as gauged by latency measurements. Tests were made using currents which were 2-5-fold threshold, and which resulted in reproducible responses to C fibre activation. Such transcutaneous electrical stimulation has been shown to induce responses very similar to those evoked by direct nerve stimulation [27]. Following a suitable number of control stimuli, strong pinches were applied to a hind paw in order to verify the ability of the C fibre evoked responses to be modulated by a heterotopic noxious stimulus using a well established paradigm 1191: one hundred single transcutaneous electrical shocks were applied to the tai1; the pinch to the hind paw was applied from trials 46 to 70; poststimulus histograms (PSHs) were built from trials 31-45 for control, from trials 56-70 for the conditioning period and from trials 71-85 and 86-100 to evaluate post-effects. The effects of i.p. acetic acid injection were investigated as follows: sequences of 50 suprathreshold stimuli were applied every 5 min. After two reproducible response sequences, acetic acid was injected via an intraperitoneal cannula previousIy inserted and held in place. Only one cell was studied per animal. For each sequence, PSHs were built for the analysis of C fibre responses. Results were expressed as percentages of the mean control responses. At the conclusion of each experiment, the recording site was marked by electrophoresis of pontamine sky blue for further histological identification. (3) Conditioning stimuli In all behavioural and electrophysiological experiments, identical acetic acid (AA) or vehicle (V) solutions were used. AA was prepared by dissolving 1 g arabic gum in 9 ml of a 1% aqueous solution of acetic acid (pH = 3.20 at 22OC), and V by dissolving 1 g arabic gum in 9 ml of distilled water (pH = 4.31 at 22’C). Each animal received 5 mg/kg of one of these solutions.
Results Both behavioural and electrophysiolo~cal responses plied to the tail were conditioned by i.p. administration
to nociceptive stimuli apof the algogenic agent AA.
(I) Behavioural experiments The responses to a nociceptive stimulus applied to the tail or paws were lowered by the i.p. AA injection but these effects varied depending upon the beharioural test used. (a) Tail-flick test (Fig. IA). Predrug tail flick latencies were not statistically different for the 2 groups of rats: 2.57 _t 0.33 and 2.43 & 0.19 set for control and
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experimental groups respectively. In individual experiments, the i.p. AA induced variable changes in tail-flick latencies, which, in any case, were of low magnitude. As shown in Fig. lA, a very transient increase (10%) was observed 5 min after the i.p. injection; during the following 55 min, differences between control and experimental groups were not significant. (6) Vocalization test (Fig. IB). Predrug vocalization thresholds were not statistically different for the 2 groups of rats: 1.96 k 0.12 and 1.71 + 0.10 mA for control and experimental groups respectively. AA injection induced a very significant increase in the threshold for vocalization, reaching a maximum at 10 min with a progressive recovery to baseline level within 1 h. (c) Hot plate test (Fig. 1 C). In this case, rats were tested only once, and therefore comparison has been made between rats receiving vehicle or acetic acid at fixed times (15 and 30 min) after i.p. injection. As shown in Fig. 1C (left), jump latency was significantly increased (98%) 15 min after i.p. AA, whereas the increase (27%)
[TAIL-FLICK TEST]
[ HOT - PLATE TEST ]
15min.
30min. JJMP
1 Bmin.
PAW
30min.
LuxuiG
Fig. 1. Time courses of the paradoxical hypoalgesic effects of i.p. AA (see text). A: tail flick test. B: vocalization test, In both cases, results are expressed as percentages of the mean pre-injection baseline values (A: latencies; B: current intensities). Dotted line: group receiving i.p. vehicle; solid line: group receiving i.p. AA; n = number of animals in each group. C: hot plate test (6OOC). Jump (left) and paw-licking (right) latencies (ordinate in set) were measured in different groups of animals 15 and 30 min after i.p. vehicle (open columns) or AA (cross-hatched columns). * P < 0.02; ** P -C0.01; *** P < 0.001; unpaired 1 test.
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observed 30 min after i.p. AA was not significant. On the other hand, hind-paw licking (Fig. lC, right) was significantly affected both 15 and 30 min after i.p. AA (37% and 34% increase respectively). (2) Electrophysiologicul experiments (a) General characteristics of cells. A total number of 16 neurones were recorded. They all exhibited the characteristics common to convergent units, responding to both noxious and innocuous stimuli applied to their excitatory receptive fields. They were located in the medial part of the dorsal horn of the second coccygeal segment, in an area corresponding to laminae III-V, as defined by the approximate laminar organization delineated by Grossman et al. [lo]. All units were activated by noxious stimuli applied to a clearly defined area, which in every case included both sides of the tail. The extent of these excitatory receptive fields was variable (rostro-caudal extension ranging from 2 to 14.5 cm) but most were large (mean rostro-caudal extension: 7.8 f 1.2 cm). In most cases (14/16). the receptive field was organized in two concentric areas with the centre responding to both noxious and innocuous stimuli and the periphery being activated only by noxious heat and noxious pinch; the mean extent of the centre was 37.1 t_ 4.0% of the total receptive field. A typical example is shown in Fig. 2 (right). In the two remaining cases the receptive field was homogenous but very small and responded to both noxious and innocuous stimuli. By applying transcutaneous electrical stimulation to the centre of the excitatory receptive field on either side of the tail (2 msec pulses; 0.6 Hz), all the neurones could be shown to respond to both Aa and C fibre inputs; some (7/16) also responded to A6 inputs. Only the C fibre evoked responses were considered in the present study. The mean threshold for obtaining a C fibre response was 6.2 f 1.6 A.A. IP-lnlectlon
Control
I
5min.
,,
15min.
25min. 1
LL
II&L
1 ! !
1 \
> \ ,‘, \),,:s il 0 ;lya
Fig. 2. Example of i.p. AA effects on C fibre evoked responses of a convergent neurone to tramcutaneous suprathreshold electrical stimulation of the tail (peripheral excitatory field depicted on the right). Poststimulus histograms (bin width: 5 msec) were built with 50 consecutive trials (see text).
261 mA,
and the current was systematically increased to a suprathreshold value (mean: 3.6 x threshold) which was sufficient to give a maximal, stable C fibre response. In these conditions, the latencies of the maximum C fibre responses (259 k 20 msec), as gauged by poststimulus histogram analysis, were found to correspond to peripheral fibres with a mean conduction velocity of 0.73 -t- 0.12 m/set. We examined the possibility that these cells were subject to diffuse noxious inhibitory controls. As previously described in the lumbar cord [19], the activity of convergent neurones with excitatory receptive fields located on the extremity of one hind paw, can be powerfully inhibited by various noxious stimuli applied to various parts of the body; for example, it has been reported that the responses of such neurones to C fibre activation are strongly inhibited by a pinch applied to the tail. In the present study, we have investigated the converse situation and found that the C fibre responses of sacro-coccygeal cells produced by transcutaneous electrical stimulation of the tail were strongly depressed by a strong pinch applied to the left hind paw; the mean inhibition was 81.2 k 6.5% and was followed by marked post-effects (62.0 k 11% inhibition during the 22.5 set period following the conditioning stimulus). All neurones recorded were therefore under the influence of DNIC. (b) Effect of i.p. AA on C fibre evoked activities. For 14/16 cells, the i.p. AA injection induced a clear inhibition of the C fibre evoked responses to supramaximal electrical stimulation of the tail: 5 min after i.p. AA, a mean inhibition of 57.3 k 8.0% was observed. Fig. 2 shows a typical individual example: note that the inhibition was very marked 5 min after the injection and that following a transient recovery at 15 min, the inhibition was strong again at 25 and 35 min, with a progressive recovery to the baseline level occurring within an hour. Eleven units were recorded for enough time to investigate the time course of the i.p. AA effects. One of these was completely inhibited for 1 h and has been excluded from the mean data shown in Fig. 3: again note the biphasic effect induced by i.p. AA. b % inhibition n=lO 50-
15
20
25
30
35
40
45
50
55
60 min.
I.P. Injection Fig. 3. Time course of the inhibitory effect induced by i.p. AA on C fibre evoked activities. Ordinate: mean percentage of inhibition with reference to the pre-injection baseline: abscissa: time. * P < 0.02; ** P < 0.01; *** P < 0.001; paired t test.
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Finally, 2/16 cells showed an atypical effect of i.p. AA: the C fibre evoked responses of one cell were transiently facilitated 5 min after the injection while, in the remaining case, they were permanently, albeit variably, facilitated. Discussion These results demonstrate that i.p. injection of acetic acid, an algogenic agent commonly used to induce writhing behaviour in rats [29] and mice [16], when used as a conditioning stimulus, is able to modify both behavioural and electrophysiological responses to nociceptive stimuli. Our experiments confirm that dull pain of visceral origin can block some behavioural responses to a heterotopic nociceptive stimulus. Winter and Flataker [36] and Kraus et al. [17,18] reported that in the rat, there was a long lasting ‘antinociceptive effect’ induced by i.p. PBQ or stearic acid when pinches or electric shocks applied to the tail were used as the conditioned stimuli. Other i.p. chemical irritants, such as croton oil, formalin or kaolin, induced similar effects on the pressure pain test in rats or hot plate test in mice [14]. In the rat, hypertonic saline, a strong painful stimulant of the viscera [4] produced an elevation in the threshold for vocalization induced by electrical stimulation of the tail and a transient (< 4 min) increase in the tail flick latency [12,15]. On the other hand, Chapman and Way [3] reported an increase of tail-flick latency in mice, which lasted up to 1 h after i.p. acetic acid. In our study in rats, the tail-flick test was less sensitive to i.p. AA than the hot plate and vocalization tests. The small (10%) short duration (5 min) increase of tail-flick latency which we observed is in good agreement with the observations by Hayes et al. [12] of a short acting effect of i.p. hypertonic saline, but is at variance with the magnitude (35%) and duration (1 h) of the effects reported by Chapman and Way [3] in mice after i.p. AA. It is most probable that the discrepancies in these observations result from species differences: indeed, for a given algogenic agent, writhing is easier to induce in mice than in rats. In addition, writhing behaviour in rats is easy to produce in light-weight animals [4,29] and such a factor could contribute to discrepancies in the results. On the other hand, there is general agreement that stressful factors. including strong nociceptive stimuli [12] are able to increase tail-flick latency and this phenomenon has been described under the term ‘stress-induced analgesia.’ In these experiments the hypoalgesic effects lasted from 10 to 30 min, depending on the type and duration of the conditioning nociceptive stimulus. Variations in hypoalgesic effects may also result from experimental conditions such as the way in which rats are restrained for testing. There are no data available concerning the effect of visceral pain on spinal reflex activities in man. However some experimental situations may well be relevant: for instance, the polysynaptic reflex response, RIII, recorded from the biceps femoris when the sural nerve is stimulated, can be depressed by the application of nociceptive stimuli to the contralateral hand [22,35]. With regard to animal studies, when tests involve processes more integrated than
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a spinal reflex, there is general agreement in the descriptions of the paradoxical hypoalgesic effects of heterotopic nociceptive stimuli. In our experiments, these effects concern both a threshold behavioural reaction (vocalization test) and responses to a suprathreshold stimulus (hot plate test). These observations are in keeping with psychophysiological data obtained in man reporting that both the pain threshold [7,9,11,21,31] and the threshold for intolerable pain [35] are elevated by heterotopic nociceptive stimuli. These experimental observations confirm earlier reports of the pain-relieving effects of counter-irritation [24,30,34; and see refs. in 221. Our present electrophysiological experiments strongly suggest that the neuronal processes involved in the above described effects originate, at least partly, from the spinal cord. Indeed we found that i.p. AA induced a long lasting inhibition of C fibre evoked responses of convergent dorsal horn neurones which most probably play an important role in the transmission of nociceptive information [see refs. in 191. In the parallel situations, i.e., the comparable conditioning visceral stimulation and conditioned tail stimulation, the time courses of effects were roughly parallel in the behavioural (vocalization test) and electrophysiological experiments. However, the time course observed for the depression of neuronal activities was biphasic with a powerful (50% inhibition) transient effect followed by a long lasting but smaller (25-30%) effect. The short lasting inhibition characterizing the first period (5-15 min) may be interpreted as a primary acute irritant action of acetic acid, i.e., a local effect upon peritoneal and visceral tissues; this may be the result of stimulation of chemoreceptors associated with free terminals of C-sympathetical afferent fibres [23,26]. The long lasting inhibition characterizing the second period (20-60 min) may be interpreted as a secondary action resulting from inflammatory processes. The mechanisms leading to abdominal contractions induced by algogenic substances are not known; Niemegeers et al. [29] have postulated a relationship of acetic acid induced writhing with prostaglandin biosynthesis, a hypothesis which appeared in accordance with the relatively long period of writhing. Such an interpretation is also supported by the latency of writhing occurring after i.p. AA, at least in rats: in a previous experiment, in rats weighing 280-320 g [22], we found that writhing began 15 min after the administration of the algogenic substance. It seems important that another factor be taken into consideration. Our study was an attempt to parallel behavioural and electrophysiological investigations. However, the experimental situations differ markedly since in the former case, rats were freely moving whereas in the latter they were anaesthetized and paralysed. It is well known that halothane induces relaxation of the gastrointestinal musculature, inhibiting its motility [25]. In a control study, we have injected the AA solution dyed with pontamine sky blue (5%) in the two experimental situations and sacrified the animals 5, 15 or 45 min after the injection. In paralysed anaesthetized animals the dye was concentrated in peritoneum and intestinal loops near the injection site, even 45 min after its administration. In freely moving animals, we observed an increasing spread of dye in the viscera with time: intestinal loops surrounding the injection site were involved at 5 min; most of the intestine was dyed at 15 min, whereas all viscera, including the liver, were marked at 45 min. These observations underline the obvious
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difficulties in comparing behavioural and electrophysiological experiments and could explain the slight dissociation in the time courses observed in the two experimental situations. In any case, our present results confirm earlier reports indicating that the short-acting algogenic agent bradykinin administered intraperitoneally induced strong inhibitions (80-90%) of C fibre evoked responses of convergent neurones recorded in the lumbar dorsal horn [19] or trigeminal nucleus caudalis [6]; in this case effects were of short duration ( -c 5 min) and easily reproducible. In our present experiments DNIC triggered by noxious pinches of the hind paw were also powerful (mean inhibition about 80%) and reproducible. By contrast, the dull pain which probably follows an i.p. AA injection, is associated with smaller inhibitory effects which, in addition, show a much greater variability. We believe that the present experimental situation is closer to clinical pain than those envisaged in our earlier reports. Our data clearly indicate that a long duration dull pain of visceral origin is associated with sustained inhibitory processes occurring at the spinal level and strong enough to induce paradoxical heterotopic hypoalgesic effects in a correlative fashion. The clinical observation that pain elevates experimental thresholds tested on body areas distant from the painful focus, strongly supports this proposition [13.28].
Acknowledgements We wish to thank Dr. S.W. Cadden for English corrections, Mr. E. Dehausse for drawing and photography and Miss M. Hoch for secretarial help. This work was supported by I’INSERM (CRL No. 82 60 29). Luis Villanueva was supported by a scholarship from the French Government.
References 1 Benoist, J.M., Kayser, V., Gautron, M. and Guilbaud, G., Letter to the Editor, Pain, 18 (1984) 410-411. 2 Carrol, M.N. and Lim, R.K.S., Observationson the neuropharmacology of morphine and morphine-like analgesia, Arch. int. Pharmacodyn., 167 (1960) 383-403. 3 Chapman, D.B. and Way, E.L., Modification of endorphin/enkephalin analgesia and stress-induced analgesia by divalent cations, a cation chelator, and an ionophore, Brit. J. Pharmacol., 75 (1982) 389-396. 4 Collier, H.O. and Schneider, C., Profiles of activity in rodents of some narcotic and narcotic antagonist drugs, Nature (Lond.), 224 (1969) 610-612. 5 D’Amour, F.E. and Smith, D.L., A method for determining loss of pain sensation, J. Pharmacol. exp. Ther., 72 (1941) 74-79. 6 Dickenson, A.H., Le Bars, D. and Besson, J.M., Diffuse noxious inhibitory controls (DNIC). Effects on trigeminal nucleus caudalis neurones in the rat, Brain Res., 200 (1980) 293-305. 7 Duncker, K., Some preliminary experiments on the mutual influence of pains, Psychol. Forsch., 21 (1937) 311-326. 8 Eddy, N.B. and Leimbach, D., Synthetic analgesics. II. Dithienylbutenyl and dithienylbutylamines, J. Pharmacol. exp. Ther., 107 (1953) 385-393. 9 Gammon, G.D. and Starr, I., Studies on the relief of pain by counter-irritation, J. clin. Invest., 20 (1941) 13-20.
271
10 Grossman, M.L., Basbaum, AI. and Fields, H.L., Afferent and efferent connections of the rat tail flick reflex (a model used to analyze pain control mechanisms), J. camp. Neurol., 206 (1982) 9-16. 11 Hardy, J.D., Wolff, H.G. and Goodell, H., Studies on pain. A new method for measuring pain threshold: observations on spatial summation of pain, J. clin. Invest., 19 (1940) 649-657. 12 Hayes, R.L., Bennett, G.J., Newlon, P.G. and Mayer, D.J., Behavioral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli, Brain Res., 155 (1978) 69-90. 13 Hazouri, L.A. and Mueller, A.D., Pain threshold studies on paraplegic patients, Arch. Neurol. Psychiat. (Chic.), 64 (1950) 607-613. 14 Hitchens, J.T., Goldstein, S., Shemano, 1. and Beiler, J.M., Analgesic effects of irritants in three models of experimentally-induced pain, Arch. int. Pharmacodyn., 169 (1967) 384-393. 15 Komisaruk, B.R. and Wallman, J., Antinociceptive effects of vaginal stimulation in rats: neurophysiological and behavioral studies, Brain Res., 137 (1977) 85-107. 16 Koster, R., Anderson, M. and De Beer, E.J., Acetic acid for analgesic screening, Fed. Proc., 18 (1959) 412. 17 Kraus, E., Le Bars, D. and Besson, J.M., Behavioral confirmation of ‘diffuse noxious inhibitory controls’ (DNIC) and evidence for a role of endogenouos opiates, Brain Res., 266 (1981) 495-499. 18 Kraus, E., Besson, J.M. and Le Bars, D., Behavioral model for diffuse noxious inhibitory controls (DNIC): potentiation by 5-hydroxytryptophan, Brain Res., 231 (1982) 461-465. 19 Le Bars, D., Dickenson, A.H. and Besson, J.M., Diffuse noxious inhibitory controls (DNIC). I. Effects on dorsal horn convergent neurones in the rat, Pain, 6 (1979) 283-304. 20 Le Bars, D., Dickenson, A.H. and Besson, J.M., Diffuse noxious inhibitory controls (DNIC). II. Lack of effect on non-convergent neurones, supraspinal involvement and theoretical implications, Pain, 6 (1979) 305-327. 21 Le Bars, D., Roby, A. and Willer, J.C., Effects of heterotopic thermal stimuli on a nociceptive flexion reflex and the related pain sensation in man, J. Physiol. (Lond.), 336 (1983) 31P-32P. 22 Le Bars, D., Calvino, B., Villanueva, L. and Cadden, S., Physiological approaches to counter-irritation phenomena. In: G. Curzon and M.D. Tricklebank (Eds.), Stress Induced Analgesia, Wiley, London, 1984, in press. 23 Lim, R.K.S., Pain, Ann. Rev. Physiol., 32 (1970) 269-288. J.G. and Alstead, S., Counter-irritants. A method of assessing their effect, Lancet, ii 24 MacArthur, (1953) 1060-1062. F.N., Pittinger, C.B. and Long, J.P., Effects of halothane on gastrointestinal motility, 25 Marshall, Anesthesiology, 22 (1961) 363-366. 26 Mei, N., La sensibilite viscerale, J. Physiol. (Paris), 77 (1981) 597-612. 27 Menetrey, D., Giesler, G.J. and Besson, J.M., An analysis of response properties of spinal cord dorsal horn neurones to non-noxious and noxious stimuli in the spinal rat, Brain Res., 27 (1977) 15-33. and neurological 28 Merskey, H. and Evans, P.R., Variation in pain complaint threshold in psychiatric patients with pain, Pain, 1 (1975) 73-79. C.J.E., Van Bruggen, J.A.A. and Janssen, P.A.J., Suprofen, a potent antagonist of 29 Niemegeers, acetic-acid induced writhing in rats, Arzneimittel-Forsch., 25 (1975) 1505-1509. 30 Parsons, C.M. and Goetzl, F.R., Effect of induced pain on pain threshold, Proc. Sot. exp. Biol. (N.Y.), 60 (1945) 327-329. 31 Pertovaara, A., Kemppainen, P., Johansson, G. and Karonen, S.L., Ischemic pain non-segmentally produces a predominant reduction of pain and thermal sensitivity in man: a selective role for endogenous opioids, Brain Res., 251 (1982) 83-92. 32 Taber, R.I., Greenhouse, D.D., Rendell, J.K. and Irwin, S., Agonist and antagonist interactions of opioids on acetic acid-induced abdominal stretching in mice, J. Pharmacol. exp. Ther., 169 (1969) 29-38. 33 Waibl, H., Zur Topographie der Medulla spinalis der Albinoratte (Rarrm noruegicus), Adv. Anat. Embryol. Cell Biol., 47 (1973) 7-42. 34 Wand-Tetley, J.I., Historical methods of counter-irritation, Ann. phys. Med., 3 (1956) 90-98. 35 Willer, J.C., Roby, A. and Le Bars, D., Psychophysical and electrophysiological approaches to the pain relieving effects of heterotopic nociceptive stimuli, Brain, (1984) in press. 36 Winter, C.A. and Flataker, L., Nociceptive thresholds as affected by parenteral administration of irritants and of various antinociceptive drugs, J. Pharmacol. exp. Ther., 148 (1965) 373-379.