Neurophysiology, Vol. 41, No.

6, 2009

Tolerance Effects Induced by NSAID Microinjections into the Central Nucleus of the Amygdala in Rats
M. G. Tsagareli,1 N. D. Tsiklauri,1 G. P. Gurtskaia,1 I. R. Nozadze,1 R. A. Kandelaki,1 and E. V. Abzianidze2
Neirofiziologiya/Neurophysiology, Vol. 41, No. 6, pp. 476-482, November-December, 2009. Received May 12, 2009.
Recent investigations have shown that microinjections of non-opioid analgesics, nonsteroidal antiinflammatory drugs, NSAIDs, into some brain areas, particularly, into the midbrain periaqueductal gray matter (PAG) and rostral ventro-medial medulla (RVM), cause antinociception with some effects of tolerance. Our preliminary findings have also shown the same effects of tolerance after intraperitoneal injections. The present study was designed to examine whether microinjections of metamizole (Analgin), ketorolac, and xefocam into the central nucleus of the amygdala (Ce) lead to the development of tolerance in rats, and to ascertain whether this nucleus is the pain-modulating pathway through PAG. Our investigation revealed that microinjections of NSAIDs into the Ce both unilaterally (the left side) and bilaterally produced antinociception, as indicated by a latency increase in tail-flick reflex (TF) compared to controls with saline, on the first experimental day for Analgin (P < 0.001), ketorolac ( P < 0.001), and xefocam ( P < 0.001). However, when these drug microinjections were repeated during subsequent days, the antinociceptive effects progressively diminished so that on the fifth experimental day the TF latency was similar to that in the rats that received repeated injections of only saline. These results show that, alongside with PAG and RVM, the Ce is an important site of the endogenous antinociceptive system, which triggers the descending pain control mechanism and thus inhibits nociceptive transmission. On the other hand, our data confirm the results of other authors that NSAIDs are closely related to endogenous opioids, and tolerance to these non-opioid drugs probably depends on opioid tolerance.

Keywords: non-opioid tolerance, morphine cross-tolerance, tail-flick reflex, Ce of the amygdala.

INTRODUCTION
It has recently been demonstrated that the pain modulation system includes the midbrain periaqueductal gray matter (PAG) and rostral ventromedial medulla (RVM). The RVM involves the midline nucl. raphe magnus and the adjacent reticular formation. The PAG is a part of the CNS circuits controlling nociceptive transmission at the level of the spinal cord, mainly through the RVM. The PAGRVM system is the central substrate for the action of opioid analgesic drugs. Endogenous opioid peptides are present in the somata and/or terminal fields of the neurons in several components of these networks. In animals, electrical stimulation of the PAG inhibits simple noxious stimulation-induced reflexes, such as
1 2

Beritashvili Institute of Physiology, Tbilisi, Georgia. Tbilisi State Medical University, Tbilisi, Georgia. Correspondence should be addressed to M. G. Tsagareli (e-mail: Tsagareli@biphysiol.ge; Tsagareli@geo.net.ge).

the tail flick (TF) or the paw withdrawal. Furthermore, this circuit contributes to opiate analgesia and opioid dependence [1]. Recent investigations have shown that in some brain areas, particularly, in the PAG and RVM, microinjections of non-opioid analgesics, metamizole, and lysine acetylsalicylate (LASA) cause antinociception and also some manifestations of tolerance [2-5]. Our preliminary findings also showed that the same effects of tolerance develop after i.p. injections of Analgin (metamizole), ketorolac, and xefocam [6-8]. Taken together, these studies support the supposition that the contribution of the downstream pain-controlling system to the tolerance effects of the above-mentioned nonsteroidal anti-inflammatory drugs (NSAIDs) directly relates to endogenous opioidergic mechanisms. The amygdala, which receives massive inputs from the hippocampus and neocortex, is a major source of afferents projecting to PAG [9]. Analgesia resulting

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0090-2977/09/4106-0404 2009 Springer Science+Business Media, Inc.

Tolerance to NSAIDs Microinjected into the Amygdalar Ce

from microinjections of opioid agonists into the basolateral amygdala is blocked by lidocaine-evoked inactivation of the PAG or after injections of opioid antagonists into this structure [10, 11]. Cortical afferents to the amygdala largely target the basolateral component of the latter. The basolateral amygdala then projects to the central nucleus of the amygdala (Ce), which, in turn, projects densely to the PAG [12]. The Ce also receives nociceptive inputs, both directly from the spinal cord and indirectly via a large projection from the dorsal horn to the parabrachial nucleus [13, 14]. Other authors provided evidence that the Ce is an integral component of the endogenous pain-modulatory circuit. This nucleus is a crucial structure responsible for systemic morphine-induced suppression of the TF nociceptive reflex [15]. Our study was designed to examine whether microinjections of Analgin, ketorolac, and xefocam into the Ce lead to the development of tolerance to these agents in rats.

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METHODS
The experiments were carried out on male albino rats bred at the Beritashvili Institute of Physiology and weighing 200 to 250 g. Guidelines of the International Association for the Study of Pain regarding animal experimentation were followed throughout. Under thiopental anesthesia (55 mg/kg, i.p., Kyivmed, Ukraine), a 12-mm-long stainless steel guide cannula (Plastic One, USA) was stereotaxically implanted unilaterally on the left side or bilaterally into the Ce of the amygdala, according to the atlas of Paxinos and Watson [16], and anchored to the cranium by dental cement. The guide cannula was plugged with a stainless steel stylet. Thereafter, the rats were handled every day for 15 min to get them to become familiar with the testing protocol and experimental environment during three days. During these episodes, the stylet was removed, and the injection cannula was inserted into the guide cannula, but no drug was injected. This helped us to habituate the rats to the injection procedure and to reduce artifacts arising from mechanical manipulation during the test days. Five days after surgery, a 10-mm tubing was attached to a 50-l Hamilton syringe (Hamilton, USA) and was then joined to the guide cannula. The drug was introduced through it, while the rat was gently restrained. The injected solutions contained 150 g Analgin (derivative of pyrazolone, metamizolum natricum, Sanitas, Lithuania), 90 g ketorolac, or 12 g xefocam dissolved in 0.3 l of

saline (Galichpharm, Ukraine). Three microliters of saline were injected as the control. Then, the guide cannula was plugged with the stainless steel stylet. Twenty minutes after microinjection, i.e., 10 min before the peak of the drug effect is normally reached, the proximal part of the tail was heat-stimulated by focused light, and the latency of the TF was measured using a specialized TF analgesia meter (IITC Life Science, USA). Similar microinjections of Analgin, ketorolac, xefocam, or saline were repeated for five consecutive days. On the 5th day, all animals received microinjection of morphine hydrochloride (3 g/2 l, Laboratoires Stella, France) into the Ce, and the TF latencies were measured 20 min thereafter. At the end of each experiment, after the 5th day, the microinjection site was marked with 2 l of a saturated solution of Pontamine Sky Blue (Sigma, USA), and the animal was euthanized with an overdose of ether. After fixation by immersion in 10% formalin, the brain was sectioned, and the microinjection site was identified according to stereotaxic maps [16]. All data are presented below as means s.e.m. Analysis of variance (ANOVA) subsequent to the TukeyKramer multiple comparison test was used for statistical evaluations. The statistical software utilized was InStat 3.05 (GraphPad Software, USA). The statistical significance was achieved if P < 0.05.

RESULTS
Only rats with sufficiently accurate microinjections into the Ce were included in the data analysis (Fig. 1). These groups consisted of 13 rats microinjected with Analgin, 13 rats with ketorolac, 12 rats with xefocam, and 15 animals with control saline injected. Animals with injection sites outside the boundaries of the Ce (shaded region in Fig. 1) were excluded from the analysis. Our investigation showed that unilateral microinjections of NSAIDs into the Ce (the left side) produced significant antinociception, as revealed by a latency increase in the TF compared to the control values (with saline microinjected into the same nucleus). On the first experimental day, the respective differences were highly significant for Analgin ( P < 0.001; Fig. 2A), ketorolac ( P < 0.001; Fig. 3A), and xefocam ( P < 0.001; Fig. 4A). Mean values of the latency of the TF reflex induced by thermal stimulation after microinjections of Analgin and ketorolac corresponded to about 250% of the control values, while the respective increase in the case of

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of NSAIDs into the amygdalar Ce (especially upon injections of Analgin and xefocam). On the first day of the experiments, the analgesic effects of bilateral microinjections were stronger than those in the unilateral group. The latencies of the TF reflex under conditions of bilateral injections were notably longer (intergroup differences were significant, P < 0.01). Therefore, we suppose that, when both sides of the Ce (left and right) are together involved in the induction of antinociceptive effects, the magnitude of analgesic reaction is greater than when only one nucleus is influenced. In bilateral microinjections of the NSAIDs used, we also observed clear crosstolerance effects to morphine. Significant analgesic effects after injections of morphine were observed only in animals of the control groups and were absent in animals that obtained repeated injections of NSAIDs (Figs. 2B-4B).
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Fig. 1. Location of the microinjection sites in the Ce of the amygdala. Only NSAID and saline injections within the shaded regions were included in the data analysis. The coronal section corresponds to the distance 5.7 mm from the interaural line and 3.3 mm from the bregma [16]. All injections fell within not more than 0.5 mm of this coronal plane.

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injections of xefocam was nearly 5-fold. However, when microinjections of these drugs were repeated during subsequent days, the antinociceptive effects progressively diminished. On the 5th experimental day, the TF latencies were practically similar to those in the rats receiving repeated injections of only saline. This phenomenon was akin to the development of tolerance to morphine administration into the PAG under similar experimental conditions [17, 18], and we shall therefore refer to it as non-opioid tolerance. As was mentioned, both experimental and control groups of rats received on the 5th day morphine hydrochloride microinjections into the same Ce sites, and only the saline-treated animals responded with antinociception to these injections ( P < 0.001). The TF latencies of the non-opioid tolerant rats were not altered by opioid (morphine) microinjections, i.e., these animals showed cross-tolerance to morphine (Figs. 2A-4A). Bilateral microinjections of NSAIDs into the Ce also increased the latency of the TF reflex compared to that in control rats on the first day. These shifts were rather similar for Analgin ( P < 0.001; Fig. 2B), ketorolac ( P < 0.001; Fig. 3B), and xefocam ( P <0.001; Fig. 4B). Nevertheless, on the second day, when microinjections were repeated, this index began to decrease as that at the time of unilateral administration. On the fourth to fifth day of the experiments, the TF latency became practically similar to that in the rats receiving bilateral injections of only saline. In this case of bilateral injections, the tolerance developed somewhat more rapidly than after unilateral injections

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Fig. 2. Mean latencies (sec) of the tail ick (TF) reex for ve consecutive experimental days with microinjections of Analgin in the central nucleus (Ce) of the amygdala followed by injection of morphine on the fth day. The numbers of experimental rats subjected to unilateral (A) and bilateral (B) injections of Analgin (2) were 6 and 7, while those at unilateral (A) and bilateral (B) control injections of physiological saline (1) were 8 and 8, respectively. In this and subsequent gures, one, two, and three asterisks show cases of signicant intergroup differences with P < 0.05, P < 0.01, and P < 0.001, respectively.

Tolerance to NSAIDs Microinjected into the Amygdalar Ce

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Fig. 3. The same as in Fig. 2, but after injections of ketorolac into the amygdalar Ce. The numbers of experimental rats were 6 at unilateral (A) and 6 at bilateral (B) injections. Other designations are the same as in Fig. 2.

Fig. 4. The same as in Figs 2 and 3, but after injections of xefocam into the amygdalar Ce. The numbers of experimental animals were 7 at unilateral (A) and 6 at bilateral (B) injections of xefocam (2) and 6 and 6 at control injections (1), respectively. Other designations are the same as in Fig. 2.

DISCUSSION
Our experiments showed that microinjections of Analgin, ketorolac, and xefocam into the amygdalar Ce induced considerable antinociception in awake rats. This observation confirms our previous results obtained on rats where these NSAIDs were introduced i.p., or those of other authors using microinjections of these agents into the PAG. More importantly, our findings [6, 7], similarly to the findings of our colleagues [3, 5], indicate that repeated microinjections of NSAIDs into the Ce and PAG induce a dramatic decrease in the antinociceptive effectiveness reminiscent of that induced by opiates. The significant involvement of the opioidergic mechanisms in the induction of tolerance effects of NSAIDs is surprising because, traditionally, the cellular and molecular actions of opioids have been considered as fundamentally different from those of non-opioid analgesics. At the same time, one interesting aspect of the effects of NSAID

administration, however, emphasizes their certain similarity to opioid analgesics, namely their common ability to induce tolerance (moreover, cross-tolerance). Indeed, microinjections of metamizole or LASA into the PAG [19] or into the Ce lead to a progressive loss of their antinociceptive effects, i.e., produce clear tolerance. Furthermore, tolerance to metamizole or LASA is accompanied by the development of cross-tolerance to morphine [3, 4], similar to that observed after opioid analgesics had been repeatedly administered. It is interesting that tolerance to the effect of PAG-microinjected metamizole can, like tolerance to morphine, be reversed by microinjection of proglumide, a cholecystokinin antagonist, into the same PAG site [5]. The latter fact should be considered additional evidence that the effects of nonopioid analgesics on the PAG are similar to those of morphine. Our recent data also demonstrated the same effects of tolerance after repeated i.p. injections of Analgin, ketorolac, and xefocam [6-8]. Our results on tolerance effects with Analgin,

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ketorolac, and xefocam microinjected into the Ce confirm the suggestion that the mechanism of their tolerance is realized through the PAG triggering the descending pain control system acting at the dorsal spinal cord level [1] and suggest that the Ce should be incorporated into current models of endogenous pain control circuitry. Thus, our results show that, alongside with the PAG and RVM, the Ce is an important site of the endogenous antinociceptive system, which triggers the descending pain control mechanism and, thus, inhibits nociceptive transmission. On the other hand, our data confirm the findings of other authors that NSAIDs in certain aspects are closely related to endogenous opioids, and tolerance to these non-opioid drugs probably depends on opioid tolerance [20].
Acknowledgement. This research was supported by a grant from Georgian National Science Foundation (GNSF/ST07/ 6-234).

M. G. Tsagareli et al.
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