- Email: [email protected]
Scratching behaviour in arthritic rats: a sign of chronic pain or itch?
123
Pain, 29 (1987) 123-131 Rlsevier
PAI 01028
Basic Section Scratching behaviour in arthritic rats: a sign of chronic pain or itch? Mauricio De Castro-Costa *, Jan Gybels, Ronnie Kupers and Johan Van Hees Department (Received
of Neurologv and Neurosurgery, University of Louvain, B-3ooO Louvain {Belgium) 8 July 1986, revised received 9 September
1986, accepted
15 September
1986)
In a previous study, it was shown that adjuvant-induced arthritic rats present an abnormal behaviour pattern up to 60 days after inoctdation with Mycv~eterium butyric~m. The purpose of the present study was to investigate (1) how long the abnormal behaviour pattern continues, and (2) whether the observed increased scratching beha+iour is a parameter of chronic pain or rather a reaction to itch. Adjuvant-induced arthritic rats were observed for up to 180 days after the inoculation and their behaviour was quantitatively analysed. The following behavioural changes persisted for more than 60 days: rearing, running and climbing were decreased while grooming, scratching, biting and freezing were increased. No behavioural changes were observed 120 days after the inoculation. The increased scratching was not influenced by an antihistamine drug (astemizole). Not only morphine but also acetylsalicylate selectively depressed the increased scratching behaviour without influencing the other behaviours. These results reinforce the notion that in arthritic rats the increased scratching is a sign of chronic pain.
S-
Key words:
Adjuvant-induced
arthritis;
Chronic
pain:
Morphine;
Acetylsalicylate;
Itch
Introduction
In a previous paper [7], it was shown that rats with ~ycob~crer~~rn buryricum-induced adjuvant arthritis presented si~fic~t changes in their behaviour. Among these, scratching fulfilled certain criteria making it a possible parameter for chronic pain behaviour. Indeed it was shown that adjuvant poiyarthritis induced an increased scratching behaviour, that morphine reversed this increase and that nalo-
* Present address: e Farmacologia,
Universidade Federal do Ceara, 6O.ooO Fortaka, Brasil.
Faculdade
CQrres~ondence to: J. Gybels, Department of Neurology Gasthuisberg, Herestraat 49, B-3000 Louvain, Belgium.
03043959/87/$03.50
Q 1987 Elsevier Science Publishers
de Medicina,
and
Departamento
Neurosurgery,
B.V. (Biomedical
Division)
U.Z.
de Fisiologia
Sint-Rafa&
124
xone blocked the effect of morphine. It was also shown that the increased scratching behaviour was still present more than 50 days after inoculation. In this study, we wanted to answer the following questions: (1) how long does it take before the abnormal behavioural pattern normalizes, and (2) what is the influence of a peripherally acting analgesic without sedative effects on the arthritic rats’ behaviour? One can argue that the increased scratching in arthritic rats might be a reaction to itch. Arthritic rats show severe signs of inflammation of the hind paws. It is known that in acute inhalator lesions histamine is released which can induce itch and hence also scratching. Arthritic rats present wounds at the onset of the disease; it has been suggested [4] that as these wounds heal around the third week, itch might be elicited. Furthermore, dermatitis, which can elicit a histamine-mediated itch [23], is a possible side effect of adjuvant polyarthritis [24]. In humans, systemic diseases such as liver failure, can also induce itch [22]. Finally, one might suggest that the increased scratching behaviour is simply a means of compensating for a lack of body cleaning function (grooming) in the acute phase of the disease [4]. Therefore, a third objective of this study was to search for evidence that scratching in arthritic rats is not a reaction to itch.
Methodology One hundred and twenty-two Wistar male rats, weighing about 250 g, were used. Sixty-four of them were inoculated intradermally in the base of the tail with 0.05 mI of a paraffin oil suspension of heat-killed ~_yc~bacter~~m butyricum (5 mg/ml) [l]. The other 58 rats, used as controls, were inoculated with only the sterile vehiculum (paraffin oil). For the time evolution of the rats’ behaviour, the animals were divided into different groups: 24 rats * were observed during the period O-60 days after the inoculation (18 arthritic rats and 6 vehiculum-inoculated rats) and 54 rats were observed 60, 120 or 180 days after the inoculation (27 arthritic rats and 27 ve~culum-inoculated rats). Lysine acetylsalicylate was injected intr~uscularly in a dose of 75 mg/kg, 30-50 days after the inoculation with Mycobacterium butyricum. Observations started 30 min after the injection. The acetylsalicylate-injected arthritic rats (N = 6) were compared with others which received no drugs (N = 6) or only the vehiculum (N = 6). To rule out the possibility that the increased scratching behaviour might be due to a histamine-mediated itch, some experiments with antihistamine drugs were ** is an Hl-~sta~ne antagonist with a miniperformed. Astemizole (Hisman~) mum of side effects on the nervous system. It was administered to arthritic rats in the post-acute (N = 6) and very late phase (N = 7) of the disease in a dose of 1 * The spinal cord was removed from these rats for histochemical analysis. The results of this analysis are presented elsewhere [26]. * * We received this drug and information on its characteristics from Janssen Pharmaceutics.
125
mg/kg. For this, a suspension of the substance was injected into the centre of the food pellets given to the 24-h fasting rats. Observations started 120 min after food intake which was well within the period of the drug’s maximal effect. The results were compared with those from the same animals receiving no drugs. Statistical analysis of the results was performed using non-paramet~c tests; P c 0.05 (two-tailed) was considered as significant. For all experiments we adopted the same methodology of quantified behavioural observation as described in a previous paper [7]. The guidelines, published in a Guest Editorial in Pain on ethical standards for investigations of experimental pain in animals, have been followed (Pain, 16 (1983) 109-110). Results
The analysis of the arthritic rats’ behaviour from 0 up to 60 days has shown the following: the arthritic rats (N = 6) of the acute phase (lo-30 days) exhibited a significant decrease (P c 0.005) in rearing, sniffing, running and climbing and a EFFECT
OF
SNIFFING
ARTHRITIS
RUNNING
ON
BEHAVIOUR
GROOMING
(N-61
SCRATCHING
500 _
.
hi 2 N : is u) 0:
cl.4 EATING
FREEZING
BITING
II
pcooo5
m
p-zoo5
Ez
D 1005
LL RESTING
Fig. 1. The behaviour of arthritic rats (immediate acute, behaviour of normal rats. Median values in xc/O.5 Mu-W~tney U-test was used to compute the level of appears in the early post-acute phase and late phase inflammation).
SLEEPING
post-acute, and late phases) is compared with the h observation of 6 rats are represented. The significance. Increased scratching behaviour only and not in the acute phase (phase of maximal
126
EFFECT
OF
ARTHRITIS
ON
BEHAVIOUR
tN:lOl
m
pc5005
fita p-zoo5 lza
Fig with level lasts
p>oos
2. The behaviour of arthritic rats of the very late phase ( > 60 days after the inoculation) is compared the behaviour of normal rats. Median values in set/0.5 h observation of 10 rats are represented. The of probability is computed by the Mann-Whitney U-test. The scratching behaviour in arthritic rats more than 60 davs after the inoculation. EFFFCT
OF
ACETYLSALICILATE (N ~6)
m m
P'OOS P4005
d 4
s 111 p >0.05
1
-IJ
5 --
Fig. 3. A: effect of acetylsalicylate (75 mg/kg i.m.) on the behaviour of arthritic rats, 30-50 days after the inoculation with M_vcohucferium hu~vrie~m (N = 6). Median values in sec/O.S h observation are represented. Level of probability is computed by the Wilcoxon matched pairs signed rank test, comparing acetylsalicylate effect with the no-drug condition. Scratching behaviour is significantly reversed (depressed) by acetylsaiicylate. B: comparison of the acetylsalicylate effect with the vehiculum effect in the same animals.
significant increase (P < 0.005) in grooming, resting and sleeping (Fig. 1). The phase (30-50 days) showed a significant arthritic rats (N = 6) in the pust-acute decrease (P -C O,.OOS>in rearing, sniffing, running and climbing, and a significant increase in scratching (P -=cO.OOS), biting (P -E0.05) and sleeping (P -C 0.005) (Fig. 1). In the late phase (50-60 days), the arthritic rats (N = 6) also exhibited significant changes in their behaviour: eating (P < O.OOS), rearing (P i 0.005), running (P < 0.005) and climbing (P -C0.05) were decreased, while grooming (P -zO.OS), scratching (P < O.OOS), biting (P -C0.05) and sleeping (P < 0.005) were increased in comparison to normal rats (Fig. 1). Some of these behavioural changes persisted fur more than 60 days: rearing (P < O.OOS), running f P < 0.005) and climbing (P -C 0.005) were still decreased, while grooming (P -=z 0.05), scratching (P c O.OOS), biting (P -=c 0.05) and freezing (P < 0.05) (Fig. 2) were still increased (N = 10). However, 120 days (N = 7) or 180 days (N = 10) after the inoculation, no more significant changes in the rats’ behaviours were present. Statistical analysis was performed using the Mann- Whitney U-test. In comparison with arthritic rats without drugs (N = S), arthritic rats which received acetylsalicylate (N = 6) ran more (P -C 0.05) and scratched Iess (P -e0.05). In comparison with rats which received only the vehiculum (N = 6), acetylsalicylate-injected animals showed more rearing and running (P < 0.05) and
EFFECT OF ASTEMIZULE
N:7
Fig. 4. Effect of astemizole (1 mg/kg p.0.) on behaviour in 2 groups of arthritic rats (N = 6, 30-50 days after inoculation; N = 7. > 60 days after inoculation). Median values in seci0.5 h observation of 6 rats are represented. Level of probability is computed by the Wilcoxon matched pairs signed rank test. The antihistamine drug induces no significant change in the behaviour of arthritic rats.
128
less scratching (P < 0.05). The Wilcoxon matched pairs signed ranks test was used for the statistical analysis (Fig. 3). The arthritic rats (N = 6, 30--50 days after inoculation. and N = 7. ) 60 days after inoculation) which received astemizole were compared with the same rats without this drug. The results showed no change in their level of scratching behaviour. Furthermore, no change in the other behaviours was observed (Fig. 4). The Wilcoxon matched pair test was used for the statistical analysis.
Discussion First of all, we will discuss the finding that scratching is not an itch-related behaviour. Then we will present some arguments which favour the hypothesis that scratching might be a chronic pain-related form of behaviour. Itch may be induced by many factors such as external physical and chemical stimuli, metabolic diseases, several skin pathologies and psychological disturbances, In some of these conditions, itch may be mediated by a release of histamine [ 16,22,23]. Generally it is assumed that itch and mild pain have a common neural substrate [16]. Both are evoked by activation of unmyelinated fibres. Anterolateral cordotomy abolishes both itch and pain. Furthermore, in patients with congenital insensitivity to pain, itch is absent. A difference is, however, that itch is elicited only from the most superficial layers of the skin, while pain can also be elicited from deep structures. Noxious stimulation of low intensity and long duration on the outermost layers of the skin may induce an itching sensation, whereas a more penetrating stimulus of higher intensity is needed to produce pain [16,21]. While pain is enhanced or induced by heating or cooling the skin, itch can be depressed by these stimuli f9,16]. In some clinical and experimental conditions [14,16,28] morphine and acetylsalicylate produce or enhance itch while these same drugs decrease pain. On the basis of our results, it can be proposed that increased scratching is not due to a histamine-mediated itch. We have indeed shown that the administration of an antihistamine substance to arthritic rats did not influence the scratching behaviour (Fig. 4) and that no increased scratching was present in the early acute inflammatory phase of the disease in which histamine is released (Fig. 1). Other arguments can be added. In arthritic rats, the increased scratching is significantly reversed by morphine [7]. This arg~ent gains even more support from the observation that in humans. morphine induces itching (pruritus) [3,16,28]. Experimental work in humans and animals [14,28] has shown that acetylsahcylate induces or exacerbates itch. In our experiments, however, acetylsalicylate induced a significant decrease in scratching (see Fig. 3). Another possible cause of itch is the presence of systemic diseases [16,22]. TO evaluate this, nerve and liver biopsies were taken from the arthritic rats. These did not show any abnormality during the period of maximal scratching (30-50 days). Nor is the increased scratching behaviour due to a lack of body cleaning function, because grooming was not decreased in any phase of the disease (Figs. I and 2). It
129
AUTHORS PARAMETERS I4,5,61
paw diameter / body weight / respif ation t temper~ure
f
vocaiisation f suprofen fentanyl [IO, 11,12, 13,19, x)1
intake / intake /
abnormal neural responses
1261
substance P # (~aminae I, II, X1 (histochem.)
[71
motility / resting I eating / rearing / running 1 scratching t
Fig. (-
5. Comparative picture ); period of maximal
of changed parameters significant changes (_
in arthritic rats. Period of significant ); total period of observation (------).
changes
has been suggested [4] that arthritic rats show wounds at the beginning of the disease and that the healing of these wounds might elicit itch. In our experiments, wounds were not frequent at the beginning of the disease. Furthermore, there was a time lag between the supposed period of healing in the third week of the disease and the period of increased scratching (more than 30 days after the inoculation). Many arguments support the hypothesis that scratching in arthritic rats might be seen as a chronic pain-related behaviour. This increased behaviour, which lasts from 30 up to more than 60 days (Figs. 1 and 2). is time-correlated with other possible pain-related behaviours such as increased vocalisation, analgesic (suprofen) intake, increased paw diameter, and decreased body weight (4-61 (see Fig. 5). Our experiments have shown that the increased scratching is not only reversed by morphine but also by acetylsalicylate (Fig. 3). This was the only behaviour that was depressed by acetylsalicylate. Other motor behaviours (rearing and running) were significantly increased, possibly due to an analgesic effect. In arthritic rats, acetylsalicylate induces a depression of thalamic responses to mechanical stimuli applied to the joint [ll] as well as a depression of the enhanced sensitivity of arthritic joint capsule receptors [12]. Recent results have shown that tricyclic antidepressants (a~t~ptyline or imipramine), which are used extensively to treat chronic pain in man, depress the scratching behaviour in arthritic rats [2].
130
The relationship between scratching and pain is further supported by experiments on substance P, a putative pain-related peptide. Substance P. in acute administration, induces scratching behaviour [8,15,18,25,27]. In mice and rats [17]. substance P antagonists block the algesic effect of substance P in the hot-pIate test. They also abolish biting and scratching behaviour observed after intrathecal administration of substance P. In mice, intracerebrai injection of substance P induces a dose-related scratching response which is prevented by analgesic narcotic agents, this effect being blocked by naloxone [27]. In addition to this role in acute pain. substance P is also involved in chronic pain. This can be suggested on the basis of the correlation between the increased scratching behaviour and the level of substance P in laminae I, II and X of the spinal cord of arthritic rats. This correlation was most pronounced 30 days after inoculation, but it was also present after 15 and 60 days 1261 (see Fig. 5). Finally, electrical stimulation of the nucleus paraventricularis thalami which contains beta-lipotropin and beta-endorphin significantly reduces the scratching behaviour of arthritic rats in a selective way and gives rise to analgesia as measured by hot-plate and tail-flick test (unpublished observations); no such effects were seen while stimulating surrounding structures. In summary, these results strongly suggest that scratching in arthritic rats is not a sign of itch but one of chronic pain.
Acknowledgements The authors are indebted to Dr. G. Guilbaud and Dr. F. Colpaert for valuable suggestions. They thank Mrs. M. Feytons-Heeren and Mr. P. De Sutter for expert technical assistance. The data presented formed part of a thesis for which the University of Louvain has awarded the degree of Doctor in Medical Sciences to M. De Castro-Costa. This work was supported by the F.G.W.O. (Grant 3.0053.83) of Belgium.
References 1 Awouters. F.. Lenaerts, P.M.H. and Niemegeers, C.J.E., Increased incidence of adjuvant arthritis in Wistar rats, Drug Res., 26 (1976) 40-43. F. and Besson. J.-M., Reduction of arthritis and pain 2 Butler, S.H., Weil-Fugazza, J., Godefroy, behaviour following chronic administration of amitriptyline or imipramine in rats with adjuvant-induced arthritis, Pain. 23 (1985) 1599175. 3 Cairns, R.J., The causes of pruritus. In: A. Rook, D.S. Wilkinson and F.J.G. Ebling (Eds.), Textbook of Dermatology, 2nd edn, Davis, Philadelphia, PA, 1972, pp. 1798-1802. 4 Colpaert, F.C., Evidence that adjuvant arthritis in the rat is associated with chronic pain, Pain, 28 (1987) 201-222. 5 Colpaert, F.C.. De Witte, Ph., Maroli, A.N., Awouters, F.. Niemegeers, C.J.E. and Janssen, R.A.J.. Self-ad~nistration induced arthritis
of the analgesic as an experimental
suprofen in arthritic rats: evidence of ~,yco&uc~e~i~~ ~ufy~~cummodel of chronic pain, Life Sci.. 27 (1980) 921-92X.
131 6 Colpaert, F.C., Meert, Th., De Witte, Ph. and Schmitt, P., Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat, Life Sci., 31 (1982) 67-75. 7 De Castro-Costa, CM., De Sutter, P., Gybels, J. and Van Hees, J., Adjuvant-induced arthritis in rats: a possible animal model of chronic pain, Pain, 10 (1981) 173-185. 8 Dobry, P.J.K., Piercey, M.F. and Schroeder, L.A., Pharmacological characterization of scratching behavior induced by intracranial injection of substance P and somatostatin, Neuropharmacology, 20 (1981) 267-272. 9 Fruhstorfer, H., Hermanns, M. and Latzke, L., The effects of thermal stimulation on clinical and experimental itch, Pain, 24 (1986) 259-269. 10 Gautron, M. and Guilbaud, G., Somatic responses of ventrobasal thalamic neurons in polyarthritic rats, Brain Res., 237 (1982) 459-471. 11 Guilbaud, G., Benoist, J.M., Gautron, M. and Kayser, V., Aspirin clearly depresses responses of ventrobasal thalamus neurons to joint stimuli in arthritic rats, Pain, 13 (1982) 153-163. 12 Guilbaud, G., Iggo, A. and Tegner, R., Sensory changes in joint-capsule receptors of arthritic rats: effect of aspirin. In: H.L. Fields et al. (Eds.), Advances in Pain Research and Therapy, Vol. 9, Raven Press, New York, 1985, pp. 81-89. 13 Guilbaud, G., Peschanski, M., Kayser, V. and Benoist, J.M., Properties of nociceptive neurons in the thalamic ventrobasal complex in normal and arthritic rats. In: Development, Organization, and Processing in Somatosensory Pathways, Liss, New York, 1985, pp. 383-391. 14 Hiigermark, O., Influence of antihistamines, sedatives and aspirin on experimental itch, Acta derrn-venereal. (Stockh.), 53 (1973) 363-367. IS Hayes, A.G. and Tyers, M.B., Effects of intrathecal and intracerebrovent~~ar injections of substance P on nociception in the rat and mouse, Brit. J. Pharmacoi., 66 (1979) 488P. 16 Hemdon, J.H., Itching: the pathophysiology of pruritus, Int. J. Dermatol., 14 (1975) 465-477. 17 Henry, J.L., Relation of substance P to pain transmission: neurophysiological evidence. In: Ciba Foundation Symposium. Substance P in the Nervous System, Pitman, London, 1982, pp. 206-224. 18 Hylden, J.L.K. and Wilcox, G.L., Intrathecal substance P elicits a caudally directed biting and scratching behaviour in mice, Brain Res., 217 (1981) 212-215. 19 Kayser, V., Benoist, J.M. and Guilbaud, G., Further evidence for a strong depressive effect of low doses of morphine on VB thalamic neuronal responses (a study on arthritic rats), Brain Res., 267 (1983) 187-191. 20 Kayser. V. and Guilbaud, G., Further evidence for changes in responsiveness of somatosensory neurons in arthritic rats: a study of the posterior intralaminar region of the thalamus, Brain Res., 323 (1984) 144147. 21 Keele, C.A. and Armstrong, D. (Eds.), Substances Producing Pain and Itch, Edward Arnold, London, 1964, pp. 288-304. 22 Madden, E.J., Clinical editorial: itch, Pain, 21 (1985) 313-314. 23 Muller, S.A., Psychocutaneous disorders and neurogenic skin diseases. In: S.L. Moschella and Hurley (Eds.), Dermatology, Vol. 2, Saunders, Philadelphia, PA, 1985, pp. 1833-1834. 24 Pearson, CM. and Wood, F.D., Studies of polyerthritis and other lesions induced in rats by injection of mycobacterial adjuvant. I. General clinical and pathologic characteristics and some modifying factors, Arthr. Rheum., 2 (1959) 440-459. 25 Piercey, M.F., Dobry, P.J.K., Schroeder, L.A. and Einspahr, F.J., Behavioral evidence that substance P may be a spinal cord sensory neurotransmitter, Brain Res., 210 (1981) 407-412. 26 Schoenen, J., Van Hees, J., Gybels, J., De Castro-Costa, CM. and Vanderhaeghen, J.J., Hist~he~cal changes of substance P, FRAP, serotonin and succinic dehydrogenase in the spinal cord of rats with adjuvant arthritis, Life Sci., 36 (1985) 1247-1254. 27 Share, N.N. and Rackham, A., Intracerebral substance P in mice: behavioral effects and narcotic agents, Brain Res., 211 (1981) 379-386. 28 Shelley, W.B. and Arthur, R.P., The neurohistology and neurophysiology of the itch sensation in man, Arch. Derm., 76 (1957) 296-323.
Pain, 29 (1987) 123-131 Rlsevier
PAI 01028
Basic Section Scratching behaviour in arthritic rats: a sign of chronic pain or itch? Mauricio De Castro-Costa *, Jan Gybels, Ronnie Kupers and Johan Van Hees Department (Received
of Neurologv and Neurosurgery, University of Louvain, B-3ooO Louvain {Belgium) 8 July 1986, revised received 9 September
1986, accepted
15 September
1986)
In a previous study, it was shown that adjuvant-induced arthritic rats present an abnormal behaviour pattern up to 60 days after inoctdation with Mycv~eterium butyric~m. The purpose of the present study was to investigate (1) how long the abnormal behaviour pattern continues, and (2) whether the observed increased scratching beha+iour is a parameter of chronic pain or rather a reaction to itch. Adjuvant-induced arthritic rats were observed for up to 180 days after the inoculation and their behaviour was quantitatively analysed. The following behavioural changes persisted for more than 60 days: rearing, running and climbing were decreased while grooming, scratching, biting and freezing were increased. No behavioural changes were observed 120 days after the inoculation. The increased scratching was not influenced by an antihistamine drug (astemizole). Not only morphine but also acetylsalicylate selectively depressed the increased scratching behaviour without influencing the other behaviours. These results reinforce the notion that in arthritic rats the increased scratching is a sign of chronic pain.
S-
Key words:
Adjuvant-induced
arthritis;
Chronic
pain:
Morphine;
Acetylsalicylate;
Itch
Introduction
In a previous paper [7], it was shown that rats with ~ycob~crer~~rn buryricum-induced adjuvant arthritis presented si~fic~t changes in their behaviour. Among these, scratching fulfilled certain criteria making it a possible parameter for chronic pain behaviour. Indeed it was shown that adjuvant poiyarthritis induced an increased scratching behaviour, that morphine reversed this increase and that nalo-
* Present address: e Farmacologia,
Universidade Federal do Ceara, 6O.ooO Fortaka, Brasil.
Faculdade
CQrres~ondence to: J. Gybels, Department of Neurology Gasthuisberg, Herestraat 49, B-3000 Louvain, Belgium.
03043959/87/$03.50
Q 1987 Elsevier Science Publishers
de Medicina,
and
Departamento
Neurosurgery,
B.V. (Biomedical
Division)
U.Z.
de Fisiologia
Sint-Rafa&
124
xone blocked the effect of morphine. It was also shown that the increased scratching behaviour was still present more than 50 days after inoculation. In this study, we wanted to answer the following questions: (1) how long does it take before the abnormal behavioural pattern normalizes, and (2) what is the influence of a peripherally acting analgesic without sedative effects on the arthritic rats’ behaviour? One can argue that the increased scratching in arthritic rats might be a reaction to itch. Arthritic rats show severe signs of inflammation of the hind paws. It is known that in acute inhalator lesions histamine is released which can induce itch and hence also scratching. Arthritic rats present wounds at the onset of the disease; it has been suggested [4] that as these wounds heal around the third week, itch might be elicited. Furthermore, dermatitis, which can elicit a histamine-mediated itch [23], is a possible side effect of adjuvant polyarthritis [24]. In humans, systemic diseases such as liver failure, can also induce itch [22]. Finally, one might suggest that the increased scratching behaviour is simply a means of compensating for a lack of body cleaning function (grooming) in the acute phase of the disease [4]. Therefore, a third objective of this study was to search for evidence that scratching in arthritic rats is not a reaction to itch.
Methodology One hundred and twenty-two Wistar male rats, weighing about 250 g, were used. Sixty-four of them were inoculated intradermally in the base of the tail with 0.05 mI of a paraffin oil suspension of heat-killed ~_yc~bacter~~m butyricum (5 mg/ml) [l]. The other 58 rats, used as controls, were inoculated with only the sterile vehiculum (paraffin oil). For the time evolution of the rats’ behaviour, the animals were divided into different groups: 24 rats * were observed during the period O-60 days after the inoculation (18 arthritic rats and 6 vehiculum-inoculated rats) and 54 rats were observed 60, 120 or 180 days after the inoculation (27 arthritic rats and 27 ve~culum-inoculated rats). Lysine acetylsalicylate was injected intr~uscularly in a dose of 75 mg/kg, 30-50 days after the inoculation with Mycobacterium butyricum. Observations started 30 min after the injection. The acetylsalicylate-injected arthritic rats (N = 6) were compared with others which received no drugs (N = 6) or only the vehiculum (N = 6). To rule out the possibility that the increased scratching behaviour might be due to a histamine-mediated itch, some experiments with antihistamine drugs were ** is an Hl-~sta~ne antagonist with a miniperformed. Astemizole (Hisman~) mum of side effects on the nervous system. It was administered to arthritic rats in the post-acute (N = 6) and very late phase (N = 7) of the disease in a dose of 1 * The spinal cord was removed from these rats for histochemical analysis. The results of this analysis are presented elsewhere [26]. * * We received this drug and information on its characteristics from Janssen Pharmaceutics.
125
mg/kg. For this, a suspension of the substance was injected into the centre of the food pellets given to the 24-h fasting rats. Observations started 120 min after food intake which was well within the period of the drug’s maximal effect. The results were compared with those from the same animals receiving no drugs. Statistical analysis of the results was performed using non-paramet~c tests; P c 0.05 (two-tailed) was considered as significant. For all experiments we adopted the same methodology of quantified behavioural observation as described in a previous paper [7]. The guidelines, published in a Guest Editorial in Pain on ethical standards for investigations of experimental pain in animals, have been followed (Pain, 16 (1983) 109-110). Results
The analysis of the arthritic rats’ behaviour from 0 up to 60 days has shown the following: the arthritic rats (N = 6) of the acute phase (lo-30 days) exhibited a significant decrease (P c 0.005) in rearing, sniffing, running and climbing and a EFFECT
OF
SNIFFING
ARTHRITIS
RUNNING
ON
BEHAVIOUR
GROOMING
(N-61
SCRATCHING
500 _
.
hi 2 N : is u) 0:
cl.4 EATING
FREEZING
BITING
II
pcooo5
m
p-zoo5
Ez
D 1005
LL RESTING
Fig. 1. The behaviour of arthritic rats (immediate acute, behaviour of normal rats. Median values in xc/O.5 Mu-W~tney U-test was used to compute the level of appears in the early post-acute phase and late phase inflammation).
SLEEPING
post-acute, and late phases) is compared with the h observation of 6 rats are represented. The significance. Increased scratching behaviour only and not in the acute phase (phase of maximal
126
EFFECT
OF
ARTHRITIS
ON
BEHAVIOUR
tN:lOl
m
pc5005
fita p-zoo5 lza
Fig with level lasts
p>oos
2. The behaviour of arthritic rats of the very late phase ( > 60 days after the inoculation) is compared the behaviour of normal rats. Median values in set/0.5 h observation of 10 rats are represented. The of probability is computed by the Mann-Whitney U-test. The scratching behaviour in arthritic rats more than 60 davs after the inoculation. EFFFCT
OF
ACETYLSALICILATE (N ~6)
m m
P'OOS P4005
d 4
s 111 p >0.05
1
-IJ
5 --
Fig. 3. A: effect of acetylsalicylate (75 mg/kg i.m.) on the behaviour of arthritic rats, 30-50 days after the inoculation with M_vcohucferium hu~vrie~m (N = 6). Median values in sec/O.S h observation are represented. Level of probability is computed by the Wilcoxon matched pairs signed rank test, comparing acetylsalicylate effect with the no-drug condition. Scratching behaviour is significantly reversed (depressed) by acetylsaiicylate. B: comparison of the acetylsalicylate effect with the vehiculum effect in the same animals.
significant increase (P < 0.005) in grooming, resting and sleeping (Fig. 1). The phase (30-50 days) showed a significant arthritic rats (N = 6) in the pust-acute decrease (P -C O,.OOS>in rearing, sniffing, running and climbing, and a significant increase in scratching (P -=cO.OOS), biting (P -E0.05) and sleeping (P -C 0.005) (Fig. 1). In the late phase (50-60 days), the arthritic rats (N = 6) also exhibited significant changes in their behaviour: eating (P < O.OOS), rearing (P i 0.005), running (P < 0.005) and climbing (P -C0.05) were decreased, while grooming (P -zO.OS), scratching (P < O.OOS), biting (P -C0.05) and sleeping (P < 0.005) were increased in comparison to normal rats (Fig. 1). Some of these behavioural changes persisted fur more than 60 days: rearing (P < O.OOS), running f P < 0.005) and climbing (P -C 0.005) were still decreased, while grooming (P -=z 0.05), scratching (P c O.OOS), biting (P -=c 0.05) and freezing (P < 0.05) (Fig. 2) were still increased (N = 10). However, 120 days (N = 7) or 180 days (N = 10) after the inoculation, no more significant changes in the rats’ behaviours were present. Statistical analysis was performed using the Mann- Whitney U-test. In comparison with arthritic rats without drugs (N = S), arthritic rats which received acetylsalicylate (N = 6) ran more (P -C 0.05) and scratched Iess (P -e0.05). In comparison with rats which received only the vehiculum (N = 6), acetylsalicylate-injected animals showed more rearing and running (P < 0.05) and
EFFECT OF ASTEMIZULE
N:7
Fig. 4. Effect of astemizole (1 mg/kg p.0.) on behaviour in 2 groups of arthritic rats (N = 6, 30-50 days after inoculation; N = 7. > 60 days after inoculation). Median values in seci0.5 h observation of 6 rats are represented. Level of probability is computed by the Wilcoxon matched pairs signed rank test. The antihistamine drug induces no significant change in the behaviour of arthritic rats.
128
less scratching (P < 0.05). The Wilcoxon matched pairs signed ranks test was used for the statistical analysis (Fig. 3). The arthritic rats (N = 6, 30--50 days after inoculation. and N = 7. ) 60 days after inoculation) which received astemizole were compared with the same rats without this drug. The results showed no change in their level of scratching behaviour. Furthermore, no change in the other behaviours was observed (Fig. 4). The Wilcoxon matched pair test was used for the statistical analysis.
Discussion First of all, we will discuss the finding that scratching is not an itch-related behaviour. Then we will present some arguments which favour the hypothesis that scratching might be a chronic pain-related form of behaviour. Itch may be induced by many factors such as external physical and chemical stimuli, metabolic diseases, several skin pathologies and psychological disturbances, In some of these conditions, itch may be mediated by a release of histamine [ 16,22,23]. Generally it is assumed that itch and mild pain have a common neural substrate [16]. Both are evoked by activation of unmyelinated fibres. Anterolateral cordotomy abolishes both itch and pain. Furthermore, in patients with congenital insensitivity to pain, itch is absent. A difference is, however, that itch is elicited only from the most superficial layers of the skin, while pain can also be elicited from deep structures. Noxious stimulation of low intensity and long duration on the outermost layers of the skin may induce an itching sensation, whereas a more penetrating stimulus of higher intensity is needed to produce pain [16,21]. While pain is enhanced or induced by heating or cooling the skin, itch can be depressed by these stimuli f9,16]. In some clinical and experimental conditions [14,16,28] morphine and acetylsalicylate produce or enhance itch while these same drugs decrease pain. On the basis of our results, it can be proposed that increased scratching is not due to a histamine-mediated itch. We have indeed shown that the administration of an antihistamine substance to arthritic rats did not influence the scratching behaviour (Fig. 4) and that no increased scratching was present in the early acute inflammatory phase of the disease in which histamine is released (Fig. 1). Other arguments can be added. In arthritic rats, the increased scratching is significantly reversed by morphine [7]. This arg~ent gains even more support from the observation that in humans. morphine induces itching (pruritus) [3,16,28]. Experimental work in humans and animals [14,28] has shown that acetylsahcylate induces or exacerbates itch. In our experiments, however, acetylsalicylate induced a significant decrease in scratching (see Fig. 3). Another possible cause of itch is the presence of systemic diseases [16,22]. TO evaluate this, nerve and liver biopsies were taken from the arthritic rats. These did not show any abnormality during the period of maximal scratching (30-50 days). Nor is the increased scratching behaviour due to a lack of body cleaning function, because grooming was not decreased in any phase of the disease (Figs. I and 2). It
129
AUTHORS PARAMETERS I4,5,61
paw diameter / body weight / respif ation t temper~ure
f
vocaiisation f suprofen fentanyl [IO, 11,12, 13,19, x)1
intake / intake /
abnormal neural responses
1261
substance P # (~aminae I, II, X1 (histochem.)
[71
motility / resting I eating / rearing / running 1 scratching t
Fig. (-
5. Comparative picture ); period of maximal
of changed parameters significant changes (_
in arthritic rats. Period of significant ); total period of observation (------).
changes
has been suggested [4] that arthritic rats show wounds at the beginning of the disease and that the healing of these wounds might elicit itch. In our experiments, wounds were not frequent at the beginning of the disease. Furthermore, there was a time lag between the supposed period of healing in the third week of the disease and the period of increased scratching (more than 30 days after the inoculation). Many arguments support the hypothesis that scratching in arthritic rats might be seen as a chronic pain-related behaviour. This increased behaviour, which lasts from 30 up to more than 60 days (Figs. 1 and 2). is time-correlated with other possible pain-related behaviours such as increased vocalisation, analgesic (suprofen) intake, increased paw diameter, and decreased body weight (4-61 (see Fig. 5). Our experiments have shown that the increased scratching is not only reversed by morphine but also by acetylsalicylate (Fig. 3). This was the only behaviour that was depressed by acetylsalicylate. Other motor behaviours (rearing and running) were significantly increased, possibly due to an analgesic effect. In arthritic rats, acetylsalicylate induces a depression of thalamic responses to mechanical stimuli applied to the joint [ll] as well as a depression of the enhanced sensitivity of arthritic joint capsule receptors [12]. Recent results have shown that tricyclic antidepressants (a~t~ptyline or imipramine), which are used extensively to treat chronic pain in man, depress the scratching behaviour in arthritic rats [2].
130
The relationship between scratching and pain is further supported by experiments on substance P, a putative pain-related peptide. Substance P. in acute administration, induces scratching behaviour [8,15,18,25,27]. In mice and rats [17]. substance P antagonists block the algesic effect of substance P in the hot-pIate test. They also abolish biting and scratching behaviour observed after intrathecal administration of substance P. In mice, intracerebrai injection of substance P induces a dose-related scratching response which is prevented by analgesic narcotic agents, this effect being blocked by naloxone [27]. In addition to this role in acute pain. substance P is also involved in chronic pain. This can be suggested on the basis of the correlation between the increased scratching behaviour and the level of substance P in laminae I, II and X of the spinal cord of arthritic rats. This correlation was most pronounced 30 days after inoculation, but it was also present after 15 and 60 days 1261 (see Fig. 5). Finally, electrical stimulation of the nucleus paraventricularis thalami which contains beta-lipotropin and beta-endorphin significantly reduces the scratching behaviour of arthritic rats in a selective way and gives rise to analgesia as measured by hot-plate and tail-flick test (unpublished observations); no such effects were seen while stimulating surrounding structures. In summary, these results strongly suggest that scratching in arthritic rats is not a sign of itch but one of chronic pain.
Acknowledgements The authors are indebted to Dr. G. Guilbaud and Dr. F. Colpaert for valuable suggestions. They thank Mrs. M. Feytons-Heeren and Mr. P. De Sutter for expert technical assistance. The data presented formed part of a thesis for which the University of Louvain has awarded the degree of Doctor in Medical Sciences to M. De Castro-Costa. This work was supported by the F.G.W.O. (Grant 3.0053.83) of Belgium.
References 1 Awouters. F.. Lenaerts, P.M.H. and Niemegeers, C.J.E., Increased incidence of adjuvant arthritis in Wistar rats, Drug Res., 26 (1976) 40-43. F. and Besson. J.-M., Reduction of arthritis and pain 2 Butler, S.H., Weil-Fugazza, J., Godefroy, behaviour following chronic administration of amitriptyline or imipramine in rats with adjuvant-induced arthritis, Pain. 23 (1985) 1599175. 3 Cairns, R.J., The causes of pruritus. In: A. Rook, D.S. Wilkinson and F.J.G. Ebling (Eds.), Textbook of Dermatology, 2nd edn, Davis, Philadelphia, PA, 1972, pp. 1798-1802. 4 Colpaert, F.C., Evidence that adjuvant arthritis in the rat is associated with chronic pain, Pain, 28 (1987) 201-222. 5 Colpaert, F.C.. De Witte, Ph., Maroli, A.N., Awouters, F.. Niemegeers, C.J.E. and Janssen, R.A.J.. Self-ad~nistration induced arthritis
of the analgesic as an experimental
suprofen in arthritic rats: evidence of ~,yco&uc~e~i~~ ~ufy~~cummodel of chronic pain, Life Sci.. 27 (1980) 921-92X.
131 6 Colpaert, F.C., Meert, Th., De Witte, Ph. and Schmitt, P., Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat, Life Sci., 31 (1982) 67-75. 7 De Castro-Costa, CM., De Sutter, P., Gybels, J. and Van Hees, J., Adjuvant-induced arthritis in rats: a possible animal model of chronic pain, Pain, 10 (1981) 173-185. 8 Dobry, P.J.K., Piercey, M.F. and Schroeder, L.A., Pharmacological characterization of scratching behavior induced by intracranial injection of substance P and somatostatin, Neuropharmacology, 20 (1981) 267-272. 9 Fruhstorfer, H., Hermanns, M. and Latzke, L., The effects of thermal stimulation on clinical and experimental itch, Pain, 24 (1986) 259-269. 10 Gautron, M. and Guilbaud, G., Somatic responses of ventrobasal thalamic neurons in polyarthritic rats, Brain Res., 237 (1982) 459-471. 11 Guilbaud, G., Benoist, J.M., Gautron, M. and Kayser, V., Aspirin clearly depresses responses of ventrobasal thalamus neurons to joint stimuli in arthritic rats, Pain, 13 (1982) 153-163. 12 Guilbaud, G., Iggo, A. and Tegner, R., Sensory changes in joint-capsule receptors of arthritic rats: effect of aspirin. In: H.L. Fields et al. (Eds.), Advances in Pain Research and Therapy, Vol. 9, Raven Press, New York, 1985, pp. 81-89. 13 Guilbaud, G., Peschanski, M., Kayser, V. and Benoist, J.M., Properties of nociceptive neurons in the thalamic ventrobasal complex in normal and arthritic rats. In: Development, Organization, and Processing in Somatosensory Pathways, Liss, New York, 1985, pp. 383-391. 14 Hiigermark, O., Influence of antihistamines, sedatives and aspirin on experimental itch, Acta derrn-venereal. (Stockh.), 53 (1973) 363-367. IS Hayes, A.G. and Tyers, M.B., Effects of intrathecal and intracerebrovent~~ar injections of substance P on nociception in the rat and mouse, Brit. J. Pharmacoi., 66 (1979) 488P. 16 Hemdon, J.H., Itching: the pathophysiology of pruritus, Int. J. Dermatol., 14 (1975) 465-477. 17 Henry, J.L., Relation of substance P to pain transmission: neurophysiological evidence. In: Ciba Foundation Symposium. Substance P in the Nervous System, Pitman, London, 1982, pp. 206-224. 18 Hylden, J.L.K. and Wilcox, G.L., Intrathecal substance P elicits a caudally directed biting and scratching behaviour in mice, Brain Res., 217 (1981) 212-215. 19 Kayser, V., Benoist, J.M. and Guilbaud, G., Further evidence for a strong depressive effect of low doses of morphine on VB thalamic neuronal responses (a study on arthritic rats), Brain Res., 267 (1983) 187-191. 20 Kayser. V. and Guilbaud, G., Further evidence for changes in responsiveness of somatosensory neurons in arthritic rats: a study of the posterior intralaminar region of the thalamus, Brain Res., 323 (1984) 144147. 21 Keele, C.A. and Armstrong, D. (Eds.), Substances Producing Pain and Itch, Edward Arnold, London, 1964, pp. 288-304. 22 Madden, E.J., Clinical editorial: itch, Pain, 21 (1985) 313-314. 23 Muller, S.A., Psychocutaneous disorders and neurogenic skin diseases. In: S.L. Moschella and Hurley (Eds.), Dermatology, Vol. 2, Saunders, Philadelphia, PA, 1985, pp. 1833-1834. 24 Pearson, CM. and Wood, F.D., Studies of polyerthritis and other lesions induced in rats by injection of mycobacterial adjuvant. I. General clinical and pathologic characteristics and some modifying factors, Arthr. Rheum., 2 (1959) 440-459. 25 Piercey, M.F., Dobry, P.J.K., Schroeder, L.A. and Einspahr, F.J., Behavioral evidence that substance P may be a spinal cord sensory neurotransmitter, Brain Res., 210 (1981) 407-412. 26 Schoenen, J., Van Hees, J., Gybels, J., De Castro-Costa, CM. and Vanderhaeghen, J.J., Hist~he~cal changes of substance P, FRAP, serotonin and succinic dehydrogenase in the spinal cord of rats with adjuvant arthritis, Life Sci., 36 (1985) 1247-1254. 27 Share, N.N. and Rackham, A., Intracerebral substance P in mice: behavioral effects and narcotic agents, Brain Res., 211 (1981) 379-386. 28 Shelley, W.B. and Arthur, R.P., The neurohistology and neurophysiology of the itch sensation in man, Arch. Derm., 76 (1957) 296-323.