Antinociceptive tolerance to NSAIDs in the rat formalin test is mediated by the opioid mechanism

Accepted Manuscript Title: Antinociceptive tolerance to NSAIDs in the rat formalin test is mediated by the opioid mechanism Author: Nana Tsiklauri Ivliane Nozadze Gulnaz Gurtskaia Merab G. Tsagareli PII: DOI: Reference:

S1734-1140(16)30252-3 http://dx.doi.org/doi:10.1016/j.pharep.2016.10.004 PHAREP 576

To appear in: Received date: Revised date: Accepted date:

28-4-2016 4-10-2016 4-10-2016

Please cite this article as: Nana Tsiklauri, Ivliane Nozadze, Gulnaz Gurtskaia, Merab G.Tsagareli, Antinociceptive tolerance to NSAIDs in the rat formalin test is mediated by the opioid mechanism, http://dx.doi.org/10.1016/j.pharep.2016.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antinociceptive Tolerance to NSAIDs in the Rat Formalin Test is Mediated by the Opioid Mechanism Nana Tsiklauri a, Ivliane Nozadze a, Gulnaz Gurtskaia a, Merab G. Tsagareli a,* a

Lab of Pain and Analgesia, Beritashvili Center for Experimental Biomedicine, 14, Gotua Street, 0160 Tbilisi, Georgia

* Corresponding author: M.G. Tsagareli Tel.: +995 32 237 1149; fax: +995 32 237 3411 E-mail addresses: [email protected] [email protected]

Number of pages: 13 Number of Figures: 5

ABSTRACT

Background: In the past decade it has been shown that tolerance develops to the antinociceptive effect of repeated systemic administration of commonly used non-steroidal anti-inflammatory drugs (NSAIDs) in acute pain models using rats. This is similar to the tolerance observed with opioid-induced analgesia. In the present study, we investigated the development of tolerance to the analgesic effects of NSAIDs diclofenac, ketorolac and xefocam in a chronic inflammatory pain model, the formalin test. Methods: Male Wistar rats receiving intraplantar formalin were tested for antinociception following intraperitoneal injection of NSAIDs in thermal paw withdrawal (Hargreaves) test and mechanical paw withdrawal (von Frey) test. Repeated measures analysis of variance with posthoc Tukey-Kramer multiple comparison tests were used for statistical evaluations. Results: Treatment with each NSAID significantly elevated the thermal paw withdrawal latency and mechanical paw withdrawal threshold on the first day, followed by a progressive decrease in the analgesic effect over a 4-day period, i.e., tolerance developed. With daily intraplantar injections of formalin, there was a trend toward reduced antinociceptive effects of diclofenac and ketorolac while xefocam exhibited a significant reduction (tolerance). It is noteworthy that the NSAID tolerant groups of rats still exhibited a strong hyperalgesia during phase I formalin following administration of each NSAID, an effect not observed in non-tolerant rats. Pretreatment with naloxone completely prevented the analgesic effects of these three NSAIDs in both behavioral assays. Conclusions: The present findings support the notion that the development of tolerance to the antinociceptive effects of NSAIDs in an inflammatory pain model is mediated via an endogenous opioid system possibly involving descending pain modulatory systems.

1

Keywords: Antinociception, descending modulation, hyperalgesia, nociception, non-opioid tolerance.

Introduction One of the vital functions of the nervous system is to provide signals about the occurrence or threat of injury; the sensation of pain contributes to this function [1,2]. It is well established that a family of non-opioid analgesics known as non-steroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the cyclooxygenases (COXs), which generate inflammatory mediators that enhance pain sensitivity [3-7]. In addition to a peripheral action on inflamed tissue, NSAIDs also act at central structures such as the spinal cord [8], the midbrain periaqueductal grey matter (PAG) [913], the central nucleus of amygdala (CeA) [5, 14], the nucleus raphe magnus (NRM), [5,15], and the dorsal hippocampus [16]. Furthermore, when microinjected into the above-noted sites, NSAIDs activate descending pain-modulatory pathways that inhibit spinal dorsal horn nociceptive neurons [5,17-20]. It is interesting that the -opioid receptor antagonist naloxone blocks analgesia induced by NSAIDs [5, 20-21]. These data suggest that NSAIDs may activate an endogenous opioid system [22-24]. Endocannabinoids are probably also involved in the antinociceptive effects of NSAIDs during inflammation [25-27]. We have recently shown that tolerance develops to the analgesic effects of the widely used NSAIDs metamizol, ketorolac and xefocam in juvenile and adult rats in models of acute pain [28]. Our data confirm that of others that tolerance to the analgesic action of NSAIDs may depend on an opiate-mediated mechanism [22-24, 26]. In the present study, we hypothesized that in a formalin model of chronic inflammatory pain, the analgesic effects of these three NSAIDs would exhibit tolerance that is mediated via endogenous opioids.

Materials and methods

Animals

Adult male Wistar rats weighing 200–250 g were used. The animals were kept under standard housing conditions (22±2 °C, 65% humidity, light from 7:00 a.m. to 8:00 p.m.) and were kept on a standard dry diet; water was freely available. Every effort was made to minimize both the number of animals used and their suffering. Six rats were used for each experimental and control groups. The local animal care committee approved the experimental protocols, adhering to the Guidelines of the International Association for the Study of Pain regarding investigation of experimental pain in conscious animals [29].

2

Drugs

Diclofenac (diclofenac sodium, 37,5 mg/kg, RotexMedica, Germany), ketorolac (ketorolac tromethamine, 12 mg/kg, Grindex, Latvia), or xefocam (lornoxicam, 1.2 mg/kg, Nycomed, Austria) were injected intraperitoneally (ip). Saline (BioPharma, Ukraine) was injected in the same volume and manner in a separate group of rats for controls. All these drugs are non-opioid medications, but they are representative of three different groups of NSAIDs. Diclofenac is a derivative of phenyl-acetic acid, ketorolac belongs to the indole group and xefocam belongs to the group of oxicames. These different groups of drugs were chosen specifically to examine the extent to which tolerance generalizes across non-opioid drugs. The antinociceptive doses of diclofenac, ketorolac and xefocam were clinically relevant, corresponding to maximal daily doses used in human patients. The doses were calculated by scaling human daily dosages to rat body weight [30]. In a separate set of control experiments, rats were pretreated with ip naloxone (1 mg/kg, Polfa Warszawa S.A., Poland) followed by behavioral tests. Twenty min after rats had been treated with ip NSAIDs at the same doses as in the first series of experiments; they were tested again in the behavioral assays. Usually, 40 min elapsed between the naloxone and the NSAIDs injections.

Behavioral testing

Before formal testing, the rats were handled for 30 min over three successive days to make them familiar with both testing protocols and the experimental environment. Each experiment was carried out over four consecutive days (Tuesday–Friday) except experiments with naloxone. Two behavioral models were used: thermal paw withdrawal reflex and mechanical paw withdrawal reflex (IITC, Woodland Hills, CA, USA). Prior to initiating the tests, baseline values were assessed for the experimental and control rats for thermal and mechanical withdrawal tests, which involved averaging five baseline measurements for the left and right hindpaws, with 5 min intervals between tests.

Thermal paw withdrawal (Hargreaves) test

Rats were first habituated over three successive daily sessions to stand on a glass surface heated to 30 ± 1°C within a ventilated Plexiglas enclosure. Before formal testing, baseline

3

latencies for paw withdrawals evoked by radiant thermal stimulation were measured five times/paw, with at least 5 min elapsing between tests for a given paw. A light beam (Plantar Test 390; IITC) was focused onto the plantar surface of the hindpaw through the glass plate from below, and the latency from the onset of light application to brisk withdrawal of the stimulated paw was measured. To prevent potential tissue damage, a cutoff time of 20 s was imposed if no paw movement occurred. Withdrawal latencies for both treated and untreated paws were measured 5, 30, and 60 min after administration of formalin or 15 and 45 min after ip injection of NSAIDs or saline.

Mechanical paw withdrawal (von Frey) test

Baseline mechanical withdrawal thresholds were assessed using an electronic Von Frey filament with 90 g range (1601C; IITC) pressed against the plantar surface of one hindpaw. This device registered the force (g) at the moment that the hindpaw was withdrawn away from the filament. After application of formalin, NSAIDs or saline, the mechanical pressure paw withdrawal thresholds were measured at the same post-application time points as mentioned above for thermal paw withdrawals.

Formalin-induced nociception test

Rats were placed in plastic cylinders on a room temperature glass surface and allowed to acclimate for approximately one hour before injection. The formalin solution was prepared at 10% in saline from a formalin stock (Sigma-Aldrich, St. Louis, MO, USA) and a unilateral intraplantar injection (right hindpaw) was made in a volume of 50 μl. The formalin stock corresponded to a 37% formaldehyde solution. In rodents, intraplantar injections of formalin produce a biphasic behavioral reaction consisting of an initial phase of paw-flinching occurring about 3-5 min after the injection, followed by a quiescent period, a then second phase of flinching beginning after 20-30 minutes. The intensities of these behaviors are dependent on the concentration of formalin that is administered [31]. We presently collected data at minute 5 post-formalin injections representing the first phase, and at minutes 15 and 60 post-formalin injections representing the second phase.

Experimental Procedure

4

Rats were tested for baseline (both paws) withdrawal latency and mechanical withdrawal threshold prior to formalin injection. Five min after formalin injection, rats were retested behaviorally during phase I. Fifteen min post-formalin, rats received ip administration of one of the NSAIDs (either diclofenac, ketorolac, or xefocam) or saline (control) and tested again behaviorally 30 and 60 min post-formalin injection. The experiment was repeated for the next 3 days without intraplantar formalin injection, but with ip injection of the same NSAID, and rats were administered the behavioral tests 15 and minute 45 post-NSAID administration. In the second set of experiments, rats received the same first-day treatment as above (formalin, followed 15 min later by NSAID), and this procedure was followed daily for the next 4 days. The only difference is that rats received daily intraplantar formalin injections in the second, but not first, experiment. In order to test the post-formalin first phase, in the third set of experiments, we developed tolerance in rats to these three ip administered NSAIDs over a 4 day period. On day 4, after NSAID administration, formalin was injected into the right hindpaw and rats tested 5-10 min later in the thermal and mechanical withdrawal plantar tests. Here we compared NSAIDstolerant groups with non-tolerant groups receiving either diclofenac, ketorolac or xefocam, respectively, for both the formalin-injected and non-injected paws, in these two behavioral assays. In the last, fourth set of experiments, rats were pretreated with naloxone (1 mg/kg, ip); 15 min later they were injected ip with one of the NSAIDs followed by the formalin test. Rats were behaviorally tested 5 and 30 min after NSAID administration on both the ipsilateral (formalininjected) and contralateral (non-injected) paws. Effective blinding was possible during the experiment, however, it was recognized that it was not always possible to be truly blind to certain test conditions (e.g., adopted position of the inflamed hindpaw).

Statistical analysis All mean control and experimental groups’ values are presented as mean ± SEM. The repeated measure of analysis of variance (rMANOVA) or ordinary ANOVA (for testing Phase I) with post-hoc Tukey–Kramer multiple comparison tests was used for statistical evaluation of comparisons between treated and saline groups and tolerant and non-tolerant groups. The statistical software utilized was InStat 3.05 (GraphPad Software, Inc, San Diego, CA, USA). Differences between means were acknowledged as statistically significant if p < 0.05.

5

Results

Tolerance to antinociceptive effect of NSAIDs in rats receiving formalin only on day 1

The first experiment tested the acute effects of the NSAIDs on thermal and mechanical paw withdrawals during phase II post-formalin. Five min following intraplantar formalin injection (phase I), prior to the injection of NSAIDs, all animals showed a significant reduction in thermal paw withdrawal latency and mechanical withdrawal threshold compared to pre-baseline values (Fig. 1A, B). The formalin caused a decrease of thermal paw withdrawal latencies for the groups of saline, t = 26.559, p < 0.001, diclofenac, t = 27.435, p < 0.001, ketorolac, t = 22.596, p < 0.001, and xefocam, t = 25.206, p < 0.001, respectively for the formalin-injected paw as well as for the non-injected paw but less (saline, t = 14.169, p < 0.001, diclofenac, t = 14.352, p < 0.001, ketorolac, t = 9.286, p < 0.001, xefocam, t = 9.437, p < 0.001, respectively) in the Hargreaves test (Fig. 1A). We observed equivalent differences in the von Frey test for the formalin injected groups (saline, t = 23.61, p < 0.001, diclofenac, t = 21.943, p < 0.001, ketorolac, t = 23.495, p < 0.001, xefocam, t = 25.05, p < 0.001, respectively) as well as for the non-injected groups (saline, t = 10.209, p < 0.001, diclofenac, t = 10.136, p < 0.001, ketorolac, t = 10.367, p < 0.001, xefocam, t = 9.206, p < 0.001, respectively) (Fig. 1B). These data show some spreading hyperalgesia from the formalin-injected paw to the non-injected paw. For each treatment group, the thermal withdrawal values on the formalin-injected side were smaller than on the non-injected side (saline, t = 6.76, p < 0.001, diclofenac, t = 4.583, p < 0.05, ketorolac, t = 7.859, p < 0.001, xefocam, t = 9.355, p < 0.001, respectively) (Fig. 1A). Fifteen minutes after formalin injection, either saline, diclofenac, ketorolac or xefocam was administered ip, and thermal and mechanical paw withdrawals were assessed again bilaterally 15 and 45 min later (i.e., at minute 30 and 60 post-formalin) during phase II. As can be seen in the saline treatment group, withdrawals recovered to near pre-formalin baseline levels. A simple comparison of pre-formalin baselines with thermal paw withdrawal latencies and threshold data at minute 30 and 60 post-formalin clearly shows antinociceptive effects of NSAIDs, especially xefocam. Each NSAID resulted in an elevation in both thermal paw withdrawal latency and mechanical paw withdrawal threshold bilaterally (Fig. 1A, B). In the thermal test, the rMANOVA revealed significant effects for diclofenac F(10,25) = 62.116, p < 0.0001, ketorolac

6

F(10,25) = 39.347, p < 0.0001, and xefocam F(10,25) = 34.009, p < 0.0001, respectively for the formalin-injected paw. Specifically, at 30 min post-formalin injection, differences between the saline group and NSAIDs groups were significant for ketorolac, t = 4.537, p < 0.05, diclofenac, t = 10.578, p < 0.001, and xefocam, t = 10.715, p < 0.001, respectively. At 60 min post-formalin, differences were still significant (ketorolac, t = 9.042, p < 0.001, diclofenac, t = 10.645, p < 0.001, and xefocam, t = 11.919, p < 0.001, respectively) (Fig. 1A). There is a clear antinociception on the non-injected paw, too, meaning that systemic NSAIDs have an antinociceptive effect in both the normal paw and in the formalin-inflamed paw. Similar effects were observed for the plantar mechanical pressure test. The rMANOVA revealed significant values for diclofenac F(10,25) = 67.767, p < 0.0001, ketorolac F(10,25) = 60.144, p < 0.0001, and xefocam F(10,25) = 50.63, p < 0.0001, respectively. Differences between formalin-injected and non-injected paw groups were significant for diclofenac, t = 7.583, p < 0.05, ketorolac, t = 8.346, p < 0.001, xefocam, t = 10.243, p < 0.001, and saline, t = 7.694, p < 0.001, respectively (Fig. 1B). Here also, subsequent ip injections of all three nonopioid analgesics resulted in a significant increase in pressure threshold compared to the saline control group for phase II, at minute 60 for the ipsilateral paw, especially for xefocam. In particular, at 60 min post-formalin, differences between saline group and NSAIDs groups were significant for diclofenac, t = 9.253, p < 0.001, ketorolac, t = 7.028, p < 0.001, and xefocam, t = 14.231, p < 0.001, respectively. At 30 min post-formalin, a significant value was only observed for xefocam, t = 10.109, p < 0.001 (Fig. 1B). We next investigated if tolerance develops to the antinociceptive effect of NSAIDs in these animals that received only one intraplantar injection of formalin on the first day (see above). These animals received daily ip administration of one of the three NSAIDs on three successive days (no intraplantar formalin) following the first-day treatment with formalin and NSAID. Fig. 2 shows averaged data for thermal (Fig. 2A) and mechanical (Fig. 2B) paw withdrawals at minute 15 and minute 45 post-NSAID administration for days 2-4. There was a progressive decrease in the antinociceptive effect of all 3 NSAIDs across treatment days, for both the paw receiving formalin on day one, and the non-injected paw. By day 4 there was no longer any significant difference in thermal paw withdrawal latencies between the saline–treated group and any of the NSAID treatment groups for both formalin-injected and non-injected paws (Fig. 2A,B). For thermal paw withdrawals, the rMANOVA showed significant effects for diclofenac, F(16,55) = 46.383, p < 0.0001, ketorolac, F(16,55) = 38.92, p < 0.0001, and xefocam, F(16,55) = 48.263, p < 0.0001, respectively. For ketorolac the difference in thermal latencies was significant

7

between the 2nd and 4th days, in the formalin-injected paw 15 min post-ketorolac (t = 8.893, p < 0.001). The difference in thermal paw withdrawal latencies 15 min post-diclofenac was significant between the 2nd and 3rd days (t = 6.962.3, p < 0.001), and more so between the 2nd and 4th days, t = 11.109, p < 0.001. For xefocam these differences were also significant between the 2nd and 3rd days, t = 5.739, p < 0.001, and the 2nd and 4th days, t = 13.905, p < 0.001 (Fig. 2A). Results were similar for the von Frey test. The rMANOVA revealed significant effects for diclofenac, F(16,55) = 11.101, p < 0.0001, ketorolac, F(16,55) = 19.126, p < 0.0001, and xefocam, F(16,55) = 16.263, p < 0.0001, respectively. For ketorolac, for example, the difference in mechanical thresholds was significant between the 2nd and 4th days in the formalin injected paw 15 min post-administration, t = 9.16, p < 0.001. For diclofenac the same index 15 min postdiclofenac was also significant between the 2nd and 4th days, t = 5.592, p < 0.05. For xefocam this difference was significant between the 2nd and 4th days, t = 8.742, p < 0.001 (Fig. 2B).

Tolerance to antinociceptive effect of NSAIDs for phase II in rats receiving daily formalin

In the next set of experiments, animals received unilateral intraplantar injection of formalin followed by ip administration of one of the NSAIDs (as in Fig. 1), with this procedure being repeated over 4 successive days. There was some reduction in antinociception for all the three NSAIDs for both formalin-injected and non-injected paws (Fig. 3). For thermal paw withdrawals, the rMANOVA revealed significant effects for ketorolac, F(28,115) = 7.93, p < 0.0001, diclofenac, F(28,115) = 15.126, p < 0.0001, and xefocam, F(28,115) = 12.706, p < 0.0001, respectively. However, there were no significant differences in thermal latencies between the 1st and 4th days for either formalin-injected or non-injected paws in groups receiving ketorolac or diclofenac. For xefocam there was a significant difference between the 1st and 4th days at 30 min post-xefocam for the formalin injected paw (t = 7.099, p < 0.001) and non-injected paw (t = 7.172, p < 0.001), but not at 60 min post-xefocam (formalin-injected paw t = 4.007, p > 0.05; non-injected paw t = 3.566, p > 0.05) (Fig. 3A,B). In the mechanical withdrawal test, similar results were obtained for ketorolac and diclofenac. The rMANOVA revealed significant effects for ketorolac, F(28,115) = 15.524, p < 0.0001, diclofenac, F(28,115) = 28.88, p < 0.0001, and xefocam, F(28,115) = 59.574, p < 0.0001, respectively. There were significant differences between the 1st and 4th days 30 min postxefocam (formalin-injected paw: t = 12.425, p < 0.001; non-injected paw t = 14.08, p < 0.001) as well as 60 min post-xefocam (formalin injected paw: t = 14.846, p < 0.001; non-injected paw: t =

8

8.916, p < 0.001) (Fig. 3 C,D). In general, thermal paw withdrawal latencies and mechanical thresholds were less in the formalin-injected paw and tolerance was only observed for the antinociceptive effect of xefocam.

Tolerance to antinociceptive effect of NSAIDs during formalin phase I

In these experiments we compared the antinociceptive effect of NSAIDs during phase I formalin behavior in naïve rats and rats that had developed tolerance. Tolerance was produced by daily i.p. administration of each NSAID over a 4 day period. On day 4, formalin was injected into the right paw 15 min after NSAID or saline administration and animals were tested in the thermal and mechanical paw withdrawal tests approximately 5 min later. Figure 4 shows that non-tolerant animals exhibited significantly greater thermal and mechanical withdrawal values (i.e., antinociception), in both formalin-injected and non-injected paws. In contrast, the tolerant animals exhibited very short thermal withdrawal latencies and mechanical thresholds indicating phase I hyperalgesia in the formalin-injected paw even in the presence of NSAIDs (Fig. 4A,C), indicating that the thermal antinociceptive or antihyperalgesic effect of NSAIDs during phase I formalin is lost in NSAIDs-tolerant animals. For thermal paw withdrawals, ANOVA revealed a significant effect for tolerant and nontolerant groups and for both formalin injected and non-injected paws: F(11,60) = 47.974, p < 0.0001). The differences between tolerant and non-tolerant groups were significant for ketorolac (t = 12.624, p < 0.001), diclofenac (t = 10.978, p < 0.001) and xefocam (t = 14.562, p < 0.001), respectively for the formalin-injected paw (Fig. 4A). Similar differences were observed the noninjected paw (ketorolac, t = 11.529, p < 0.001; diclofenac, t = 12.341, p < 0.001; xefocam, t = 11.255, p < 0.001, respectively) (Fig. 4B). Similar results were obtained for mechanical withdrawals. ANOVA revealed a significant effect for tolerant and non-tolerant groups in both formalin-injected and non-injected paws (F(11,60) = 53.828, p < 0.0001). There were significant differences between tolerant and nontolerant groups with each NSAID (ketorolac: t = 17.399, p < 0.001; diclofenac: t = 11.504, p < 0.001; xefocam: t = 13.562, p < 0.001, respectively) for the formalin-injected paw (Fig. 4C) as well as the non-injected paw (ketorolac: t = 15.691, p < 0.001; diclofenac: t = 9.185, p < 0.001; xefocam: t = 13.216, p < 0.001, respectively) (Fig. 4D). These findings indicate that the mechanical antinociceptive effect of NSAIDs during phase I formalin is lost in NSAID-tolerant animals.

9

Pretreatment with naloxone prevents NSAIDs-induced antinociception

In the fourth set of experiments, we tested if pretreatment with naloxone would prevent NSAIDs-induced antinociception in post-formalin phases I and II. Rats received naloxone, followed 15 min later by ip administration of one of the NSAIDs or saline, followed 10 min later by unilateral intraplantar injection of formalin. Pretreatment with naloxone completely prevented any thermal or mechanical antinociceptive or antihyperalgesic effect of all three NSAIDs during phase I in the formalin-injected paw (Fig. 5A, B, ipsi). In the non-injected paw, there appeared to be a weak antinociceptive effect of all three NSAIDs during phase I for thermal (Fig. 5A) but not mechanical paw withdrawals (Fig. 5B). Antinociceptive effects of NSAIDs were weak or absent during phase II for both paws and tests. The rMANOVA revealed significant effects for ketorolac, F(8,15) = 32.263, p < 0.001, diclofenac, F(8,15) = 51.659, p < 0.001, and xefocam, F(8,15) = 32.263, p < 0.001, respectively. There were significant differences between ipsilateral and contralateral paws for ketorolac at minutes 5 (t = 8.973, p < 0.001) and 30 (t = 6.308, p < 0.01); for xefocam at minute 5 (t = 9.546, p < 0.001) and 30 (t = 6.522, p < 0.01), and for diclofenac only at minute 5 (t = 10.414, p < 0.001). Similar results were obtained for mechanical paw withdrawals (Figure 5B). The rMANOVA revealed significant effects for ketorolac (F(8,15) = 23.38, p < 0.001), diclofenac (F(8,15) = 60.347, p < 0.001) and xefocam (F(8,15) = 28.257, p < 0.001), respectively. Significant differences between ipsilateral and contralateral paws were observed at minute 5 of postformalin injection for ketorolac (t = 8.687, p < 0.001) and xefocam, t = 6.41, p < 0.01), respectively. For diclofenac these differences were significant at minutes 5 (t = 6.436, p < 0.01), and 30 (t = 11.207, p < 0.001), respectively (Fig. 5B).

Discussion

The present study has shown that injection of commonly used NSAIDs (diclofenac, ketorolac and xefocam) induces antinociception in an inflammatory pain model induced by intraplantar injection of formalin into one (right) hindpaw of rats. These findings are in line with the results of our and other colleagues’ previous investigations in an acute pain model with tail-flick and hot plate tests, and in which metamizol, xefocam, ketorolac or lysine-acetylsalicylate were given systemically or microinjected into the periaqueductal gray matter [9,10-13,21, 28], into the CeA [5,14], the NRM [5,15], and the dorsal hippocampus [16].

10

More importantly, our data indicate that the repeated administration of these non-opioid analgesics over a period of 4 days results in decreased antinociceptive efficacy, i.e. development of tolerance, reminiscent of that induced by opiates [5,13,21,32,33]. It is well known that longterm administration of opioids eventually leads to a dose ceiling that is attributable to the development of tolerance with undesirable side effects of respiratory depression, nausea and decreased gastrointestinal motility [34]. The present data confirm our previous results in which development of tolerance to the analgesic effects of metamizol, ketorolac and xefocam was observed in juvenile and adult rats. After injection of each drug, a progressive decrease in tail-flick latency (i.e., tolerance) was noticed over the 5-day period, as well as cross-tolerance to morphine-induced analgesia [13]. Furthermore, it is noteworthy that the NSAID tolerant groups of rats exhibited a strong hyperalgesia unlike non-tolerant groups. According to our data, naloxone completely prevented the analgesic effects of diclofenac, ketorolac and xefocam in both ipsilateral and contralateral paws. These findings confirm our previous evidence where pretreatment with naloxone prevented antinociceptive effects of analgin, ketorolac and xefocam in tail-flick and hot plate tests in juvenile and adult rats. Moreover, in morphine-tolerant juvenile and adult rats we revealed effects of cross-tolerance to metamizol, ketorolac and xefocam [13]. These and the present data also confirm previous results that antinociception induced by systemic metamizol (dipyrone) involves endogenous opioids that can be blocked by naloxone at the levels of the PAG, the NRM and the spinal dorsal horn [22], as well as other findings that endogenous opioids are involved in the potentiation of analgesia observed with a combination of morphine plus dipyrone [23]. These findings suggest a role for endogenous opioidergic descending pain control circuits. The latter consists of the brainstem pain modulatory network with critical links in the PAG as well as the rostral ventro-medial medulla (RVM) [18,19,26,35,36]. The precise molecular mechanism of NSAIDs tolerance is not yet known. According to our and other colleagues’ data, NSAIDs tolerance mimics opioid tolerance. At present we cannot precisely determine the cellular and molecular mechanisms of such similarities. However, it is well established that organization of opioid adaptations in the nervous system include receptor tolerance at the μ-opioid receptor itself showing loss in the coupling of μ-opioid receptor to major cellular effectors, such as the G-protein-regulated inwardly rectifying potassium channel. [19,37,38].

Conclusions

11

In summary, systemic, ip administration of diclofenac, ketorolac and xefocam, widely used non-opioid, NSAID analgesics, at clinically relevant doses, induces antinociception in the formalin test in rats. When administered repeatedly, tolerance develops to the antinociceptive effects of these drugs. The present findings support the notion that the development of tolerance to the antinociceptive effects of NSAIDs is mediated via an endogenous opioid system possibly involving descending pain modulatory systems. Funding The work was supported by an intramural grant of Beritashvili Exp BMC.

Conflict of interest Authors have declared that no conflicting interests exist. Acknowledgements We wish to express our gratitude to Dr. E. Carstens for his kind assistance with English edition of the manuscript and useful notes and suggestions. The work was supported by an intramural grant of Beritashvili Exp BMC.

References [1]

Ringkamp M, Raja SN, Campbell JN, Meyer RA. Peripheral mechanisms of cutaneous nociception. In: McMahon SB, Koltzenburg M, Tracey I, Turk DC (Eds.) Wall & Melzack’s Textbook of Pain. Elsevier, Philadelphia 2013; pp. 1-30.

[2]

Hodkinson, DJ, Khawaja N, O’Daly O, Thacker MA, Zelaya FO, Wooldridge CL et al. Cerebral analgesic response to nonsteroidal anti-inflammatory drug ibuprofen. Pain 2015;156(7):1301-10.

[3]

Pierre SC, Schmidt R, Brenneis C, Michaelis M, Geisslinger G, Scholich K. Inhibition of cyclooxygenases by dipyrone. Brit J Pharmacol 2007;151(4):494-503.

[4]

Vergne-Salle P, Beneytout J-L. Targeting the cyclooxygenase pathway. In: Beaulieu P, Lussier D, Porreca F, Dickenson AH (Eds.) Pharmacology of Pain. IASP Press, Seattle 2010; pp. 43-64.

[5]

Tsagareli MG, Tsiklauri N. Behavior Study of ‘Non-opioid Tolerance’. Nova Biomedical, New York, 2012.

[6]

Dawes JM, Andersson DA, Bennett DLH, Bevan S, McMahon S.B. Inflammatory mediators and modulators of pain. In: McMahon SB, Koltzenburg M, Tracey I, Turk DC (Eds.) Wall & Melzack’s Textbook of Pain. Elsevier, Philadelphia, 2013; pp. 48-67.

[7]

Zeilhofer HU, Brune K. Cyclooxygenase inhibitors: Basic aspects. In: McMahon SB, Koltzenburg M, Tracey I, Turk DC (Eds.) Wall & Melzack’s Textbook of Pain. Elsevier, Philadelphia 2013; pp. 444-54.

[8]

Vanegas H, Schaible H-G. Prostaglandins and cyclooxygenases in the spinal cord. Prog Neurobiol 2001;64(4):327-63.

[9]

Tortorici V, Vanegas H. Opioid tolerance induced by metamizol (dipyrone) microinjections into the periaqueductal gray of rats. Eur J Neurosci 2000;12(11):4074-80.

12

[10] Vanegas H, Tortorici V. The periaqueductal gray as critical site for antinociception and tolerance induced by non-steroidal anti-inflammatory drugs. In: Maione S, Di Marzo V (Eds.) Neurotransmission in the Antinociceptive Descending Pathway. Kerala: Research Signpost 2007; pp. 69-80. [11] Tortorici V, Aponte Y, Acevedo H, Nogueira L, Vanegas H. Tolerance to non-opioid analgesics in PAG involves unresponsiveness of medullary pain-modulating neurons in male rats. Eur J Neurosci 2009;29(6):1188-96. [12] Tsiklauri N, Nozadze I, Gurtskaia G, Abzianidze E, Tsagareli MG. Tolerance induced by non-opioid analgesic microinjections into rat's periaqueductal gray and nucleus raphe. Georgian Med News 2010;3(180):47-55. [13] Tsiklauri N, Viatchenko-Karpinsky V, Voitenko N, Tsagareli MG. Non-opioid tolerance in juvenile and adult rats. Eur J Pharmacol 2010;629(1-3):68-72. [14] Tsagareli MG, Tsiklauri N, Gurtskaia G, Nozadze I. The central nucleus of amygdala is involved in tolerance to the antinociceptive effect of NSAIDs. Health 2010;2(1):64-8. [15] Tsagareli MG, Nozadze I, Tsiklauri N, Gurtskaia G. Tolerance to non-opioid analgesics is opioid-sensitive in nucleus raphe magnus. Front Neurosci 2011;5(7):92- 6 (doi: 10.3389/ fnins.2011. 00092). [16] Gurtskaia G, Tsiklauri N, Nozadze I, Nebieridze M, Tsagareli MG. Antinociceptive tolerance to NSAIDs microinjected into dorsal hippocampus. BMC Pharmacol Toxicol 2014;15, art 10 (doi: 10.1186/2050-6511-15-10). [17] Vanegas H, Tortorici V. Opioidergic effects of non-opioid analgesics on the central nervous system. Cell Mol Neurobiol 2002;22(5-6):655-61. [18] Vazquez E, Escobar W, Ramirez C, Vanegas H. A non-opioid analgesic acts upon the PAG-RVM axis to reverse inflammatory hyperalgesia. Eur J Neurosci 2007;25(2):471-9. [19] Heinricher MM, Ingram SL. The brainstem and nociceptive modulation. In: Basbaum AI, Bushnell MC (Eds.) Science of Pain. Elsevier, San Diego 2009; pp. 593-626. [20] Tsagareli MG, Tsiklauri N, Nozadze I, Gurtskaia G. Tolerance effects of NSAIDs microinjected into central amygdala, periaqueductal grey, and nucleus raphe: Possible cellular mechanism. Neural Regen Res 2012;7(13):1029-39. [21] Pernia-Andrade AJ, Tortorici V, Vanegas H, Induction of opioid tolerance by lysine-acetylsalicylate in rats. Pain 2004;111 (1-20):191–200. [22] Vazquez E, Hernandez N, Escobar W, Vanegas H. Antinociception induced by intravenous dipyrone (metamizol) upon dorsal horn neurons: involvement of endogenous opioids at the periaqueductal gray matter, the nucleus raphe magnus, and the spinal cord in rats. Brain Res 2005;1048(1-2):211–7. [23] Hernández-Delgadillo GP, Cruz SL. Endogenous opioids are involved in morphine and dipyrone analgesic potentiation in the tail flick test in rats. Eur J Pharmacol 2006;546(1):54-9. [24] Wessendorf MW, Vaughan CW, Vanegas H. Rethinking the PAG and RVM: supraspinal modulation of nociception by opioids and non-opioids. In: Flor H, Kalso E, Dostrovsky JO (Eds.) Proc. 11th World Congress on Pain. IASP Press, Seattle 2006; pp. 311-20. [25] Telleria-Diaz A, Schmidt M, Kreusch S, Neubert AK, Schache F, Vazquez E et al. Spinal antinociceptive effects of cyclooxygenase inhibition during inflammation: Involvement of prostaglandins and endocannabinoids. Pain 2010;148(1): 26-35. [26] Vanegas H, Vazques E, Tortorici V. NSAIDs, opioids, cannabinoids and the control of pain by the central nervous system. Pharmaceuticals 2010;3:1335-47 (doi:10.3390/ph3051335). [27] Escobar W, Ramirez K, Avila C, Limongi R, Vanegas H, Vazquez E. Metamizol, a non-opioid analgesic, acts via endocannabinoids in the PAG-RVM axis during inflammation in rats. Eur J Pain 2012;16(5):676-89. [28] Tsiklauri N, Gurtskaia G, Tsagareli MG. Study of non-opioid analgesic tolerance in young and adult rats. Georgian Med News 2008; 5(158):40-4. [29] Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16(2):109–10. [30] Pong SF, Demuth SM, Kinney CM, Deegan P. Prediction of human analgesic dosages of non-steroidal anti-inflammatory drugs (NSAIDs) from analgesic ED50 values in mice. Arch Int Pharmacodyn Ther 1985;273(2):212–20.

13

[31] Le Bars M, Gozariu M, Cadden SW. Animal models of nociception. Pharmacol Rev 2001;53(4):597–652. [32] Tortorici V, Nogueira L, Salas R, Vanegas H. Involvement of local cholecystokinin in the tolerance induced by morphine microinjections into the periaqueductal gray of rats. Pain 2003;102(1-2):9–16. [33] Tortorici V, Nogueira L, Aponte Y, Vanegas H. Involvement of cholecystokinin in the opioid tolerance induced by dipyrone (metamizol) microinjections into the periaqueductal gray matter of rats. Pain 2004;112(1-2):113–20. [34] Dumas EO, Pollack GM. Opioid tolerance development: A pharmacokinetic/pharmacodynamic perspective. The AAPS Journal 2008;10(4):537-51. [35] Heinricher MM, Fields HL. Central nervous system mechanisms of pain modulation. In: McMahon SB, Koltzenburg M, Tracey I, Turk DC (Eds.) Wall & Melzack’s Textbook of Pain. Elsevier, Philadelphia 2013; pp. 129-42. [36] Heinricher MM, Tavares I, Leith JL, Lumb BM. Descending control of nociception: Specificity, recruitment and plasticity. Brain Res Rev 2009;60(1):204-25. [37] Christie MJ. Cellular neuroadaptations to chronic opioids: tolerance, withdrawal and addiction. Brit J Pharmacol 2008; 154(2):384-96. [38] Niikura K, Narita M, Butelman ER, Kreek MJ, Suzuki T. Neuropathic and chronic pain stimuli down-regulate central muopioid and dopaminergic transmission. Trends Pharmacol Sci (2010);31(7):299-305. [39] Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci 2002;25(6): 319-25.

14

Legends to Figures: Fig. 1. Latencies of the thermal paw withdrawal reflex (sec) (A) and thresholds of the mechanical paw withdrawal reflex (gram) (B) after intraplantar formalin injection to one (right) paw on the first experimental day. The vertical open arrow indicates the time of formalin injection. Vertical black arrows indicate the time of i.p. NSAIDs administration. Note analgesics result in a significant increase in latencies and thresholds compared to the saline control for post-formalin phase II, for both formalin injected and non-injected paws. BL – pre-formalin baseline for the thermal paw withdrawal latency and mechanical paw withdrawal threshold for both paws together.

15

Fig. 2. Latencies of the thermal paw withdrawal reflex (sec) (A,B) and thresholds of the mechanical paw withdrawal reflex (gram) (C,D) after i.p. NSAIDs administration for three consecutive days. Note in subsequent 2-4 days antinociception decreased gradually for formalin injected (A,C) and non-injected (B.D) paws, respectively, i.e., developed tolerance.

16

Fig. 3. Latencies of the thermal paw withdrawal reflex (sec) (A,B) and thresholds of the mechanical paw withdrawal reflex (gram) (B,D) after daily intraplantar formalin injection to one (right) paw for four consecutive days. Vertical black arrows indicate a moment of i.p. NSAIDs administration. Note there are some significances of the reduction of antinociception for all the three NSAIDs and for both formalin injected (A,C) and non-injected (B,D) paws, respectively.

17

Fig. 4. Differences between tolerant and non-tolerant groups of rats in latencies of the thermal paw withdrawal reflex (sec) (A,B) and thresholds of the mechanical paw withdrawal reflex (gram) (C,D) in post-formalin phase I. Note strong significant differences between tolerant and non-tolerant groups of rats for all the three NSAIDs and for both formalin injected (A,C) and non-injected (B.D) paws, respectively. Significance levels are: ***p<0.001.

18

Fig. 5. Naloxone pretreatment completely prevents analgesic effects of NSAIDs in ipsilateral (formalin injected) paw compared to contralateral (non-injected paw) in latencies of the thermal paw withdrawal reflex (sec) (A) and thresholds of the mechanical paw withdrawal reflex (gram) (B) for post-formalin phase I (5 min) and phase II (30 min), respectively.

19

20