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Are mechanical and cold allodynia in mononeuropathic and arthritic rats relieved by systemic treatment with calcitonin or guanethidine?
Pain, 52 (1993) 41-47 8 1993 Elsevier Science
41 Publishers
B.V. All rights reserved
0304-3959/93/$06.00
PAIN 02188
Are mechanical and cold allodynia in mononeuropathic and arthritic rats relieved by systemic treatment with calcitonin or guanethidine? S. Perrot,
N. Attal, D. Ardid and G. Guilbaud
UnitP de Rrcherches de Physiopharmacologie du Syst&nr Neweux (INSERM U 161), 75014 Paris (Frunce) (Received
17 March
1992, revision received
12 August
1992, accepted
19 August
1992)
Summary The putative antinociceptive action of guanethidine and calcitonin systemically injected has been compared in 2 rat models of persistant experimental pain: Freund’s adjuvant-induced arthritis (n = 29) and mononeuropathy induced by 4 loose ligatures around the sciatic nerve (n = 24). Guanethidine (30 mg/kg, i.v.) and calcitonin (0.125 mg, s.c.) were injected once a day over 1 week, when hyperalgesia was fully developed. The antinociceptive action was gauged using nociceptive tests based on mechanical or cold stimuli (vocalization threshold to paw pressure and struggle latency to 10°C respectively), and the score of spontaneous pain-related behavior was measured on the basis of the abnormal hind paw position. No antinociceptive action was observed in calcitonin-compared to saline-injected rats, either in arthritic or neuropathic animals. Guanethidine treatment was ineffective on hyperalgesia exhibited in arthritic rats but was able to reduce reliably and even suppress the abnormal reactions to cold stimulus in neuropathic animals. The lack of hypoalgesic action of calcitonin versus its beneficial action in bone repair, as well as the possible role(s) of the sympathetic system in neuropathic versus arthritic pain and in hyperalgesia versus physical signs of inflammation, are discussed.
Key words: Adjuvant arthritis; Inflammatory
pain; Neuropathic
Introduction
Clinical observations (Leriche 1939; Procacci and Maresca 1987; see Refs. in Stanton-Hicks et al. 1990; JCnig et al. 1991) and experimental studies (Devor 1989; see Refs. in Besson and Guilbaud 1991) have demonstrated involvement of the sympathetic system in the pathophysiology of some painful chronic and acute syndromes. Experimental studies have confirmed the efficacy of antisympathetic treatment in different animal models of pain (Coderre et al. 1991; Neil et al. 1991). Over the last 5 years a rat model of experimental mononeuropathy producing abnormal pain-related behaviors to mechanical and thermal stimuli has been extensively investigated (Bennett and Xie 1988; see Refs. in Besson and Guilbaud 1991). The abnormal sensitivity of these rats to cold stimulus was especially
Correspondence to: Serge Perrot, 75014 Paris, France.
INSERM
U 161.2 Rue d’Alesia,
pain; Calcitonin; Guanethidine;
(Rat)
striking. This mononeuropathy is associated with abnormality of sympathetic efferents and of local cutaneous temperature (Wakisaka et al. 1991). In this model, sympatholytic treatment with guanethidine decreases the behavioral sensitization to cold and warm stimuli (Neil et al. 1991). In an electrophysiological study of sciatic nerve fibers in such a model, Xie and Xiao (1990) demonstrated that abnormal firing of injured fibers was increased by stimulation of the sympathetic system and depressed by phentolamine, an alpha-adrenoreceptor blocker. These data strongly suggest that the mononeuropathic model could be a good model of causalgia and sympathetic maintained pain, for which hyperalgesia to cooling stimuli seems to be a main symptom (Frost et al. 1988). In another model of chronic pain, the Freund’s adjuvant arthritic rat, the efficiency of antisympathetic drugs has been proved based on clinical and radiological symptoms (Coderre et al. 1991) but to our knowledge there is no report of measure of antinociceptive activity of sympatholytic treatment in arthritis. It appeared to be of interest to test and compare the
antinociceptive efficacy of sympatholytic treatment in chronic arthritis to that observed in the neuropathic model. As in an initial study in the same model of neuropathy (Neil et al. 1991), we used guanethidine as a sympatholytic drug. It is commonly employed for chemical experimental sympathectomy to deplete peripheral stores of noradrenaline (Furst 1987) and in human therapy for sympathetic blockade. Another drug, calcitonin, has also been used in sympathetic maintained pain (Gennari et al. 1984; Tanguy and Peyron 19X8> but its antalgic efficiency in human pathology seemed to be observed mainly after central injection in pain specially due to metastatic bone lesion (Miralles et al. 1987). Recently, it has been proved that it could also have an antalgic action on phantom limb pain either after central or i.v. injections (Kessel and Worz 1987; Jaeger and Maier 1992). In animal experiments, calcitonin has been mostly administered by intracerebral or Lt. injections; in addition, results differ according to the test, route of administration and species (Welch et al. 1986; Spampinato et al. 1988; Guidobono et al. 1991). Thus, finally, the analgesic effect of calcitonin is somewhat controversial, especially when systemically injected. It appeared to be of interest to test its putative antinociceptive effect in the rat models of mononeuropathy and arthritis using S.C. injections, as commonly used in human therapy. Thus, in the present study the putative antinociceptive of chronic guanethidine and calcitonin were gauged in parallel in the 2 models of clinical pain, with tests previously used to demonstrate antinociceptive activity of opioids (Kayser and Guilbaud 1990), non-steroidal anti-inflammatory agents (Attal et al. 1988) and antidepressant drugs (Ardid and Guilbaud 1992) in the 2 pain models.
(1988) were
and Attal considered
et al. (1990)
but, as explained
in the final
data analysis. Male
River,
arrival.
kept in cages with light and free access to food and
were
for I week before
after a first behavioral sodium The
200-225
g (7 weeks old) on
surgery. They were operated
I week later
testing session. Rats were anesthetized
pentobarbital
loosely (with about
weighing
Sprague-Dawley
rats (Charles water
France).
later. only 24 rats
(St1 mg/kg.
i.p.)
and 4 Iigatureb
was
same dissection
performed
on the opposite
side without
Treatment procedure We used guanethidine tonin (Cihacalcine)
monosulphate
The drugs were injected adjuvant
and human cnlci-
injection
for the arthritic
old when treated.
week after surgery
i.v. in arthritic
hy several studies (Wall
peripheral
S.C. at 0.125
WB~ injected
(n = 17) rats at 30 mg/kg
to the dose reported totally to deplete
I
model and
model. Thus, all the rats were Y-IO weeks
Guanethidine
and mononeuropathic
France).
for 1 week starting 3 weeks after Freund’s
for the mononeuropathic
jected
(Ismeline)
given generously by Dr. Sallikres (CIBA,
sympathetic
mg each day (i.e..
usually given in humans)
in arthritic
(n = Y)
each day according
et al. 1979) that seems
stores. C’alcitonin was in-
25% of the total daily dose
(n = 11) and mononeuropathic
rats (n = 12). Control
animals received the same volume of O.Y% NaCt i.v. daily
for I week: n = Y for arthritic Six additional
and n = IS for mononeuropathic
normal rats were treated with calcitonin as detailed
not administered
to normal animals since it was previously shown to
he ineffective
above. A guanethidine
rats.
under the
same condition>
treatment
was
(Neil et al. lY91b).
Testing procedure Rats were tested in ~1quiet room by the same person, blind to the drug used. Rats were randomly
arranged
in groups of 4 or S for a
eerie:, of tests. All the groups were tested before and after I week of treatment,
then every week
neuropathic
over 4 and 8 weeks for arthritic
The antinociceptive tion threshold
effect WB~ tested by measuring
uaing the Basile Analgcsymeter compounds
the vocaliza-
elicited by pressure on the right hind paw of each rat, Centrdlly
This response,
particularly
Sensitization
(Apelex)
integrated.
Kayser and Guilbaud
measuring
and
rats. respectively.
until a squeak was audible.
is especially sensitive to analgesic
In thcac 2 pain models (Attal
et al. 1990;
IYYO).
to an otherwise
determined.
innocuous cold stimulus (cold test)
as extensively
described
elsewhere
et al. 1990), by plunging the hind paw into a 10°C water
and methods
tied
l-mm spacing) around the common sciatic nrtve.
ligating the sciatic nerve (sham procedure).
was similarly
Materials
with
were
(Attal
bath and
the latency to a struggle response occurring with a cut-off
time of IS sec.
Animul models Male
In addition.
Sprague-Dawley
arthritic
and normal
rats (Charles
River.
ethical standards lefinited
were made
for investigations
by the IASP (1983).
according
tn the guidelines
of experimental
regard to housing conditions.
in order to avoid or minimize
fort to the animals:
which were
housed pre-operatively
on
pain in animals
Great care was taken, particularly
animals
to undergo
with
discom-
surgery were
6 to a cage then 1 to a cage for at least 5 days
after surgery. Cages floors were covered with sawdust, and food and fluid were available Arthritis were
then
radiological
River,
tested
by injection
France)
3 weeks
arthritic
according
of Freund‘s
at 6-7
later
was produced
to the method
few minutes
before
periods. Six different from
0 r= normal
the test, and observed
for a 5-min
positions of the lesioned hind paw were rated
to 5 = most
adapted
from that described
Dennis
1977).
For
for up to three
each
ahnormal
position.
tar the formalin
S-min
period
the
This
scale was
test (Dubuisson score
according to the formula [Tl + T2 + T3 + T4 + TS]/300 the time. expressed in seconds, spent in position
and
was calculated (Tl.
TI?,... was
I. 2.... over a total
at the maximum
into the
of clinical
et al. 19X7: Colpaert
and 1987):
g.
calculated
for each particular
rat.
in detail
Stutisticul unalysis Results of arthritic calcitonin
in 39 rats on the
described
adjuvant
weeks of age (n = 2Y). The)
symptoms (Calvin0
at this time their weight was 200-225 Neuropathy
et al. 1990). Each
of 5 min). Then, using 3 consecutive score values. the mean score was
ad lihitum.
was induced
rat tail (Charles
pain in the mononeuro-
animal was placed in a plexiglass cylinder. allowed to habituate
France) were used for this study. All the experiments
the bcort: of spontaneous
pathic rat was rated as described previously (Attal
right
hy Bennett
hind
paw’
and Xir
or
and mononeuropathic
guanethidine
were
control arthritic
and neuropathic
volume
the
during
same
time,
rats treated either with
compared
to those
obtained
in
animals receiving NaCl in the same using
l-way
analysis
of variance
43 TABLE
TABLE
I
THRESHOLD VALUES OF THE DIFFERENT TESTS IN ARTHRITIC RATS (3 weeks after injection of Freund’s adjuvant in the tail) AND IN MONONEUROPATHIC RATS (1 week after sciatic nerve ligature) PRIOR TO ANY TREATMENT Non-operated rats were those devoted to receive a sciatic nerve ligature; the values were comparable to those obtained in different studies. Vocalization to paw pressure expressed in grams (pressure test); struggle latency to paw immersion in a 10°C water bath expressed in seconds (cold test). Pressure
test
151+
270+
12.9kO.7
**
**
10.3+0.6
***
7
Observation period from W, to W, (i.e., 7 weeks after arthritis inoculation). No significant difference between the 3 groups of rats with ANOVA, Tukey test. Saline (n = 9)
7 ***
245+10
IN ARTHRITIC RATS, VOCALIZATION THRESHOLD TO PAW PRESSURE (in grams) IN DIFFERENT GROUPS OF RATS, TREATED DAILY WITH SALINE, CALCITONIN OR GUANETHIDINE FROM W,, (3 weeks after Freund’s adjuvant inoculation) TO W,
Cold test (set)
(g) Arthritic (n = 29) Mononeuropathic (n = 24) Non-operated rats (n = 24)
II
Week Week Week Week Week
0 1 2 3 4
155* 144* 156+ 163+10 176k
7 7 9 7
Calcitonin (n = 11)
Guanethidine (n=9)
152k6 150+7 159+8 166+6 164k5
147+ 165 + 138k 152k 157k
8 12 8 8 6
14.9 f 0.9
* * P < 0.01; ** * P < 0.001. Difference between arthritic or mononeuropathic compared to non-operated rats. Differences between arthritic and mononeuropathic rats P < 0.01 or 0.05 for the pressure and cold tests, respectively; ANOVA.
(ANOVA) with the Tukey test. Results were expressed in absolute values (in grams or seconds) or as percentage of initial threshold values determined just before the beginning of treatment. Differences at P < 0.05were considered significant. S.E.M. was calculated by first getting a single score for each rat.
Results
In normal rats (data not shown) calcitonin injected S.C. has no effect on vocalization threshold to paw pressure nor on the struggle’ latency to 10 “C water bath, as has already been shown after guanethidine treatment (Neil et al. 1991a,b). General results in the 2 pain models prior to any treatment (Table I)
(a> As in previous studies (Kayser and Guilbaud 1990), the vocalization thresholds to the mechanical stimulus 3 weeks after Freund’s adjuvant inoculation were much lower in arthritic (n = 29) than in normal (n = 24) rats prior to nerve ligature: the mean threshold for hind-paw pressure was 151 k 7 g vs. 270 + 7 g (P < 0.001). The decrease observed for neuropathic rats, although significant compared to presurgery values (245 f 10 g, n = 24, P < 0.011, was less marked than in arthritic rats (significant difference between the 2 models: P < 0.01). (b) The latency for the struggle reaction to the cold stimulus (cold test) of 12.9 & 0.7 sqc in arthritic rats was significantly decreased compared to normal rats (P < O.Ol), but was less than in neuropathic rats (10.3
f 0.6 set (P < 0.001) (significant difference between the 2 models P < 0.05). (c) The mean score of tonic pain gauged by the hind-paw position in neuropathic rats was 1.84 _t 0.18 (n = 241, slightly below the mean score initially described (Attal et al. 1990; Ardid and Guilbaud 1992). Results in treated arthritic rats (n = 29)
(a) In saline-treated arthritic rats (n = 9), the vocalization threshold to paw pressure (155 f. 7 g at week 0) remained at a low level over the observation period (Table II) and started to recover toward normal values at the end of the observation period, i.e., 7 weeks after arthritis induction, in agreement with a previous study (Calvin0 et al. 1987). The latency of the struggle reaction to 10°C (cold test) (12.6 + 1 set at week 0) did not change significantly over the course of the observation period (Table III): 7 weeks after arthritis induction the rats were still abnormally sensitive to cold (in another study not shown
TABLE
III
IN ARTHRITIC RATS, STRUGGLE LATENCY TO PAW IMMERSION IN A 10°C WATER BATH (in seconds, with a cut-off at 15 set) IN DIFFERENT GROUPS OF RATS, TREATED DAILY WITH SALINE, CALCITONIN OR GUANETHIDINE, FROM W, (3 weeks after Freund’s adjuvant inoculation) TO W, Observation period from W, to W, (i.e., 7 weeks after arthritis inoculation). No significant difference between the 3 groups of rats with ANOVA, Tukey test.
Week Week Week Week Week
0 1 2 3 4
Saline (n = 6)
Calcitonin (n = 9)
Guanethidine (n = 9)
12.6 f 1 14.2 + 0.5 13.6 * 0.7 11.8+0.8 12.0& 1.0
13.2 + 13.8 f 14.1* 14.5 * 13.1 f
13.8 f 0.4 13.6 f 0.5 14.OkO.5 14.4kO.2 13.6 f 0.5
0.8 0.7 0.4 0.2 0.9
hcrc. the recovery to a normal value was observed 10 weeks after Freund’s adjuvant injection). (h) In calcitonin treated arthritic rats (n = 11). there was no modification of the vocalization threshold to paw pressure over the observation period f 152 i: 6 vs. 150 i 7 g before and at the end of treatnlcnt, rcspectively) nor change in the latency of the cold test (13.11 f 0.8 vs. 13.8 I: 0.7 set before and at the end of treatment, respectively). There was no significant difference between this group of rats and those injected with saline (Tables II and III). (cl In guanethidine-treated arthritic rats (n = 91, there was a slight increase in the vocalization threshold to paw pressure, at the end of treatment (147 h 8 at week 0 vs. 165 & 12 g at week 1) but it was not significant and only transient (Table II). The cold test was not modified at all by ~uanethidine trcatmcnt (13.X i 0.4 vs. 13.6 i: 0.5 set before and at the end of treatment). There was no significant difference between this group of rats and the saline-injected animals (Table III>. Thus, with the 2 tests, there was no significant difference between the saline-, calcitoninand guanethidine-treated arthritic rats. kwlt.~
in treated mononeuropathic
ruts fn = 24)
Initially 39 rats were prepared with loose sciatic nerve ligatures, then tested and pretreated. Fifteen rats (6 saline-, 4 calcitonin- and 5 guanethidine-treated animals) exhibited self-mutilation lesions of the 4th and 5th digits. They were mostly minor lesions. limited to nails, but we chose to sacrifice these rats prior to the subsequent tests. and only 24 rats were included in the data analysis. (a) In saline-injected mononeuropathic rats (n = 41 (Fig. I), the vocalization threshold to paw pressure (227 + 10 g) was already decreased compared to the presurgery value when treatment started and exhibited a further decrease over the following 2 weeks (i.c.. until the 3rd week following ligature). Then, recovery toward normal threshold values was observed, roughly in agreement with the initial study (Attal et al. 1990). The decrease in struggle latency to the cold test (11.1 + 0.6 see) observed 1 week after nerve ligation was maintained for several weeks after the end of treatment (Fig. 2). Recovery started after the 6th week following surgery, in good agreement with the initial investigation. The score of the tonic pain-related behavior (mean = 1.84 + 0.1X), slightly below the mean score of other studies (Attal et al. 1990: Ardid and Guilbaud 19021 at week 1 after nerve ligation, then regularly decreased over the observation period (Fig. 3). ’ (b) In calcitonin-treated rats (n = 8), the vocalization thresholds to paw pressure and the struggle latency to cold stimulus which were reduced prior to treatment
NEUROPATtiiC pressure
RATS test
. guanethidlne 0 saltne
I ,_.-_..T.._
.... . . . - T...-” .,----T.‘-T-__
0123456 treatment
7 weeks
Fig. I. Time court of thr bocaliratmn thresholif to pa% pr~~.xir~ usprcssed in yr;m,s (+S.E.M.) c~hservcit in guanethidinc- anii s;tlincinjected neuropathic r’ntb from W,, (1 week after IIL‘TVCligature) to W, (8 weeks after hurerrq). Thr preaurgcryvalur wx, mean = 170-i IO g. Kats were trcatcd saline
with
from W. to W, Jaily (II0 mg/kg
the hame volumtl.
guanethidine
i.v.1. *= * P <: 0.001 (ANOVA,
or
Tukey
test I.
(222 5 X g and 11.0 i: 0.7 SW, respectively) did not not significantly differ thereafter in the same group, compared to the values obtained in saline-injected rats.
NEUROPATHIC
20 i
RATS
45 NEUROPATHIC 2
0
salme
.
gvanethldme
RATS
0
OJ I 0
I
2
I
4
1
I
6
6 weeks
treatment Fig. 3. Time course of the spontaneous pain-related behavior of guanethidineand saline-injected neuropathic rats, rated with a scale determined on the basis of the hind-paw position from W,, to W,, i.e., from 1 to 9 weeks after nerve ligature. Rats were treated daily from W,, to W, as in Figs. 1 and 2 (guanethidine at 30 mg/kg, i.v., or saline with the same volume). There was no significant difference between the 2 curves (ANOVA, Tukey test).
The score of the spontaneous pain-related behavior was not significantly modified either by comparison to the saline-treated animals. This lack of effect was especially clear just at the end of treatment (i.e., 2 weeks after sciatic nerve ligation) (Table IV). (c) In guanethidine-treated rats (n = 7) (Fig. 11, the vocalization threshold to paw pressure was especially decreased for this group of rats (215 & 10 g> 1 week after nerve ligature when the treatment started, but was significantly increased (P < O.OOl), reaching 150% of the control value (324 _t 26 g) at the end. This
TABLE
IV
IN MONONEUROPATHIC SALINEAND CALCITONINTREATED RATS, THE VOCALIZATION THRESHOLD TO PAW PRESSURE, STRUGGLE LATENCY TO PAW IMMERSION IN A 10°C WATER BATH AND SCORE OF SPONTANEOUS PAIN-RELATED BEHAVIOR, JUST AFTER TREATMENT, i.e.. 2 weeks after sciatic nerve ligature (no significant differences compared to pre-treatment values) No significant difference was observed at this or any other time point between the 2 groups of rats (ANOVA, Tukey test). Vocalization
(d Calcitonin (n = 8) Saline (n = 6)
205*
1.5
214+ 10
10°C bed 9.9 + 1 11.3kO.9
Spontaneous
I
kO.3
1.3f0.2
pain
reflected a genuine, but not prolonged, antinociceptive effect; indeed, 1 week later, 3 weeks after surgery, the vocalization threshold of these guanethidine-treated animals was comparable to that of saline-injected rats. In these rats, the latency of the struggle reaction to a 10°C cold stimulus of 10.3 * 0.6 set at the beginning of treatment increased to a final value of 14.9 ) 0.5 set (P < O.OOl), so regaining the presurgery value (Fig. 2). The normalized responses seen in the guanethidinetreated group were observed throughout the observation period, i.e., until week 8 after ligatures, a time when normally the abnormal reaction to cold has spontaneously disappeared. This treatment, therefore, induced a persistant clear antinociceptive effect. In sharp contrast, there was no significant difference between the time course of the score of spontaneous pain-related behavior in saline- and guanethidine-injetted rats (Fig. 3). Thus, in mononeuropathic rats, calcitonin did not exhibit any antinociceptive action whatever the test considered. In sharp contrast, guanethidine treatment induced a significant but transient increase of the vocalization threshold to paw pressure and reestablished the struggle latency to a 10°C cold stimulus to a normal value.
Discussion
Before any treatment, animals in the 2 models of pain (arthritic and mononeuropathic) exhibited abnormal sensitivity to mechanical and cold stimulus, with nociceptive reactions to stimuli that are not usually in the noxious range, thus exhibiting allodynia according to the IASP definition (Merskey et al. 1986). Nevertheless, the mechanical allodynia, previously described in these 2 models (Calvin0 et al. 1987; Bennett and Xie 1988; Attal et al. 1990; Kayser and Guilbaud 1990) is more marked for arthritic rats. Inversely, allodynia to cold as described in neuropathic rats (Bennett and Xie 1988; Attal et al. 1990) was more pronounced in these animals than in arthritic rats. In these 2 models, calcitonin treatment for 1 week was ineffective on the thresholds of the tests based on mechanical or cold stimulus and used to assess an antinociceptive action. This treatment also did not modify the spontaneous pain-related behavior based on the abnormal position of the paw in neuropathic rats. It seems unlikely that this absence of effect could be due to test sensitivity, since it has been used to demonstrate antinociceptive activity of several agents such as opioid, antidepressant and non-steroidal antiinflammatory drugs in the same models of clinical pain (Attal et al. 1988, 1990; Kayser and Guilbaud 1990; Ardid and Guilbaud 1992). In fact, although calcitonin has been used to treat chronic neuropathic pains, such
as causalgia and sympathetic maintained pain (Procacci and Maresca 1987; Tanguy and Peyron 198% its antalgic action has been mainly shown after it. injection either on phantom limb pain or pain related to visceral cancer (Miralles et al. 1987) with the exception of the last study of Jaeger and Maier (1992) who used systemic injection at a dose of 200 IU. In animals, the antinociceptive effect of calcitonin has mostly been revealed after intracerebroventricular injection (Welch et al. 1986; Spampinato et al. 1988) and after a local injection in 1 recent investigation (~uidobono et al. 1991). Since in the present study, using tests in the 2 models sensitive to various analgesics, systemically administered calcitonin had no evident antinociceptive action, it could be interesting to seek an action after longer treatment periods and with multiple daily injections. In sharp contrast, we found that guanethidine had a marked effect in the mononeuropathic model, on the vocalization threshold elicited by hind-paw pressure, but mainly on the reaction time to cold stimuli. Antino~i~eptive actions tested with the mechanical test were not observed in previous experiments conducted by Neil et al. (1991). This could be due to the more prolonged treatment applied in the present study. However, antinociception is not observed either after surgical sympatectomy (Desmeules et al. 1992). In any case, this relieving action of guanethidine on mechanical allodynia was only transient. The total lack of antalgic effect on the spontaneous pain-related behavior could be due to the complexity of the test, making it less sensitive to pharmacological manipulation. In sharp contrast, and as others who also found an effect of chemical and surgical s~pathe~tomy on the abnormal sensitivity to heat (Neil et al. 1991; Desmeules et al. 19921, we noted an important antinociceptive effect of guanethidine treatment on abnormal reaction to cold stimulus. There was, indeed, a recovery to the normal struggle latency which persisted over the entire obervation period, i.e., until the 8th week after nerve ligature, the usual time of spontaneous recovery (Attal et al. 1990). Since guanethidine has a peripheral sympatholytic action in adult rats (Furst 1987), this data seem to confirm that abnormal sensibility to cold in this model is mainly mediated by the sympathetic system (N&I et al. 1991). I As discussed initially (Neil et al. 19911, effectiveness of guanethidine treatment on the cold test in this neuropathic model can be related to the fact that allodynia to cold stimulus is found to be a major sign in sympathetic maintained pain (Frost et al. 1988). This fits well with the abnormal sensitivity to thermai stimuli observed for injured afferent fibers (Matzner and Devor 19871, especially in the same model of sciatic ligature by Xie and Xiao (19901, and strongly suggests
that this rat model of mononeuropathy could be relevant to the study of causalgia and sympathetic maintained pain mechanisms. The lack of effect of guanethidine treatment on the 2 tests performed in arthritic rats was relatively surprising at first sight, according to the various obse~ations reported with this mode1 (Levine et al. 1986 a,b; Coderre et al. 1991). It could be that our treatment was too short to induce antinociceptive effects but it has been demonstrated that guanethidine treatment for 4 days at 30 mg/kg was efficient for at least 1 month (Wall et al. 1979). In addition, we have recently demonstrated (Weil-Fugazza et al. unpublished) that i.v. injections of guanethidine (30 mg/kg during 4 days) in the normal rat reduced the level of noradrenaline on the distal part of the sciatic nerve by 90% (n = 5). It could also be argued that we treated arthritic rats at the time of maximum pain state. For this reason, we treated an additional group of rats (n = 6) at week 2 after induction of arthritis, just when nociceptive behavior starts (Calvin0 et al. 1987). In this group of rats (data not shown here) guanethidine did not induce any relief of the abnormal sensitivity to cold and mechanical stimuli. In fact, the studies of Levine et al. f1986b) clearly showed that either pharmacological or surgical sympathectomy relieved or prevented clinical and radiological inflammatory symptoms. It has also been widely demonstated that sympathetic efferents contribute to the severity of joint injury (Coderre et al. 1990; Bjerknes et al. 1991). However, in these studies, the antalgic action of sympathectomy has not clearly been tested. ‘There are also some controversies concerning the role of sympathetic mediators in nociceptive processing. Koltzenburg et al. (1992) have recently demonstrated that sympathetic post-ganglionic neurons do not contribute to heat sensitization of nociceptors by bradykinin. Thus, the role of the sympathetic system in nociception appears to be even more complex than expected. According to our present data, it seems important to dissociate nociceptive thresholds from in~ammato~ symptoms because physical and sensory symptoms of arthritis seem not to be dependent on the peripheral sympathetic system to the same extent. Finally, in this parallel study of putative analgesic action of 2 treatments in 2 different pain models, calcitonin had no antinoci~eptive effect, while the sympatholytic drug was more effective against nociceptiverelated behaviors in the neuropathic than in the arthritic model, at least at the stages of disease considered in this study. Even in the same model, allodynia to I stimulus modality, cold, was more sensitive to the sympatholyti~ treatment than the tests based on mechanical stimulus. These data sharply emphasize the importance of different nociceptivc tests in different models of pain, in particular for analyzing the involvment of the sympathetic system in pain processing.
47
Aknowledgements
This study has been performed with a support from La Fondation pour la Recherche MCdicale to S. Perrot and support from Laboratoires Ciba, France. The authors wish to thank Dr. A.H. Dickenson for English revision, and Mme. M. Gautron for illustrations.
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activity and side effects of different calcitonins in man, Proc. Int. Symp. Calcitonin, Milan, 1984, Excerpta Med., 1985, pp. 183-188. Guidobono, F., Netti, C., Villani, P., Bettica, P., Pecile, A., Antinociceptive activity of eel calcitonin, injected into the inflamed paw in rats, Neuropharmacology, 30 (1991) 1275-1278. Hu, S. and Zhu, J., Sympathetic facilitation of sustained discharges of polymodal nociceptors, Pain, 38 (1989) 85-90. Jaeger, H. and Maier, C., Calcitonin in phantom limb pain: a double-blind study, Pain, 48 (1992) 21-27. Janig, W., Blumberg, H., Boas, R.A. and Campbell, N., The reflex sympathetic dystrophy syndrome: consensus statement and general recommendations for diagnosis and clinical research. In: M.R. Bond, J.E. Charlton and C.J. Woolf (Eds.), Proc. VIth Congress on Pain, Elsevier, Amsterdam, 1991, pp. 373-376. Kayser, V. and Guilbaud, G., Differential effects of various doses of morphine and naloxone on two nociceptive test thresholds in arthritic and normal rats. Pain, 41 (1990) 353-363. Koltzenburg, M., Kreb, M. and Reeh, P.W., Sympathetic post-ganglionic neurons do not contribute to the heat sensitization of nociceptors by bradykinin, J. Neurosci., 1992, in press. Leriche, R., La chirurgie de la douleur, Masson, Paris, 1939. Levine, J.D., Taiwo, Y.O., Collins, S.D. and Tam, J.K., Noradrenaline hyperalgesia is mediated through interaction with sympathetic postganglionic neurone terminals rather than activation of primary afferent nociceptors, Nature, 323 (1986a) 158-160. Levine, J.D., Dardick, S.J., Roizen, M.F., Helms, C. and Basbaum, AI., Contribution of sensory afferents and sympathetic efferents to joint injury in experimental arthritis, J. Neurosci., 6 (1986b) 3423-3429. Matzner, 0. and Devor, M., Contrasting thermal sensitivity of spontaneously active A- and C-fibers in experimental nerve-end neuromas, Pain, 30 (1987) 373-384. Merskey, H., Lindblom, U., Mumford, J.M., Nathan, P.W., Noordenbos, W. and Sunderland, S., Pain terms. A current list with definitions and notes on usage, Pain, Suppl. 3 (1986) S217-S221. Miralles, F.S., Lopez-Soriano, F., Puig, M.M. and Lopez-Rodriguez, F., Postoperative analgesia induced by subarachnoid lidocaine plus calcitonin, Anesth. Analg.. 66 (1987) 615-618. Neil, A., Attal, N. and Guilbaud, G., Effects of guanethidine on sensitization to natural stimuli and self-mutilating behaviour in rats with a peripheral neuropathy, Brain Res., 565 (1991) 237-246. Procacci, P. and Maresca, M., Reflex sympathetic dystrophies and algodystrophies: historical and pathogenic consideration, Pain, 3 (19871 137-146. Seltzer, Z., Dubner, R. and Shir, R.A., A novel behavioural model of neuropathic pain disorders produced in rats by partial sciatic nerve injury, Pain, 43 (1990) 205-218. Spampinato, S., Romualdi, P., Candeletti, S., Cavicchini, E. and Ferri, S., Distinguishable effects of intrathecal dynorphins, somatostatin, neurotensin and S-calcitonin on nociception and motor function in the rat, Pain, 35 (1988) 95-104. Stanton-Hicks, M., Janig, W. and Boas, R.A., Reflex Sympathetic Dystrophy (1990). Kluwer, Dordrecht. Tanguy, G. and Peyron, R., Traitement de I’algodystrophie par la calcitonine humaine: resultats d’une etude sur 124 patients, Vie Med., 69 (1988) 646-649. Wakisaka, S., Kajander, K.C. and Bennett, G.J., Abnormal skin temperature and abnormal symapthetic vasomotor innervation in an experimental painful peripheral neuropathy, Pain, 46 (1991) 299-314. Wall, P.D., Scadding, J.W. and Tomkiewicz, M.M., The production and prevention of experimental anesthesia dolorosa, Pain, 6 (1979) 175-182. Welch, S.P., Cooper, C.W. and Dewey, W.L., Antinociceptive activity of salmon calcitonin injected intraventricularly in mice: modulation of morphine antinociception, J. Pharmacol. Exp. Ther., 237 (1986) 54-58. Xie, Y.K. and Xiao, W.H., Electrophysiological evidence for hyperalgesia in the peripheral neuropathy, Sci. China, 33 (1990) 663-672.
41 Publishers
B.V. All rights reserved
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PAIN 02188
Are mechanical and cold allodynia in mononeuropathic and arthritic rats relieved by systemic treatment with calcitonin or guanethidine? S. Perrot,
N. Attal, D. Ardid and G. Guilbaud
UnitP de Rrcherches de Physiopharmacologie du Syst&nr Neweux (INSERM U 161), 75014 Paris (Frunce) (Received
17 March
1992, revision received
12 August
1992, accepted
19 August
1992)
Summary The putative antinociceptive action of guanethidine and calcitonin systemically injected has been compared in 2 rat models of persistant experimental pain: Freund’s adjuvant-induced arthritis (n = 29) and mononeuropathy induced by 4 loose ligatures around the sciatic nerve (n = 24). Guanethidine (30 mg/kg, i.v.) and calcitonin (0.125 mg, s.c.) were injected once a day over 1 week, when hyperalgesia was fully developed. The antinociceptive action was gauged using nociceptive tests based on mechanical or cold stimuli (vocalization threshold to paw pressure and struggle latency to 10°C respectively), and the score of spontaneous pain-related behavior was measured on the basis of the abnormal hind paw position. No antinociceptive action was observed in calcitonin-compared to saline-injected rats, either in arthritic or neuropathic animals. Guanethidine treatment was ineffective on hyperalgesia exhibited in arthritic rats but was able to reduce reliably and even suppress the abnormal reactions to cold stimulus in neuropathic animals. The lack of hypoalgesic action of calcitonin versus its beneficial action in bone repair, as well as the possible role(s) of the sympathetic system in neuropathic versus arthritic pain and in hyperalgesia versus physical signs of inflammation, are discussed.
Key words: Adjuvant arthritis; Inflammatory
pain; Neuropathic
Introduction
Clinical observations (Leriche 1939; Procacci and Maresca 1987; see Refs. in Stanton-Hicks et al. 1990; JCnig et al. 1991) and experimental studies (Devor 1989; see Refs. in Besson and Guilbaud 1991) have demonstrated involvement of the sympathetic system in the pathophysiology of some painful chronic and acute syndromes. Experimental studies have confirmed the efficacy of antisympathetic treatment in different animal models of pain (Coderre et al. 1991; Neil et al. 1991). Over the last 5 years a rat model of experimental mononeuropathy producing abnormal pain-related behaviors to mechanical and thermal stimuli has been extensively investigated (Bennett and Xie 1988; see Refs. in Besson and Guilbaud 1991). The abnormal sensitivity of these rats to cold stimulus was especially
Correspondence to: Serge Perrot, 75014 Paris, France.
INSERM
U 161.2 Rue d’Alesia,
pain; Calcitonin; Guanethidine;
(Rat)
striking. This mononeuropathy is associated with abnormality of sympathetic efferents and of local cutaneous temperature (Wakisaka et al. 1991). In this model, sympatholytic treatment with guanethidine decreases the behavioral sensitization to cold and warm stimuli (Neil et al. 1991). In an electrophysiological study of sciatic nerve fibers in such a model, Xie and Xiao (1990) demonstrated that abnormal firing of injured fibers was increased by stimulation of the sympathetic system and depressed by phentolamine, an alpha-adrenoreceptor blocker. These data strongly suggest that the mononeuropathic model could be a good model of causalgia and sympathetic maintained pain, for which hyperalgesia to cooling stimuli seems to be a main symptom (Frost et al. 1988). In another model of chronic pain, the Freund’s adjuvant arthritic rat, the efficiency of antisympathetic drugs has been proved based on clinical and radiological symptoms (Coderre et al. 1991) but to our knowledge there is no report of measure of antinociceptive activity of sympatholytic treatment in arthritis. It appeared to be of interest to test and compare the
antinociceptive efficacy of sympatholytic treatment in chronic arthritis to that observed in the neuropathic model. As in an initial study in the same model of neuropathy (Neil et al. 1991), we used guanethidine as a sympatholytic drug. It is commonly employed for chemical experimental sympathectomy to deplete peripheral stores of noradrenaline (Furst 1987) and in human therapy for sympathetic blockade. Another drug, calcitonin, has also been used in sympathetic maintained pain (Gennari et al. 1984; Tanguy and Peyron 19X8> but its antalgic efficiency in human pathology seemed to be observed mainly after central injection in pain specially due to metastatic bone lesion (Miralles et al. 1987). Recently, it has been proved that it could also have an antalgic action on phantom limb pain either after central or i.v. injections (Kessel and Worz 1987; Jaeger and Maier 1992). In animal experiments, calcitonin has been mostly administered by intracerebral or Lt. injections; in addition, results differ according to the test, route of administration and species (Welch et al. 1986; Spampinato et al. 1988; Guidobono et al. 1991). Thus, finally, the analgesic effect of calcitonin is somewhat controversial, especially when systemically injected. It appeared to be of interest to test its putative antinociceptive effect in the rat models of mononeuropathy and arthritis using S.C. injections, as commonly used in human therapy. Thus, in the present study the putative antinociceptive of chronic guanethidine and calcitonin were gauged in parallel in the 2 models of clinical pain, with tests previously used to demonstrate antinociceptive activity of opioids (Kayser and Guilbaud 1990), non-steroidal anti-inflammatory agents (Attal et al. 1988) and antidepressant drugs (Ardid and Guilbaud 1992) in the 2 pain models.
(1988) were
and Attal considered
et al. (1990)
but, as explained
in the final
data analysis. Male
River,
arrival.
kept in cages with light and free access to food and
were
for I week before
after a first behavioral sodium The
200-225
g (7 weeks old) on
surgery. They were operated
I week later
testing session. Rats were anesthetized
pentobarbital
loosely (with about
weighing
Sprague-Dawley
rats (Charles water
France).
later. only 24 rats
(St1 mg/kg.
i.p.)
and 4 Iigatureb
was
same dissection
performed
on the opposite
side without
Treatment procedure We used guanethidine tonin (Cihacalcine)
monosulphate
The drugs were injected adjuvant
and human cnlci-
injection
for the arthritic
old when treated.
week after surgery
i.v. in arthritic
hy several studies (Wall
peripheral
S.C. at 0.125
WB~ injected
(n = 17) rats at 30 mg/kg
to the dose reported totally to deplete
I
model and
model. Thus, all the rats were Y-IO weeks
Guanethidine
and mononeuropathic
France).
for 1 week starting 3 weeks after Freund’s
for the mononeuropathic
jected
(Ismeline)
given generously by Dr. Sallikres (CIBA,
sympathetic
mg each day (i.e..
usually given in humans)
in arthritic
(n = Y)
each day according
et al. 1979) that seems
stores. C’alcitonin was in-
25% of the total daily dose
(n = 11) and mononeuropathic
rats (n = 12). Control
animals received the same volume of O.Y% NaCt i.v. daily
for I week: n = Y for arthritic Six additional
and n = IS for mononeuropathic
normal rats were treated with calcitonin as detailed
not administered
to normal animals since it was previously shown to
he ineffective
above. A guanethidine
rats.
under the
same condition>
treatment
was
(Neil et al. lY91b).
Testing procedure Rats were tested in ~1quiet room by the same person, blind to the drug used. Rats were randomly
arranged
in groups of 4 or S for a
eerie:, of tests. All the groups were tested before and after I week of treatment,
then every week
neuropathic
over 4 and 8 weeks for arthritic
The antinociceptive tion threshold
effect WB~ tested by measuring
uaing the Basile Analgcsymeter compounds
the vocaliza-
elicited by pressure on the right hind paw of each rat, Centrdlly
This response,
particularly
Sensitization
(Apelex)
integrated.
Kayser and Guilbaud
measuring
and
rats. respectively.
until a squeak was audible.
is especially sensitive to analgesic
In thcac 2 pain models (Attal
et al. 1990;
IYYO).
to an otherwise
determined.
innocuous cold stimulus (cold test)
as extensively
described
elsewhere
et al. 1990), by plunging the hind paw into a 10°C water
and methods
tied
l-mm spacing) around the common sciatic nrtve.
ligating the sciatic nerve (sham procedure).
was similarly
Materials
with
were
(Attal
bath and
the latency to a struggle response occurring with a cut-off
time of IS sec.
Animul models Male
In addition.
Sprague-Dawley
arthritic
and normal
rats (Charles
River.
ethical standards lefinited
were made
for investigations
by the IASP (1983).
according
tn the guidelines
of experimental
regard to housing conditions.
in order to avoid or minimize
fort to the animals:
which were
housed pre-operatively
on
pain in animals
Great care was taken, particularly
animals
to undergo
with
discom-
surgery were
6 to a cage then 1 to a cage for at least 5 days
after surgery. Cages floors were covered with sawdust, and food and fluid were available Arthritis were
then
radiological
River,
tested
by injection
France)
3 weeks
arthritic
according
of Freund‘s
at 6-7
later
was produced
to the method
few minutes
before
periods. Six different from
0 r= normal
the test, and observed
for a 5-min
positions of the lesioned hind paw were rated
to 5 = most
adapted
from that described
Dennis
1977).
For
for up to three
each
ahnormal
position.
tar the formalin
S-min
period
the
This
scale was
test (Dubuisson score
according to the formula [Tl + T2 + T3 + T4 + TS]/300 the time. expressed in seconds, spent in position
and
was calculated (Tl.
TI?,... was
I. 2.... over a total
at the maximum
into the
of clinical
et al. 19X7: Colpaert
and 1987):
g.
calculated
for each particular
rat.
in detail
Stutisticul unalysis Results of arthritic calcitonin
in 39 rats on the
described
adjuvant
weeks of age (n = 2Y). The)
symptoms (Calvin0
at this time their weight was 200-225 Neuropathy
et al. 1990). Each
of 5 min). Then, using 3 consecutive score values. the mean score was
ad lihitum.
was induced
rat tail (Charles
pain in the mononeuro-
animal was placed in a plexiglass cylinder. allowed to habituate
France) were used for this study. All the experiments
the bcort: of spontaneous
pathic rat was rated as described previously (Attal
right
hy Bennett
hind
paw’
and Xir
or
and mononeuropathic
guanethidine
were
control arthritic
and neuropathic
volume
the
during
same
time,
rats treated either with
compared
to those
obtained
in
animals receiving NaCl in the same using
l-way
analysis
of variance
43 TABLE
TABLE
I
THRESHOLD VALUES OF THE DIFFERENT TESTS IN ARTHRITIC RATS (3 weeks after injection of Freund’s adjuvant in the tail) AND IN MONONEUROPATHIC RATS (1 week after sciatic nerve ligature) PRIOR TO ANY TREATMENT Non-operated rats were those devoted to receive a sciatic nerve ligature; the values were comparable to those obtained in different studies. Vocalization to paw pressure expressed in grams (pressure test); struggle latency to paw immersion in a 10°C water bath expressed in seconds (cold test). Pressure
test
151+
270+
12.9kO.7
**
**
10.3+0.6
***
7
Observation period from W, to W, (i.e., 7 weeks after arthritis inoculation). No significant difference between the 3 groups of rats with ANOVA, Tukey test. Saline (n = 9)
7 ***
245+10
IN ARTHRITIC RATS, VOCALIZATION THRESHOLD TO PAW PRESSURE (in grams) IN DIFFERENT GROUPS OF RATS, TREATED DAILY WITH SALINE, CALCITONIN OR GUANETHIDINE FROM W,, (3 weeks after Freund’s adjuvant inoculation) TO W,
Cold test (set)
(g) Arthritic (n = 29) Mononeuropathic (n = 24) Non-operated rats (n = 24)
II
Week Week Week Week Week
0 1 2 3 4
155* 144* 156+ 163+10 176k
7 7 9 7
Calcitonin (n = 11)
Guanethidine (n=9)
152k6 150+7 159+8 166+6 164k5
147+ 165 + 138k 152k 157k
8 12 8 8 6
14.9 f 0.9
* * P < 0.01; ** * P < 0.001. Difference between arthritic or mononeuropathic compared to non-operated rats. Differences between arthritic and mononeuropathic rats P < 0.01 or 0.05 for the pressure and cold tests, respectively; ANOVA.
(ANOVA) with the Tukey test. Results were expressed in absolute values (in grams or seconds) or as percentage of initial threshold values determined just before the beginning of treatment. Differences at P < 0.05were considered significant. S.E.M. was calculated by first getting a single score for each rat.
Results
In normal rats (data not shown) calcitonin injected S.C. has no effect on vocalization threshold to paw pressure nor on the struggle’ latency to 10 “C water bath, as has already been shown after guanethidine treatment (Neil et al. 1991a,b). General results in the 2 pain models prior to any treatment (Table I)
(a> As in previous studies (Kayser and Guilbaud 1990), the vocalization thresholds to the mechanical stimulus 3 weeks after Freund’s adjuvant inoculation were much lower in arthritic (n = 29) than in normal (n = 24) rats prior to nerve ligature: the mean threshold for hind-paw pressure was 151 k 7 g vs. 270 + 7 g (P < 0.001). The decrease observed for neuropathic rats, although significant compared to presurgery values (245 f 10 g, n = 24, P < 0.011, was less marked than in arthritic rats (significant difference between the 2 models: P < 0.01). (b) The latency for the struggle reaction to the cold stimulus (cold test) of 12.9 & 0.7 sqc in arthritic rats was significantly decreased compared to normal rats (P < O.Ol), but was less than in neuropathic rats (10.3
f 0.6 set (P < 0.001) (significant difference between the 2 models P < 0.05). (c) The mean score of tonic pain gauged by the hind-paw position in neuropathic rats was 1.84 _t 0.18 (n = 241, slightly below the mean score initially described (Attal et al. 1990; Ardid and Guilbaud 1992). Results in treated arthritic rats (n = 29)
(a) In saline-treated arthritic rats (n = 9), the vocalization threshold to paw pressure (155 f. 7 g at week 0) remained at a low level over the observation period (Table II) and started to recover toward normal values at the end of the observation period, i.e., 7 weeks after arthritis induction, in agreement with a previous study (Calvin0 et al. 1987). The latency of the struggle reaction to 10°C (cold test) (12.6 + 1 set at week 0) did not change significantly over the course of the observation period (Table III): 7 weeks after arthritis induction the rats were still abnormally sensitive to cold (in another study not shown
TABLE
III
IN ARTHRITIC RATS, STRUGGLE LATENCY TO PAW IMMERSION IN A 10°C WATER BATH (in seconds, with a cut-off at 15 set) IN DIFFERENT GROUPS OF RATS, TREATED DAILY WITH SALINE, CALCITONIN OR GUANETHIDINE, FROM W, (3 weeks after Freund’s adjuvant inoculation) TO W, Observation period from W, to W, (i.e., 7 weeks after arthritis inoculation). No significant difference between the 3 groups of rats with ANOVA, Tukey test.
Week Week Week Week Week
0 1 2 3 4
Saline (n = 6)
Calcitonin (n = 9)
Guanethidine (n = 9)
12.6 f 1 14.2 + 0.5 13.6 * 0.7 11.8+0.8 12.0& 1.0
13.2 + 13.8 f 14.1* 14.5 * 13.1 f
13.8 f 0.4 13.6 f 0.5 14.OkO.5 14.4kO.2 13.6 f 0.5
0.8 0.7 0.4 0.2 0.9
hcrc. the recovery to a normal value was observed 10 weeks after Freund’s adjuvant injection). (h) In calcitonin treated arthritic rats (n = 11). there was no modification of the vocalization threshold to paw pressure over the observation period f 152 i: 6 vs. 150 i 7 g before and at the end of treatnlcnt, rcspectively) nor change in the latency of the cold test (13.11 f 0.8 vs. 13.8 I: 0.7 set before and at the end of treatment, respectively). There was no significant difference between this group of rats and those injected with saline (Tables II and III). (cl In guanethidine-treated arthritic rats (n = 91, there was a slight increase in the vocalization threshold to paw pressure, at the end of treatment (147 h 8 at week 0 vs. 165 & 12 g at week 1) but it was not significant and only transient (Table II). The cold test was not modified at all by ~uanethidine trcatmcnt (13.X i 0.4 vs. 13.6 i: 0.5 set before and at the end of treatment). There was no significant difference between this group of rats and the saline-injected animals (Table III>. Thus, with the 2 tests, there was no significant difference between the saline-, calcitoninand guanethidine-treated arthritic rats. kwlt.~
in treated mononeuropathic
ruts fn = 24)
Initially 39 rats were prepared with loose sciatic nerve ligatures, then tested and pretreated. Fifteen rats (6 saline-, 4 calcitonin- and 5 guanethidine-treated animals) exhibited self-mutilation lesions of the 4th and 5th digits. They were mostly minor lesions. limited to nails, but we chose to sacrifice these rats prior to the subsequent tests. and only 24 rats were included in the data analysis. (a) In saline-injected mononeuropathic rats (n = 41 (Fig. I), the vocalization threshold to paw pressure (227 + 10 g) was already decreased compared to the presurgery value when treatment started and exhibited a further decrease over the following 2 weeks (i.c.. until the 3rd week following ligature). Then, recovery toward normal threshold values was observed, roughly in agreement with the initial study (Attal et al. 1990). The decrease in struggle latency to the cold test (11.1 + 0.6 see) observed 1 week after nerve ligation was maintained for several weeks after the end of treatment (Fig. 2). Recovery started after the 6th week following surgery, in good agreement with the initial investigation. The score of the tonic pain-related behavior (mean = 1.84 + 0.1X), slightly below the mean score of other studies (Attal et al. 1990: Ardid and Guilbaud 19021 at week 1 after nerve ligation, then regularly decreased over the observation period (Fig. 3). ’ (b) In calcitonin-treated rats (n = 8), the vocalization thresholds to paw pressure and the struggle latency to cold stimulus which were reduced prior to treatment
NEUROPATtiiC pressure
RATS test
. guanethidlne 0 saltne
I ,_.-_..T.._
.... . . . - T...-” .,----T.‘-T-__
0123456 treatment
7 weeks
Fig. I. Time court of thr bocaliratmn thresholif to pa% pr~~.xir~ usprcssed in yr;m,s (+S.E.M.) c~hservcit in guanethidinc- anii s;tlincinjected neuropathic r’ntb from W,, (1 week after IIL‘TVCligature) to W, (8 weeks after hurerrq). Thr preaurgcryvalur wx, mean = 170-i IO g. Kats were trcatcd saline
with
from W. to W, Jaily (II0 mg/kg
the hame volumtl.
guanethidine
i.v.1. *= * P <: 0.001 (ANOVA,
or
Tukey
test I.
(222 5 X g and 11.0 i: 0.7 SW, respectively) did not not significantly differ thereafter in the same group, compared to the values obtained in saline-injected rats.
NEUROPATHIC
20 i
RATS
45 NEUROPATHIC 2
0
salme
.
gvanethldme
RATS
0
OJ I 0
I
2
I
4
1
I
6
6 weeks
treatment Fig. 3. Time course of the spontaneous pain-related behavior of guanethidineand saline-injected neuropathic rats, rated with a scale determined on the basis of the hind-paw position from W,, to W,, i.e., from 1 to 9 weeks after nerve ligature. Rats were treated daily from W,, to W, as in Figs. 1 and 2 (guanethidine at 30 mg/kg, i.v., or saline with the same volume). There was no significant difference between the 2 curves (ANOVA, Tukey test).
The score of the spontaneous pain-related behavior was not significantly modified either by comparison to the saline-treated animals. This lack of effect was especially clear just at the end of treatment (i.e., 2 weeks after sciatic nerve ligation) (Table IV). (c) In guanethidine-treated rats (n = 7) (Fig. 11, the vocalization threshold to paw pressure was especially decreased for this group of rats (215 & 10 g> 1 week after nerve ligature when the treatment started, but was significantly increased (P < O.OOl), reaching 150% of the control value (324 _t 26 g) at the end. This
TABLE
IV
IN MONONEUROPATHIC SALINEAND CALCITONINTREATED RATS, THE VOCALIZATION THRESHOLD TO PAW PRESSURE, STRUGGLE LATENCY TO PAW IMMERSION IN A 10°C WATER BATH AND SCORE OF SPONTANEOUS PAIN-RELATED BEHAVIOR, JUST AFTER TREATMENT, i.e.. 2 weeks after sciatic nerve ligature (no significant differences compared to pre-treatment values) No significant difference was observed at this or any other time point between the 2 groups of rats (ANOVA, Tukey test). Vocalization
(d Calcitonin (n = 8) Saline (n = 6)
205*
1.5
214+ 10
10°C bed 9.9 + 1 11.3kO.9
Spontaneous
I
kO.3
1.3f0.2
pain
reflected a genuine, but not prolonged, antinociceptive effect; indeed, 1 week later, 3 weeks after surgery, the vocalization threshold of these guanethidine-treated animals was comparable to that of saline-injected rats. In these rats, the latency of the struggle reaction to a 10°C cold stimulus of 10.3 * 0.6 set at the beginning of treatment increased to a final value of 14.9 ) 0.5 set (P < O.OOl), so regaining the presurgery value (Fig. 2). The normalized responses seen in the guanethidinetreated group were observed throughout the observation period, i.e., until week 8 after ligatures, a time when normally the abnormal reaction to cold has spontaneously disappeared. This treatment, therefore, induced a persistant clear antinociceptive effect. In sharp contrast, there was no significant difference between the time course of the score of spontaneous pain-related behavior in saline- and guanethidine-injetted rats (Fig. 3). Thus, in mononeuropathic rats, calcitonin did not exhibit any antinociceptive action whatever the test considered. In sharp contrast, guanethidine treatment induced a significant but transient increase of the vocalization threshold to paw pressure and reestablished the struggle latency to a 10°C cold stimulus to a normal value.
Discussion
Before any treatment, animals in the 2 models of pain (arthritic and mononeuropathic) exhibited abnormal sensitivity to mechanical and cold stimulus, with nociceptive reactions to stimuli that are not usually in the noxious range, thus exhibiting allodynia according to the IASP definition (Merskey et al. 1986). Nevertheless, the mechanical allodynia, previously described in these 2 models (Calvin0 et al. 1987; Bennett and Xie 1988; Attal et al. 1990; Kayser and Guilbaud 1990) is more marked for arthritic rats. Inversely, allodynia to cold as described in neuropathic rats (Bennett and Xie 1988; Attal et al. 1990) was more pronounced in these animals than in arthritic rats. In these 2 models, calcitonin treatment for 1 week was ineffective on the thresholds of the tests based on mechanical or cold stimulus and used to assess an antinociceptive action. This treatment also did not modify the spontaneous pain-related behavior based on the abnormal position of the paw in neuropathic rats. It seems unlikely that this absence of effect could be due to test sensitivity, since it has been used to demonstrate antinociceptive activity of several agents such as opioid, antidepressant and non-steroidal antiinflammatory drugs in the same models of clinical pain (Attal et al. 1988, 1990; Kayser and Guilbaud 1990; Ardid and Guilbaud 1992). In fact, although calcitonin has been used to treat chronic neuropathic pains, such
as causalgia and sympathetic maintained pain (Procacci and Maresca 1987; Tanguy and Peyron 198% its antalgic action has been mainly shown after it. injection either on phantom limb pain or pain related to visceral cancer (Miralles et al. 1987) with the exception of the last study of Jaeger and Maier (1992) who used systemic injection at a dose of 200 IU. In animals, the antinociceptive effect of calcitonin has mostly been revealed after intracerebroventricular injection (Welch et al. 1986; Spampinato et al. 1988) and after a local injection in 1 recent investigation (~uidobono et al. 1991). Since in the present study, using tests in the 2 models sensitive to various analgesics, systemically administered calcitonin had no evident antinociceptive action, it could be interesting to seek an action after longer treatment periods and with multiple daily injections. In sharp contrast, we found that guanethidine had a marked effect in the mononeuropathic model, on the vocalization threshold elicited by hind-paw pressure, but mainly on the reaction time to cold stimuli. Antino~i~eptive actions tested with the mechanical test were not observed in previous experiments conducted by Neil et al. (1991). This could be due to the more prolonged treatment applied in the present study. However, antinociception is not observed either after surgical sympatectomy (Desmeules et al. 1992). In any case, this relieving action of guanethidine on mechanical allodynia was only transient. The total lack of antalgic effect on the spontaneous pain-related behavior could be due to the complexity of the test, making it less sensitive to pharmacological manipulation. In sharp contrast, and as others who also found an effect of chemical and surgical s~pathe~tomy on the abnormal sensitivity to heat (Neil et al. 1991; Desmeules et al. 19921, we noted an important antinociceptive effect of guanethidine treatment on abnormal reaction to cold stimulus. There was, indeed, a recovery to the normal struggle latency which persisted over the entire obervation period, i.e., until the 8th week after nerve ligature, the usual time of spontaneous recovery (Attal et al. 1990). Since guanethidine has a peripheral sympatholytic action in adult rats (Furst 1987), this data seem to confirm that abnormal sensibility to cold in this model is mainly mediated by the sympathetic system (N&I et al. 1991). I As discussed initially (Neil et al. 19911, effectiveness of guanethidine treatment on the cold test in this neuropathic model can be related to the fact that allodynia to cold stimulus is found to be a major sign in sympathetic maintained pain (Frost et al. 1988). This fits well with the abnormal sensitivity to thermai stimuli observed for injured afferent fibers (Matzner and Devor 19871, especially in the same model of sciatic ligature by Xie and Xiao (19901, and strongly suggests
that this rat model of mononeuropathy could be relevant to the study of causalgia and sympathetic maintained pain mechanisms. The lack of effect of guanethidine treatment on the 2 tests performed in arthritic rats was relatively surprising at first sight, according to the various obse~ations reported with this mode1 (Levine et al. 1986 a,b; Coderre et al. 1991). It could be that our treatment was too short to induce antinociceptive effects but it has been demonstrated that guanethidine treatment for 4 days at 30 mg/kg was efficient for at least 1 month (Wall et al. 1979). In addition, we have recently demonstrated (Weil-Fugazza et al. unpublished) that i.v. injections of guanethidine (30 mg/kg during 4 days) in the normal rat reduced the level of noradrenaline on the distal part of the sciatic nerve by 90% (n = 5). It could also be argued that we treated arthritic rats at the time of maximum pain state. For this reason, we treated an additional group of rats (n = 6) at week 2 after induction of arthritis, just when nociceptive behavior starts (Calvin0 et al. 1987). In this group of rats (data not shown here) guanethidine did not induce any relief of the abnormal sensitivity to cold and mechanical stimuli. In fact, the studies of Levine et al. f1986b) clearly showed that either pharmacological or surgical sympathectomy relieved or prevented clinical and radiological inflammatory symptoms. It has also been widely demonstated that sympathetic efferents contribute to the severity of joint injury (Coderre et al. 1990; Bjerknes et al. 1991). However, in these studies, the antalgic action of sympathectomy has not clearly been tested. ‘There are also some controversies concerning the role of sympathetic mediators in nociceptive processing. Koltzenburg et al. (1992) have recently demonstrated that sympathetic post-ganglionic neurons do not contribute to heat sensitization of nociceptors by bradykinin. Thus, the role of the sympathetic system in nociception appears to be even more complex than expected. According to our present data, it seems important to dissociate nociceptive thresholds from in~ammato~ symptoms because physical and sensory symptoms of arthritis seem not to be dependent on the peripheral sympathetic system to the same extent. Finally, in this parallel study of putative analgesic action of 2 treatments in 2 different pain models, calcitonin had no antinoci~eptive effect, while the sympatholytic drug was more effective against nociceptiverelated behaviors in the neuropathic than in the arthritic model, at least at the stages of disease considered in this study. Even in the same model, allodynia to I stimulus modality, cold, was more sensitive to the sympatholyti~ treatment than the tests based on mechanical stimulus. These data sharply emphasize the importance of different nociceptivc tests in different models of pain, in particular for analyzing the involvment of the sympathetic system in pain processing.
47
Aknowledgements
This study has been performed with a support from La Fondation pour la Recherche MCdicale to S. Perrot and support from Laboratoires Ciba, France. The authors wish to thank Dr. A.H. Dickenson for English revision, and Mme. M. Gautron for illustrations.
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