Vol.2, No.1, 62-66 (2010)
doi:10.4236/health.2010.21010
SciRes
Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
The central nucleus of amygdala is involved in tolerance
to the antinociceptive effect of NSAIDs
Merab G. Tsagareli1, Nana Tsiklauri1, Gulnazi Gurtskaia1, Ivliane Nozadze1, Elene Abzianidze2
1Beritashvili Institute of Physiology, Tbilisi, Georgia; tsagareli@biphysiol.ge
2State Medical University of Tbilisi, Tbilisi, Georgia
Received 12 October 2009; revised 20 November 2009; accepted 27 November 2009.
ABSTRACT
Aim: Repeated microinjections of non-opioid an-
algesics into the midbrain periaqueductal gray
matter and rostral ventro-medial medulla induce
antinociception with development of tolerance.
Antinociception following systemic administra-
tion of non-steroidal anti-inflammatory drugs (N
SAIDs) also exhibit tolerance. Presently our aim
was to investigate the development of tolerance
to the antinociceptive effects of NSAIDs analgine,
ketorolac, and xefocam microinjected into cen-
tral nucleus of amygdala (Ce) in rats. Methods:
Under anesthesia with thiopental a stainless steel
guide cannula was stereotaxically implanted uni-
laterally or bilaterally into the Ce using stereo-
taxic atlas coordinates, and anchored to the cra-
nium by dental cement. Five days after surgery,
3 µl of these NSAIDs were injected via the injec-
tion cannula while the rat was gently restrained.
Twenty min post microinjection, i.e. 10-min be-
fore the peak of the drugs’ effect is normally rea-
ched, animals were tested with tail flick (TF) and
hot plate (HP) tests. On the 5th experimental day
all animals received a Ce microinjection of mor-
phine. Results: Daily microinjection of NSAIDs
into the Ce uni- or bilaterally, produced antino-
ciception with development of complete toler-
ance over a 5-day period. Following the treat-
ment period, morphine microinjection into the
Ce failed to elicit antinociception, indicating cro-
ss-tolerance to the antinociceptive effect of N
SAIDs. In other words, the “non-opioid tolerant”
rats showed cross-tolerance to morphine. Con-
clusions: Our data confirmed the suggestion that
NSAIDs interact with endogenous opioid systems,
which likely play a key role in the development of
tolerance to the antinociceptive effects of NSA
IDs.
Keywords: Descending Inhibition; Morphine
Cross-Tolerance; Nociception
1. INTRODUCTION
Microinjection of non-opioid analgesics metamizol, and
lysine-acetylsalicylate (LASA) into certain brain areas,
including the midbrain periaqueductal gray matter (PAG)
and rostral ventro-medial medulla (RVM), produces an-
tinociception with development of some degree of tol-
erance [1-4]. We have also observed tolerance to the an-
tinociceptive effects of analgine (metamizol), ketorolac,
and xefocam administered systemically [5-7]. These stu-
dies are consistent with the possibility that endogenous
opioidergic mechanisms associated with descending pain
modulation may partly mediate the tolerance observed
with non-steroidal anti-inflammatory drugs (NSAIDs) [8].
The amygdala receives massive input from the hippo-
campus and the neocortex and provides a major source of
afferents to PAG [9]. Analgesia resulting from microin-
jection of opioid agonists into the basolateral amygdala
is blocked by lidocaine inactivation of, or opioid antago-
nist injection into, the PAG [10-12]. Cortical afferents to
the amygdala largely target its basolateral component.
The basolateral amygdala then projects to the central
nucleus of amygdala (Ce), which in turn projects densely
to the PAG [13]. The Ce also receives nociceptive input,
both directly from the spinal cord, and indirectly via a
large projection from the dorsal horn to the parabrachial
nucleus [14,15]. The Ce is an integral component of the
endogenous pain-modulatory circuit and is critical for
systemic morphine-induced suppression of spinal noci-
ceptive reflexes [16].
The present study reports that microinjection of an-
algine, ketorolac, and xefocam into the Ce of rats elicits
antinociception with the development of tolerance.
2. MATERIALS AND METHODS
The experiments were carried out using male Wistar rats,
200-250g in body weight, bred at the Beritashvili Insti-
tute of Physiology. The animals were kept under standard
housing conditions (22±2 C, 65% humidity, light from
M. G. Tsagareli et al. / Health 2 (2010) 62-66
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63
7:00 a.m. to 8:00 p.m.) and were fed a standard dry diet;
water was freely available. Experiments were performed
during the light phase of the circle between 10:00 and
14:00 clock. Guidelines of the International Association
for the Study of Pain regarding animal experimentation
and Guide for the Care and Use of Laboratory Animals
(National Academy Press, Washington, DC, 1996) were
followed throughout.
Under anesthesia with thiopental (55 mg/kg, i.p. “Kiev-
med” Ukraine) a 12-mm stainless steel guide cannula (Pla-
stic One, Inc., USA) was stereotaxically implanted uni-
laterally (left side) or bilaterally into the Ce amygdala us-
ing coordinates from the atlas of Paxinos G. & Watson C.
[17], and anchored to the cranium by dental cement. The
guide cannula was plugged with a stainless steel stylet.
Thereafter, the rats were handled in 3 daily 15 min peri-
ods to become habituated to the experimental environ-
ment and test protocol that involved removal of the stylet
and insertion of injection cannula (10 mm length of PE
tubing attached to a 50 µl Hamilton syringe; Hamilton,
Inc., USA) without drug injection. Five days after surgery,
three µl of drug was injected via the injection cannula
while the rat was gently restrained. Drugs were: Analgine
(metamizol sodium, 1.5mg/3µl, “Sanitas”, Ltd, Lithua-
nia), ketorolac (ketorolac tromethamine, 90µg/3µl, “Zee
Drugs”, India), xefocam (lornoxicam, 12µg/3µl, “Ny-
comed”, GmbH, Austria), or saline (3µl) (“Galichpharm”
Ltd. Ukraine). Twenty min post microinjection, i.e. 10-
min before the peak of the drugs’ effect is normally rea-
ched, animals were tested with TF or HP. For the TF test,
the distal part of the tail was stimulated with a light beam
(IITC #33, IITC Life science, Inc., Woodland Hills, CA,
USA) and the latency measured until the tail was reflex-
ively flicked away from the beam. For the HP test, the
rat was placed on a 52ºC hot plate (IITC #39) and the la-
tency to lick paw or jump was measured. The cut-off
time was 20 s for both TF and HP latencies. Each rat was
tested with both tail flick (TF) and hot plate (HP) laten-
cies in the same session. The same procedure was fol-
lowed to deliver repeated microinjections of each drug (an-
algine, ketorolac, xefocam) or vehicle (saline) over five
consecutive days. On the 5th experimental day all ani-
mals received a Ce microinjection of morphine hydro-
chloride (3µg/2µl, “Laboratoires Stella”, France) and TF
and HP latencies were measured 20 min thereafter. At
the conclusion of experiment on the fifth day, the micro-
injection site was marked with 2 µl of a saturated solu-
tion of Pontamine Sky Blue (Sigma Chemical Co.,USA),
and the animal was sacrificed by ester inhalation. After
fixation by immersion in 10% formalin the brain was
sectioned and the microinjection site was identified with
the aid of Paxinos & Watson’ stereotaxic atlas [17].
All data are presented as meanS.E.M. Analysis of
variance (ANOVA) with post-hoc Tukey-Kramer multi-
ple comparisons were used for statistical evaluations. The
statistical software utilized was InStat 3.05 (GraphPad
Software, Inc, USA). Statistical significance was ac-
knowledged if P<0.05.
3. RESULTS
Only rats with microinjections into Ce were included for
data analysis. Histological location of microinjection
sites is shown in simplified drawing section from the
Paxinos and Watson atlas [17] (Figure 1). These data
consisted of 13 rats microinjected with analgine (6 uni-
and 7 bilaterally), 13 with ketorolac (6 and 7), 12 with
xefocam (6 and 6), and 15 with control saline (8 and 7),
respectively. Injection sites outside the boundaries of the
Ce (the shaded region in Figure 1) were not included in
data analysis. In special control experiments with inten-
tionally microinjections of NSAIDs out of Ce we did not
reveal significant changes in TF and HP latencies (data
not shown).
On the first test day, unilateral microinjection of each
NSAID into the Ce produced antinociception as revealed
by significant increases in latency for TF [ANOVA:
F(3,20)=21.251; P<0.001] and HP [ANOVA: F(3,20)=
15.872; P<0.001] compared to saline controls (P<0.001
for all drugs) (Figure 2). However, on successive days,
microinjection of each NSAID had a progressively
weaker antinociceptive effect such that on the fourth
and/or fifth experimental days the TF and HP latencies
were not significantly different compared to saline injec-
tions. This was similar to the development of tolerance
to morphine administration to PAG in similar prepara-
tions [18,19], and we therefore refer to it as “non-opioid
tolerance”. Note, however, that tolerance to the antino-
ciceptive effect of xefocam was slower compared to an-
algine and ketorolac. On day 5, both experimental and
Figure 1. Location of microinjection sites in the Ce.
Only NSAIDs and saline injections within the shaded
regions were included in data analysis. Coronal sec-
tions are taken from the atlas of Paxinos and Watson
(1998). The Distances from interaural line ±5.7 mm
and from the bregma -3.3 mm respectively. All injec-
tions fell within ±0.5 mm of this coronal plane.
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64
control groups received a morphine microinjection at the
same Ce sites, and only the saline group exhibited anti-
nociception (P<0.001). The latencies of the non-opioid
tolerant rats were not altered by the morphine microin-
jections, i.e. they showed cross-tolerance to morphine
(Figure 2).
Bilateral microinjections into the Ce also increased
latency of TF [ANOVA: F(3,20)=8.873; P=0.006] and
HP [ANOVA: F(3,20)=11.933; P<0.001] compared to
control rats on the first day for these NSAIDs, in TF
(P<0.01) and in HP (P<0.001) respectively (Figure 3).
Similar to the unilateral drug injections, there was a pro-
gressive decline in the antinociceptive effect elicited by
bilateral injection of each drug over the 5-day period,
0
5
10
15
20
25
1 23 4 5morphine
DAYS
Tail flick latency (sec)
Saline
Analgine
Ketorol ac
Xefocam
*** ***
**
***
**
*
A
0
5
10
15
20
25
12 3 45morphine
DAYS
Hot plate latency (sec)
Sal i n e
Analgi ne
Ketoro la c
Xe fo cam
***
**
*
B
Figure 2. Response latencies in unilateral microinjections. Mean
response latencies following unilateral microinjections of each
NSAIDs are plotted over the 5-day period, followed by mor-
phine, for TF (A) and HP (B) tests. Cutoff latency (20 sec)
was reached in some animals for xefocam and morphine mi-
croinjections. Asterisks mark significant difference of latency
in comparison to saline control as analyzed by post hoc tests
(*p<0.05, **p<0.01, ***p<0.001).
0
5
10
15
20
25
1 2 34 5morphine
DAYS
Tail flick latency (sec)
Sal in e
Analgine
Ketor o l a c
Xefocam
** ***
*
**
A
0
5
10
15
20
25
12345morphine
DAYS
Hot plate latency (sec)
Saline
Analgine
Ketorol a c
Xefocam
***
***
**
*
***
*
B
Figure 3. Response latencies in bilateral microinjections. Mean
response latencies following bilateral microinjections of each
NSAIDs are plotted over the 5-day period, followed by mor-
phine, for TF (A) and HP (B) tests. Cutoff latency (20 sec)
was reached in some animals for xefocam and morphine mi-
croinjections. Asterisks mark significant difference of latency
in comparison to saline control as analyzed by post hoc tests
(*p<0.05, **p<0.01, ***p<0.001).
such that TF and HP latencies were not significantly
different from saline controls after the 4th-5th day (Fig-
ure 3). Again, xefocam exhibited a slower time course
for development of tolerance. Bilateral microinjection of
each NSAIDs also exhibited cross-tolerance to morphine
as compared with saline controls (P<0.001) (Figure 3).
4. DISCUSSION
The present study revealed that microinjection of an-
algine, ketorolac, and xefocam into the Ce induced anti-
nociception in awake rats. This confirmed our previous
results with systemic (i.p.) administration of NSAIDs
M. G. Tsagareli et al. / Health 2 (2010) 62-66
SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
65
[5-7], and results of others using microinjection of the
same NSAIDs into the PAG [2-4]. Importantly, repeated
microinjections of NSAIDs into the Ce resulted in a pro-
gressive decrease in antinociceptive effectiveness (tol-
erance) similar to that observed with intra-PAG injec-
tions [2-4], and reminiscent of the effect of opiates.
A major involvement of opioidergic mechanisms in
tolerance to the analgesic effect of NSAIDs was surpris-
ing, because traditionally the cellular and molecular ac-
tions of opioids were thought to differ from those of
nonopioid analgesics. One interesting aspect of NSAIDs
administration, namely tolerance, emphasizes their simi-
larity to opioid analgesics. Indeed, microinjection of meta-
mizol [3,4,20], or LASA [2,20] into PAG or into Ce, pro-
gressively led to a loss of their antinociceptive effects,
i.e. produced tolerance. Furthermore, tolerance to meta-
mizol or LASA was accompanied by cross-tolerance to
morphine [2-4] as if opioid analgesics had been repeat-
edly administered. Interestingly, tolerance to the effect of
PAG-microinjected metamizol can, like tolerance to mor-
phine, be reversed by microinjection of proglumide, a
cholecystokinin antagonist, at the same PAG site [3]. The
latter finding constituted additional evidence that the PAG
effects of non-opioid analgesics are similar to those of
morphine. Moreover, the data suggested that Ce should be
incorporated into current models of endogenous pain con-
trol circuitry [21].
It is well known that morphine injection after admini-
stration of NSAIDs or in combination, morphine plus
NSAIDs usually potentiates their own analgesic effects
[8]. We have recently tested each of NSAIDs for cross-
tolerance to morphine given over a 5-day period in two
age groups of rats. There was a significant difference be-
tween adult and juvenile rat groups for the degree of mor-
phine analgesia, which was most marked on the first and
second experimental days. Furthermore, morphine-tole-
rant rats exhibited cross-tolerance to analgine, ketorolac,
and xefocam for both TF and HP tests, respectively (data
not shown).
In conclusion, our data confirmed previous studies in-
dicating that the antinociceptive action of NSAIDs may
be closely related to that of endogenous opioids, includ-
ing the development of tolerance. In addition, the Ce
along with PAG and RVM represents an important com-
ponent of the endogenous antinociceptive system.
5. ACKNOWLEDGEMENTS
The authors would like to thank Professor E. Carstens for helpful
comments and English revisions of the manuscript. This research was
supported by the grant from Georgian National Science Foundation
(GNSF/ST07/ 6-234).
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