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The orofacial formalin test in rats: effects of different formalin concentrations
Pain, 62 (1995) 295-301
295
Elsevier Science B.V.
PAIN 2781
The orofacial formalin test in rats: effects of different formalin concentrations Pierre Clavelou a, R a d h o u a n e Dallel a, Thierry Orliaguet b, Alain W o d a a and Patrick Raboisson a,* a Laboratoire de Physiologie Oro-Faciale and b D~partement de Biologie, Facult~ de Chirurgie Dentaire, Clermont-Ferrand 63000 (France)
(Received 30 September 1994, revision received 5 December 1994, accepted 7 December 1994)
Summary In this study of the orofacial formalin test in rats, the effects of different formalin concentrations (0.2%, 0.5%, 1.5%, 2.5%, 5% and 10%) on the behavioural nociceptive response (face rubbing) was investigated. The histological responses of the skin were also evaluated. Increasing the concentration of formalin caused a parallel aggravation of histological signs of tissue inflammation and injury. All concentrations provoked an early phase of nociceptive response, but its intensity was not concentration-dependent. The 2nd phase of response to formalin only occurred for concentrations of 1.5% and higher. A positive relationship between the formalin concentration and the amplitude of the rubbing activity measured between 12 and 45 min after injection could be observed until 2.5% but with the highest concentrations (5 and 10%), the amplitude of the response decreased. Our findings indicate that the orofacial formalin test should be carried out using concentration between 0.5 and 2.5%. This is essential to assess increase as well as decrease in pain intensity. Moreover, this will have the effect of minimizing the suffering of the experimental animal. Key words: Nociception; Formalin test; Inflammation; Formalin concentration; Orofacial region; (Rat)
Introduction The subcutaneous (s.c.) injection of dilute formalin is now a widely used model of nociception. It generates behavioural as well as electrophysiological responses that last from several minutes up to more than 1 h and which are sensitive to a wide range of analgesics. The typical time course of the response to formalin is biphasic, with an early and short-lasting 1st phase followed, after a quiescent period, by a 2nd prolonged (tonic) phase. In this respect, formalin tests have been regarded to be a more satisfactory model of clinical pain than tests producing phasic pain like the hot-plate or tail-flick tests (e.g., Abbott et al. 1981). Mice and rats are the most frequently used animals but other species including cats, rabbits, primates, guinea pigs, naked mole-rats have also been employed (Tj¢lsen et al. 1992; for review see, Porro and Cavazzuti 1993). * Corresponding author: Dr. Patrick Raboisson, Laboratoire de
Physiologic Oro-Faciale, Facult6 de Chirurgie Dentaire, 11 Blvd. Charles de Gaulle, 63000 Clermont-Ferrand, France. Tel.: (33) 7343-6414; FAX: (33) 7343-6409. SSDI 0304-3959(94)00273-8
Volumes and concentrations of formalin solutions vary according to the studies from 20 to 150 /~1 and from 0.02 to 15%, respectively. Many methods for assessing pain after formalin have been published. For instance, after injection into the dorsal or plantar surface of a fore- or hindpaw, some authors rate single parameters, such as flinching/shaking/jerking (e.g., WheelerAceto and Cowan 1991) or licking (Rosland et al. 1990) the paw, while others use several categories of nociceptive behaviours to derive weighted scores (e.g., Dubuisson and Dennis 1977; Coderre et al. 1993). Other methods score behavioural responses related or not to pain (e.g., Berge et al. 1991; Kristensen et al. 1994). The advantage of the orofacial formalin test is that it uses as a single 'pain' indicator, the amount of time spent rubbing the injected area (upper lip). This was shown to be a reliable method for assessing pain in the trigeminal region (Clavelou et al. 1989) and has been used with success in several recent studies (Klein et al. 1991; Aigouy et al. 1992; Cadet et al. 1993; Eisenberg et al. 1993). The present study was undertaken to provide a better understanding of the orofacial formalin test. In
296
particular, we wanted to check if there was a positive relationship between the amount of time the rats spent rubbing their lip and the concentration of the formalin solution. We also investigated the histological changes caused by different concentrations of formalin. From these results, some practical suggestions for standardising the orofacial formalin test are proposed.
Material and methods
Animals Seventy male Sprague-Dawley rats weighing 170-220 g (IffaCredo, France) were used in this experiment. Rats were housed in plastic cages with soft bedding (4 per cage) with free acces to food and water. They were maintained in climate- (23_+ I°C) and lightcontrolled (12-h dark/light cycle with light on at 08:00 h) protected units (Iffa-Credo, France) for at least 1 week before the experiments. Testing sessions took place during the light phase between 1 I:00 and 19:00 h in a quiet room maintained at 23-24°C. The test box had dimension of 30 x 30 x 30 cm with 3 mirrored sides. Each animal was first placed in this box for a 10-min habituation period to minimise stress. Rats did not have access to food or water during the test. Experiments conformed to Ethical Guidelines recommended by the International Association for the Study of Pain for experimental pain in conscious animals ( Z i m m e r m a n n 1983).
Testing procedure Rats were randomly assigned to 7 groups (n = 10 per group) and received a 50/xl subcutaneous (s.c.) injection of diluted formalin or saline (0.0%) into the right upper lip, just lateral to the nose. Solutions were prepared from commercially available stock formalin further diluted in isotonic saline to 0.2%, 0.5%, 1.5%, 2.5%, 5% and 10%. Stock formalin is an aqueous solution of 37% formaldehyde.
70
-
Following injection, the rat was immediately returned to the test box for a 45-min observation period. Analysis of behaviour was made by an investigator who was blinded to the animal's group assignment. The recording time was divided into 15 blocks of 3 min and a pain score was determined for each block by measuring the number of seconds (amplitude of the response) that the animal spent rubbing the injected area with the ipsilateral fore- or hindpaw. Although this behaviour resembles face washing, it has been shown that a prolonged asymmetrical distribution of paw strokes to the face does not occur spontaneously in normal rats (Berridge 1990). However, we previously pointed out that rubbing by the ipsilateral forepaw is often accompanied by similar movements of the contralateral one (Clavelou et al. 1989). Therefore, a control group received saline instead of formalin to quantify bilateral face washing behaviour in the absence of formalin.
Tissue preparation In each group, 2 animals were killed 1 h, 4 h, 24 h, 5 days or Ill days after the formalin injection by an overdose of pentobarbital. The injected area (right upper lip near the vibrissae pad) was removed and immediately fixed in acetic acid-formalin-ethanol for 48 h and embedded in paraffin. In addition, the left upper lip of 1 rat killed 10 days after injection of saline was removed and treated according to the same procedure to serve as control. Sections (15 /zm) were stained with haematoxylin-eosin, and with toluidine blue. The preparations were made and analysed by 3 observers unaware of the used concentration and survival time. The inflammatory response was evaluated in terms of mast cell degranulation, oedema, the and invasion of granulocytes. The cicatrization was measured by the level of fibrin production. A 5-point scale was used for oedema and fibrin production and a 4-point scale for the quantification of the mast cell degranulation. The results of the 3 observers were compared and averaged.
Statistical analysis W h e n tested with a Iog-ANOVA test, data with homogeneity of variance were analysed using 1-way analysis of variance (ANOVA).
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Fig. 1. Time course of the face rubbing activity observed after s.c. injection of saline (0.0%) or different concentrations of formalin. The mean n u m b e r of seconds that rats (n = 10 per concentration) spent rubbing is plotted for each 3-min block over the 45 min of post-injection observation period. Solid symbols indicate significant differences ( P < 0.05) compared to the saline (0.0%) group (Dunnett's t test, subsequent to A N O V A for block 1; Newman-Keuls test subsequent to Kruskal-Wallis test for the others).
297 Multiple post-hoc comparisons were performed using Dunnett's t test (control group= 0.0%) or Newman-Keuls test. Data without homogeneity of variance were analysed using the Kruskal-Wallis H test and multiple post-hoc comparisons were made using non-parametric Newman-Keuls test. For all tests the level of significancewas set at P < 0.05. Data are expressed as means + SEM.
A
First Phase 70605O 40
Results
Effect of formalin concentration on the nociceptive response The time course of the nociceptive responses to the administration of the different concentrations of formalin is presented in Fig. 1. All formalin concentrations from 0.2% to 10% induced the 1st phase of the response which did not last beyond the 3rd minute after injection (Fig. 1). The amplitude of the response seemed to reach a maximum at a concentration of 1.5% (Fig. 2A) and to decrease above but there was no significant difference between concentrations (Newman-Keuls test subsequent to ANOVA). The 2nd phase of response only appeared with formalin concentrations of 1.5% and higher (Fig. 1). It started 12 min after the formalin injection (block 5) for concentrations between 1.5 and 5%. For the highest concentration (10%), the onset of the rubbing activity was delayed until 15 min (block 6) and remained significantly lower than the response to 1.5% (until block 6) and to 2.5 and 5% (until block 8) (Newman-Keuls test subsequent to Kruskal-Wallis test, P < 0.05). Thirty minutes after formalin injection (block 11), there was no longer any significant difference between responses to 1.5% formalin and saline. By the end of the experiment (45 min), the rubbing level had returned to baseline for 2.5% but was still significantly elevated for the higher concentrations (Newman-Keuls test subsequent to Kruskal-Wallis test, P < 0.05). The graph shown in Fig. 2B suggests that there was a positive relationship between the amplitude of the global rubbing activity measured between 12 and 45 min after the formalin injection and the concentration of formalin until 2.5%. At higher concentration (5 and 10%), the amplitude of the response decreased. (Fig. 1 and 2B). The mean amplitude of the rubbing activity was significantly higher at 2.5% concentration, compared to 1.5% and 10% ( P < 0.01 and P < 0.05, respectively; Newman-Keuls test subsequent to A N O V A ) (Fig. 2B).
Tissue changes caused by formalin Extravasated cells, mainly granulocytes, were only seen with formalin. All mast cells were degranulated until at least 4 h with concentration of 1.5% and higher (Table IA). At the lowest formalin concentrations (0.2%), the oedema was not different from that observed after injection of saline but the higher concen-
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Fig. 2. Effect of increasing concentrations of formalin on (A) the ]st phase (0-3 min post-formalin) and (B) the 2nd phase (12-45 rain post-formalin) of rubbing activity. Solid symbols (e) indicated significant differences (P < 0.0]) compared to the saline group (0.0%, open symbols: ©). Significant differences from the 2.5% group are indicated by * P < 0.05; * * P < 0.0! (Dunnctt's t test, subsequent to
ANOVA). trations caused an increase in both the size and the duration of the oedema. With concentrations of 1.5% and higher, the oedema was still present 24 h after formalin injection and residual traces persisted at 5 days (Table IA). The concentration of formalin had an effect on the fibrin production; cicatrization was achieved 10 days after injection with concentrations below or equal to 1.5%, while it was incomplete or just beginning whith higher concentrations (Table IB and Fig. 3).
Discussion
The rubbing of the formalin-injected upper lip with the ipsilateral fore- or hindpaw is a characteristic and
298
consistent response in the rat. This response is sensitive to both narcotic and non-narcotic analgesic drugs at doses that do not impair motor activity (Clavelou et al. 1989). Similar rubbing (or grooming, see Vos et al. 1994 for review) has also been observed after noxious electrical, thermal or mechanical stimulation applied to the face (Rosenfeld et al. 1978; Morris et al. 1982; Dallel et al. 1989; Cahusac et al. 1990) or following experimental neuropathy induced by the ligation of the infraorbital nerve in rat (Vos and Maciewicz 1991; Vos et al. 1994). The present study confirms that s.c. formalin but not saline evokes sustained episodes of stereotyped rubbing of the injected site (a similar inefficiency of saline at inducing nociceptive behaviours has already been noted in the paw test; Fanselow and Baackes 1982; Wheeler-Aceto et al. 1990). As it has previously been reported by Rosland et al. (1990), histological data show good agreement between the importance of tissue damages and the concentration of formalin. In our experiment, except a certain degree of swelling, no marked alteration was observed with saline. On the contrary, the mast cell degranulation, which is one of the earliest histological changes
signalling tissue injury, as well as the extravasation of granulocytes, were seen even at the lowest formalin concentration (0.2%). The inflammatory signs progressively intensified when the formalin concentrations increased and, 10 days after the injection of formalin, the cicatrization was not achieved for concentration above 1.5%. Since the amplitude of the rubbing activity paralleled the degree of tissue damage caused by rising concentrations of formalin (only to a certain extent; this point will be discussed further), our data support the assumption that persistent face rubbing, focused on the application site of a noxious stimulus, is a behaviour signalling facial pain. The data reported here indicate that all formalin concentrations, from 0.2% to 10% (and for a 50 ~1 volume), provoke an early behavioural nociceptive response (upper lip rubbing) the duration of which did not exceed 3 min after injection. Although the amplitude of this response was maximal in the 1.5% group, there was no significant difference between concentrations. Rosland et al. (1990) noted a similar ceiling effect in mice when paw licking was used as the behavioural nociceptive response. In rats, Wheeler-Aceto
TABLE 1 H I S T O L O G I C A L C H A N G E S A C C O R D I N G TO T H E F O R M A L I N C O N C E N T R A T I O N A T D I F F E R E N T T I M E I N T E R V A L S A F T E R INJECTION 1h
4h
24 h
5 days
10 days
A: oedema (1st time) and mast cell degranulation (2nd time) Saline (0.0%)
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2.5% Formalin
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5% Formalin
[ ** + + +
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10% Formalin
[ ,+ + +
,+~+ +
++ 0 ++ 0 ++ 0 +++ 0
+/0 +/0 + 0 +/0
0 0 0 0 0 0 0 0
+ + + + + + +
+ + + + + + +
0 = no oedema or no mast cell degranulation. Oedema: + / - , residual; + , discrete; + + , important; + + + , very important. Mast cell degranulation: * / - , residual; *, partial; * *, complete fibrin production; 0, absence; + / - , in progress; + , partial; + + , incomplete; + + + , completed. B: cicatrization Saline (0.0%) 0.2% Formalin 0.5% Formalin 1.5% Formalin 2.5% Formalin 5% Formalin 10% Formalin
0 0 0 0 0 0 0
Fibrin production: 0, absence; + / - ,
0 +/ +/ +/ 0 0 0
+ + + + +/ + +/ -
in progress; + , partial; + + , incomplete; + + + , completed.
+ + + +
+ + + + + +
+ + + +
299
and Cowan (1993) observed that the time spent licking the injected paw during the 1st phase decreased above 1.5% and was even absent at the highest concentration tested (15%). Thus, a single nociceptive indicator such as face rubbing or paw licking may not be suitable for measuring pain intensity during the 1st phase, at formalin concentrations higher than 0.2%. Other behaviours (e.g., freezing) may supersede rubbing (see below). The 2nd phase of rubbing activity only appeared at formalin concentrations above 0.5%. Using the paw formalin test in the rat, Coderre et al. (1993) obtained similar findings for score 2 (paw elevation) and 3 (licking, biting of the paw). In mice, Rosland et al. (1990) noted a 2nd phase at formalin concentrations higher than 0.2%. In our study, the total time spent rubbing the face (measured between 12 and 45 min after the formalin injection) increased in a concentration-dependent fashion to reach a maximum at 2.5% (Fig. 2B). In the paw formalin test, a similar positive r
relationship between formalin concentration and the amount of time spent licking was also observed in rats (Coderre et al. 1993; Wheeler-Aceto and Cowan 1993) and mice (Rosland et al. 1990) up to concentrations of 5%. However, the administration of the highest formalin concentrations were associated with a decrease in face rubbing (at 5 and 10% in our study), paw licking (15%, Wheeler-Aceto and Cowan 1993) or paw elevation (5%, Coderre et al. 1993). In their studies, Rosland et al. (1990) and Coderre et al. (1993) observed a ceiling effect rather than a reduction for paw licking or for score 3 but they did not use concentrations above 5%. One explanation for these paradoxical decreases or ceiling effects is that high formalin concentrations induce other behavioural reactions, rarely seen at low concentrations, that may interfere with the primary behaviour (Fanselow and Baackes 1982). For example, in our experiments, long periods of freezing during which the rat stood immobile, sometimes with its forepaws applied on the face, could be seen with
~ , i ¸~
Fig. 3. Photomicrographs of upper lip sections. A: untreated animal (magnification: x 80 × 6.3). B: 4 h after 1.5% formalin: acute inflammatory phase with oedema, dissociated connective tissue, invasion of granulocytes and diapedesis of ieukocytes through congestive capillary blood vessels (magnification: x 80 x 6.3). C: 10 days after 1.5% formalin: terminal phase of cicatrization with accumulation of fibrin, newgrown muscle fibres and some areas of young connective tissue (magnification: × 80 x 6.3). D: 10 days after 10% formalin: partial cicatrization with large areas of loose connective tissue and presence of numerous fibroblasts (magnification: x 30 x 2.5). Abbreviations: A, artery; CC, congestive capillary blood vessel; CT, connective tissue; DCT, dissociated connective tissue; F, fibrin; FB, fibroblasts; GN, granulocytes; LCT, loose connective tissue; MF, muscle fibres; N, nerve fibres; NMF, newgrown muscle fibres; OE, oedema; YCT, young connective tissue.
300 formalin concentrations above 2.5%. Rosland et al. (1990) also noted that, in mice, the highest formalin concentrations induced other reactions along with licking or biting. Since the several modalities that compose pain may elicit different behavioural reactions, some authors argue that techniques taking into account more than one behaviour are more valid measures of pain intensity than single-parameter m e t h o d s (e.g., Tj¢lsen et al. 1992; C o d e r r e et al. 1993). However, the use of various behaviours reflecting supposedly different levels of nociceptive activity, does not resolve the problem. For instance, the weighted pain intensity score during the 2nd phase reaches a ceiling at 5% (Coderre et al. 1993). In any case, we believe that the use of high concentrations of formalin can lead to paradoxical conclusions. For instance, a score could rise when an analgesic was given because a conflicting behaviour was suppressed (e.g., freezing) while a pronociceptive drug may have the opposite action (or no apparent action when the concentration-response function reaches a ceiling). Thus, when high formalin concentrations are employed, the decrease of a pain score may not be sufficient to d e m o n s t r a t e antinociception. O n the contrary, by using low concentrations, it becomes possible to trustfully assess increases as well as decreases in pain intensity. In line with this, Rosland et al. (1990) noted that low doses of indomethacin are more potent at reducing the 2nd phase of paw licking induced with 1% formalin than with 5% and C o d e r r e and Melzack (1992a,b) found that the hyperalgesic effects of intrathecal excitatory amino acids or calcium ionophore a p p e a r e d when using 1% but not 5% formalin. Whatever the test, the choice of a given formalin concentration may then not be without consequence. To avoid ambiguous changes in the total pain score, its is necessary to work on the rising slope of the concentrationresponse function. A n o t h e r a r g u m e n t against the use of high formalin concentrations is given by histological data. Analysing the fibrin production showed that, 10 days after injection, the cicatrization was not completed at formalin concentrations of 2 . 5 - 1 0 % , indicating persistent damages. Since a robust tonic phase of rubbing, associated with less severe peripheral injury, appears at 1.5%, the use of higher concentrations does not seem justified in the orofacial formalin test, unless the aim of the experiment requires maximal amplitude of the nociceptive response or the existence of a lengthy inflammatory focus. T h e main advantages of single-parameter methods, such as the 'orofacial formalin test', is the simplicity of the scoring technique. T h e observations reported here help to refine the model by showing that it will be most sensitive if formalin concentrations between 0.5 and
2.5% are used. This is essential when the effects of a treatment are unknown but also to reveal the effects of weak analgesics and to make possible the detection of hyperalgesia. Moreover, this will have the effect of minimizing the suffering of the experimental animal.
Acknowledgements The authors are grateful to Drs. O.-G. Berge and J.P. L u n d for critically reviewing the manuscript. They also thank Dr. C. D a r c h a t (Laboratoire d ' A n a t o m i e Pathologique, H o t e l - D i e u - C H R U , C l e r m o n t - F e r r a n d ) for assistance in analysing histological preparations and A.-M. Gaydier for secretarial help.
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Elsevier Science B.V.
PAIN 2781
The orofacial formalin test in rats: effects of different formalin concentrations Pierre Clavelou a, R a d h o u a n e Dallel a, Thierry Orliaguet b, Alain W o d a a and Patrick Raboisson a,* a Laboratoire de Physiologie Oro-Faciale and b D~partement de Biologie, Facult~ de Chirurgie Dentaire, Clermont-Ferrand 63000 (France)
(Received 30 September 1994, revision received 5 December 1994, accepted 7 December 1994)
Summary In this study of the orofacial formalin test in rats, the effects of different formalin concentrations (0.2%, 0.5%, 1.5%, 2.5%, 5% and 10%) on the behavioural nociceptive response (face rubbing) was investigated. The histological responses of the skin were also evaluated. Increasing the concentration of formalin caused a parallel aggravation of histological signs of tissue inflammation and injury. All concentrations provoked an early phase of nociceptive response, but its intensity was not concentration-dependent. The 2nd phase of response to formalin only occurred for concentrations of 1.5% and higher. A positive relationship between the formalin concentration and the amplitude of the rubbing activity measured between 12 and 45 min after injection could be observed until 2.5% but with the highest concentrations (5 and 10%), the amplitude of the response decreased. Our findings indicate that the orofacial formalin test should be carried out using concentration between 0.5 and 2.5%. This is essential to assess increase as well as decrease in pain intensity. Moreover, this will have the effect of minimizing the suffering of the experimental animal. Key words: Nociception; Formalin test; Inflammation; Formalin concentration; Orofacial region; (Rat)
Introduction The subcutaneous (s.c.) injection of dilute formalin is now a widely used model of nociception. It generates behavioural as well as electrophysiological responses that last from several minutes up to more than 1 h and which are sensitive to a wide range of analgesics. The typical time course of the response to formalin is biphasic, with an early and short-lasting 1st phase followed, after a quiescent period, by a 2nd prolonged (tonic) phase. In this respect, formalin tests have been regarded to be a more satisfactory model of clinical pain than tests producing phasic pain like the hot-plate or tail-flick tests (e.g., Abbott et al. 1981). Mice and rats are the most frequently used animals but other species including cats, rabbits, primates, guinea pigs, naked mole-rats have also been employed (Tj¢lsen et al. 1992; for review see, Porro and Cavazzuti 1993). * Corresponding author: Dr. Patrick Raboisson, Laboratoire de
Physiologic Oro-Faciale, Facult6 de Chirurgie Dentaire, 11 Blvd. Charles de Gaulle, 63000 Clermont-Ferrand, France. Tel.: (33) 7343-6414; FAX: (33) 7343-6409. SSDI 0304-3959(94)00273-8
Volumes and concentrations of formalin solutions vary according to the studies from 20 to 150 /~1 and from 0.02 to 15%, respectively. Many methods for assessing pain after formalin have been published. For instance, after injection into the dorsal or plantar surface of a fore- or hindpaw, some authors rate single parameters, such as flinching/shaking/jerking (e.g., WheelerAceto and Cowan 1991) or licking (Rosland et al. 1990) the paw, while others use several categories of nociceptive behaviours to derive weighted scores (e.g., Dubuisson and Dennis 1977; Coderre et al. 1993). Other methods score behavioural responses related or not to pain (e.g., Berge et al. 1991; Kristensen et al. 1994). The advantage of the orofacial formalin test is that it uses as a single 'pain' indicator, the amount of time spent rubbing the injected area (upper lip). This was shown to be a reliable method for assessing pain in the trigeminal region (Clavelou et al. 1989) and has been used with success in several recent studies (Klein et al. 1991; Aigouy et al. 1992; Cadet et al. 1993; Eisenberg et al. 1993). The present study was undertaken to provide a better understanding of the orofacial formalin test. In
296
particular, we wanted to check if there was a positive relationship between the amount of time the rats spent rubbing their lip and the concentration of the formalin solution. We also investigated the histological changes caused by different concentrations of formalin. From these results, some practical suggestions for standardising the orofacial formalin test are proposed.
Material and methods
Animals Seventy male Sprague-Dawley rats weighing 170-220 g (IffaCredo, France) were used in this experiment. Rats were housed in plastic cages with soft bedding (4 per cage) with free acces to food and water. They were maintained in climate- (23_+ I°C) and lightcontrolled (12-h dark/light cycle with light on at 08:00 h) protected units (Iffa-Credo, France) for at least 1 week before the experiments. Testing sessions took place during the light phase between 1 I:00 and 19:00 h in a quiet room maintained at 23-24°C. The test box had dimension of 30 x 30 x 30 cm with 3 mirrored sides. Each animal was first placed in this box for a 10-min habituation period to minimise stress. Rats did not have access to food or water during the test. Experiments conformed to Ethical Guidelines recommended by the International Association for the Study of Pain for experimental pain in conscious animals ( Z i m m e r m a n n 1983).
Testing procedure Rats were randomly assigned to 7 groups (n = 10 per group) and received a 50/xl subcutaneous (s.c.) injection of diluted formalin or saline (0.0%) into the right upper lip, just lateral to the nose. Solutions were prepared from commercially available stock formalin further diluted in isotonic saline to 0.2%, 0.5%, 1.5%, 2.5%, 5% and 10%. Stock formalin is an aqueous solution of 37% formaldehyde.
70
-
Following injection, the rat was immediately returned to the test box for a 45-min observation period. Analysis of behaviour was made by an investigator who was blinded to the animal's group assignment. The recording time was divided into 15 blocks of 3 min and a pain score was determined for each block by measuring the number of seconds (amplitude of the response) that the animal spent rubbing the injected area with the ipsilateral fore- or hindpaw. Although this behaviour resembles face washing, it has been shown that a prolonged asymmetrical distribution of paw strokes to the face does not occur spontaneously in normal rats (Berridge 1990). However, we previously pointed out that rubbing by the ipsilateral forepaw is often accompanied by similar movements of the contralateral one (Clavelou et al. 1989). Therefore, a control group received saline instead of formalin to quantify bilateral face washing behaviour in the absence of formalin.
Tissue preparation In each group, 2 animals were killed 1 h, 4 h, 24 h, 5 days or Ill days after the formalin injection by an overdose of pentobarbital. The injected area (right upper lip near the vibrissae pad) was removed and immediately fixed in acetic acid-formalin-ethanol for 48 h and embedded in paraffin. In addition, the left upper lip of 1 rat killed 10 days after injection of saline was removed and treated according to the same procedure to serve as control. Sections (15 /zm) were stained with haematoxylin-eosin, and with toluidine blue. The preparations were made and analysed by 3 observers unaware of the used concentration and survival time. The inflammatory response was evaluated in terms of mast cell degranulation, oedema, the and invasion of granulocytes. The cicatrization was measured by the level of fibrin production. A 5-point scale was used for oedema and fibrin production and a 4-point scale for the quantification of the mast cell degranulation. The results of the 3 observers were compared and averaged.
Statistical analysis W h e n tested with a Iog-ANOVA test, data with homogeneity of variance were analysed using 1-way analysis of variance (ANOVA).
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I 12 (33-36)
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Time after injection [ blocks, (min) ]
Fig. 1. Time course of the face rubbing activity observed after s.c. injection of saline (0.0%) or different concentrations of formalin. The mean n u m b e r of seconds that rats (n = 10 per concentration) spent rubbing is plotted for each 3-min block over the 45 min of post-injection observation period. Solid symbols indicate significant differences ( P < 0.05) compared to the saline (0.0%) group (Dunnett's t test, subsequent to A N O V A for block 1; Newman-Keuls test subsequent to Kruskal-Wallis test for the others).
297 Multiple post-hoc comparisons were performed using Dunnett's t test (control group= 0.0%) or Newman-Keuls test. Data without homogeneity of variance were analysed using the Kruskal-Wallis H test and multiple post-hoc comparisons were made using non-parametric Newman-Keuls test. For all tests the level of significancewas set at P < 0.05. Data are expressed as means + SEM.
A
First Phase 70605O 40
Results
Effect of formalin concentration on the nociceptive response The time course of the nociceptive responses to the administration of the different concentrations of formalin is presented in Fig. 1. All formalin concentrations from 0.2% to 10% induced the 1st phase of the response which did not last beyond the 3rd minute after injection (Fig. 1). The amplitude of the response seemed to reach a maximum at a concentration of 1.5% (Fig. 2A) and to decrease above but there was no significant difference between concentrations (Newman-Keuls test subsequent to ANOVA). The 2nd phase of response only appeared with formalin concentrations of 1.5% and higher (Fig. 1). It started 12 min after the formalin injection (block 5) for concentrations between 1.5 and 5%. For the highest concentration (10%), the onset of the rubbing activity was delayed until 15 min (block 6) and remained significantly lower than the response to 1.5% (until block 6) and to 2.5 and 5% (until block 8) (Newman-Keuls test subsequent to Kruskal-Wallis test, P < 0.05). Thirty minutes after formalin injection (block 11), there was no longer any significant difference between responses to 1.5% formalin and saline. By the end of the experiment (45 min), the rubbing level had returned to baseline for 2.5% but was still significantly elevated for the higher concentrations (Newman-Keuls test subsequent to Kruskal-Wallis test, P < 0.05). The graph shown in Fig. 2B suggests that there was a positive relationship between the amplitude of the global rubbing activity measured between 12 and 45 min after the formalin injection and the concentration of formalin until 2.5%. At higher concentration (5 and 10%), the amplitude of the response decreased. (Fig. 1 and 2B). The mean amplitude of the rubbing activity was significantly higher at 2.5% concentration, compared to 1.5% and 10% ( P < 0.01 and P < 0.05, respectively; Newman-Keuls test subsequent to A N O V A ) (Fig. 2B).
Tissue changes caused by formalin Extravasated cells, mainly granulocytes, were only seen with formalin. All mast cells were degranulated until at least 4 h with concentration of 1.5% and higher (Table IA). At the lowest formalin concentrations (0.2%), the oedema was not different from that observed after injection of saline but the higher concen-
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Fig. 2. Effect of increasing concentrations of formalin on (A) the ]st phase (0-3 min post-formalin) and (B) the 2nd phase (12-45 rain post-formalin) of rubbing activity. Solid symbols (e) indicated significant differences (P < 0.0]) compared to the saline group (0.0%, open symbols: ©). Significant differences from the 2.5% group are indicated by * P < 0.05; * * P < 0.0! (Dunnctt's t test, subsequent to
ANOVA). trations caused an increase in both the size and the duration of the oedema. With concentrations of 1.5% and higher, the oedema was still present 24 h after formalin injection and residual traces persisted at 5 days (Table IA). The concentration of formalin had an effect on the fibrin production; cicatrization was achieved 10 days after injection with concentrations below or equal to 1.5%, while it was incomplete or just beginning whith higher concentrations (Table IB and Fig. 3).
Discussion
The rubbing of the formalin-injected upper lip with the ipsilateral fore- or hindpaw is a characteristic and
298
consistent response in the rat. This response is sensitive to both narcotic and non-narcotic analgesic drugs at doses that do not impair motor activity (Clavelou et al. 1989). Similar rubbing (or grooming, see Vos et al. 1994 for review) has also been observed after noxious electrical, thermal or mechanical stimulation applied to the face (Rosenfeld et al. 1978; Morris et al. 1982; Dallel et al. 1989; Cahusac et al. 1990) or following experimental neuropathy induced by the ligation of the infraorbital nerve in rat (Vos and Maciewicz 1991; Vos et al. 1994). The present study confirms that s.c. formalin but not saline evokes sustained episodes of stereotyped rubbing of the injected site (a similar inefficiency of saline at inducing nociceptive behaviours has already been noted in the paw test; Fanselow and Baackes 1982; Wheeler-Aceto et al. 1990). As it has previously been reported by Rosland et al. (1990), histological data show good agreement between the importance of tissue damages and the concentration of formalin. In our experiment, except a certain degree of swelling, no marked alteration was observed with saline. On the contrary, the mast cell degranulation, which is one of the earliest histological changes
signalling tissue injury, as well as the extravasation of granulocytes, were seen even at the lowest formalin concentration (0.2%). The inflammatory signs progressively intensified when the formalin concentrations increased and, 10 days after the injection of formalin, the cicatrization was not achieved for concentration above 1.5%. Since the amplitude of the rubbing activity paralleled the degree of tissue damage caused by rising concentrations of formalin (only to a certain extent; this point will be discussed further), our data support the assumption that persistent face rubbing, focused on the application site of a noxious stimulus, is a behaviour signalling facial pain. The data reported here indicate that all formalin concentrations, from 0.2% to 10% (and for a 50 ~1 volume), provoke an early behavioural nociceptive response (upper lip rubbing) the duration of which did not exceed 3 min after injection. Although the amplitude of this response was maximal in the 1.5% group, there was no significant difference between concentrations. Rosland et al. (1990) noted a similar ceiling effect in mice when paw licking was used as the behavioural nociceptive response. In rats, Wheeler-Aceto
TABLE 1 H I S T O L O G I C A L C H A N G E S A C C O R D I N G TO T H E F O R M A L I N C O N C E N T R A T I O N A T D I F F E R E N T T I M E I N T E R V A L S A F T E R INJECTION 1h
4h
24 h
5 days
10 days
A: oedema (1st time) and mast cell degranulation (2nd time) Saline (0.0%)
[+0
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+
0 0
0 0
0 0
0.2% Formalin
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0.5% Formalin
[ , +, +
+
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0 o
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1.5% Formalin
[ ,+. +
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2.5% Formalin
[ ** + + +
**++ +
5% Formalin
[ ** + + +
**++ +
10% Formalin
[ ,+ + +
,+~+ +
++ 0 ++ 0 ++ 0 +++ 0
+/0 +/0 + 0 +/0
0 0 0 0 0 0 0 0
+ + + + + + +
+ + + + + + +
0 = no oedema or no mast cell degranulation. Oedema: + / - , residual; + , discrete; + + , important; + + + , very important. Mast cell degranulation: * / - , residual; *, partial; * *, complete fibrin production; 0, absence; + / - , in progress; + , partial; + + , incomplete; + + + , completed. B: cicatrization Saline (0.0%) 0.2% Formalin 0.5% Formalin 1.5% Formalin 2.5% Formalin 5% Formalin 10% Formalin
0 0 0 0 0 0 0
Fibrin production: 0, absence; + / - ,
0 +/ +/ +/ 0 0 0
+ + + + +/ + +/ -
in progress; + , partial; + + , incomplete; + + + , completed.
+ + + +
+ + + + + +
+ + + +
299
and Cowan (1993) observed that the time spent licking the injected paw during the 1st phase decreased above 1.5% and was even absent at the highest concentration tested (15%). Thus, a single nociceptive indicator such as face rubbing or paw licking may not be suitable for measuring pain intensity during the 1st phase, at formalin concentrations higher than 0.2%. Other behaviours (e.g., freezing) may supersede rubbing (see below). The 2nd phase of rubbing activity only appeared at formalin concentrations above 0.5%. Using the paw formalin test in the rat, Coderre et al. (1993) obtained similar findings for score 2 (paw elevation) and 3 (licking, biting of the paw). In mice, Rosland et al. (1990) noted a 2nd phase at formalin concentrations higher than 0.2%. In our study, the total time spent rubbing the face (measured between 12 and 45 min after the formalin injection) increased in a concentration-dependent fashion to reach a maximum at 2.5% (Fig. 2B). In the paw formalin test, a similar positive r
relationship between formalin concentration and the amount of time spent licking was also observed in rats (Coderre et al. 1993; Wheeler-Aceto and Cowan 1993) and mice (Rosland et al. 1990) up to concentrations of 5%. However, the administration of the highest formalin concentrations were associated with a decrease in face rubbing (at 5 and 10% in our study), paw licking (15%, Wheeler-Aceto and Cowan 1993) or paw elevation (5%, Coderre et al. 1993). In their studies, Rosland et al. (1990) and Coderre et al. (1993) observed a ceiling effect rather than a reduction for paw licking or for score 3 but they did not use concentrations above 5%. One explanation for these paradoxical decreases or ceiling effects is that high formalin concentrations induce other behavioural reactions, rarely seen at low concentrations, that may interfere with the primary behaviour (Fanselow and Baackes 1982). For example, in our experiments, long periods of freezing during which the rat stood immobile, sometimes with its forepaws applied on the face, could be seen with
~ , i ¸~
Fig. 3. Photomicrographs of upper lip sections. A: untreated animal (magnification: x 80 × 6.3). B: 4 h after 1.5% formalin: acute inflammatory phase with oedema, dissociated connective tissue, invasion of granulocytes and diapedesis of ieukocytes through congestive capillary blood vessels (magnification: x 80 x 6.3). C: 10 days after 1.5% formalin: terminal phase of cicatrization with accumulation of fibrin, newgrown muscle fibres and some areas of young connective tissue (magnification: × 80 x 6.3). D: 10 days after 10% formalin: partial cicatrization with large areas of loose connective tissue and presence of numerous fibroblasts (magnification: x 30 x 2.5). Abbreviations: A, artery; CC, congestive capillary blood vessel; CT, connective tissue; DCT, dissociated connective tissue; F, fibrin; FB, fibroblasts; GN, granulocytes; LCT, loose connective tissue; MF, muscle fibres; N, nerve fibres; NMF, newgrown muscle fibres; OE, oedema; YCT, young connective tissue.
300 formalin concentrations above 2.5%. Rosland et al. (1990) also noted that, in mice, the highest formalin concentrations induced other reactions along with licking or biting. Since the several modalities that compose pain may elicit different behavioural reactions, some authors argue that techniques taking into account more than one behaviour are more valid measures of pain intensity than single-parameter m e t h o d s (e.g., Tj¢lsen et al. 1992; C o d e r r e et al. 1993). However, the use of various behaviours reflecting supposedly different levels of nociceptive activity, does not resolve the problem. For instance, the weighted pain intensity score during the 2nd phase reaches a ceiling at 5% (Coderre et al. 1993). In any case, we believe that the use of high concentrations of formalin can lead to paradoxical conclusions. For instance, a score could rise when an analgesic was given because a conflicting behaviour was suppressed (e.g., freezing) while a pronociceptive drug may have the opposite action (or no apparent action when the concentration-response function reaches a ceiling). Thus, when high formalin concentrations are employed, the decrease of a pain score may not be sufficient to d e m o n s t r a t e antinociception. O n the contrary, by using low concentrations, it becomes possible to trustfully assess increases as well as decreases in pain intensity. In line with this, Rosland et al. (1990) noted that low doses of indomethacin are more potent at reducing the 2nd phase of paw licking induced with 1% formalin than with 5% and C o d e r r e and Melzack (1992a,b) found that the hyperalgesic effects of intrathecal excitatory amino acids or calcium ionophore a p p e a r e d when using 1% but not 5% formalin. Whatever the test, the choice of a given formalin concentration may then not be without consequence. To avoid ambiguous changes in the total pain score, its is necessary to work on the rising slope of the concentrationresponse function. A n o t h e r a r g u m e n t against the use of high formalin concentrations is given by histological data. Analysing the fibrin production showed that, 10 days after injection, the cicatrization was not completed at formalin concentrations of 2 . 5 - 1 0 % , indicating persistent damages. Since a robust tonic phase of rubbing, associated with less severe peripheral injury, appears at 1.5%, the use of higher concentrations does not seem justified in the orofacial formalin test, unless the aim of the experiment requires maximal amplitude of the nociceptive response or the existence of a lengthy inflammatory focus. T h e main advantages of single-parameter methods, such as the 'orofacial formalin test', is the simplicity of the scoring technique. T h e observations reported here help to refine the model by showing that it will be most sensitive if formalin concentrations between 0.5 and
2.5% are used. This is essential when the effects of a treatment are unknown but also to reveal the effects of weak analgesics and to make possible the detection of hyperalgesia. Moreover, this will have the effect of minimizing the suffering of the experimental animal.
Acknowledgements The authors are grateful to Drs. O.-G. Berge and J.P. L u n d for critically reviewing the manuscript. They also thank Dr. C. D a r c h a t (Laboratoire d ' A n a t o m i e Pathologique, H o t e l - D i e u - C H R U , C l e r m o n t - F e r r a n d ) for assistance in analysing histological preparations and A.-M. Gaydier for secretarial help.
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