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Morphine deteriorates spatial memory in sodium salicylate treated rats
European Journal of Pharmacology 704 (2013) 1–6
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Morphine deteriorates spatial memory in sodium salicylate treated rats Mehdi Sadegh a, Yaghoub Fathollahi a,n, Nasser Naghdi b, Saeed Semnanian a a b
Department of Physiology, School of Medical Sciences, Tarbiat Modares University, PO Box 14115-111, Tehran, Iran Department of Physiology & Pharmacology, Pasteur Institute, Tehran, Iran
a r t i c l e i n f o
abstract
Article history: Received 15 November 2012 Received in revised form 5 February 2013 Accepted 7 February 2013 Available online 24 February 2013
Tolerance and cross-tolerance for the effects of morphine (M) and sodium salicylate on nociception and learning were examined. The anti-nociceptive effects were measured by using the classic tail flick (TF) and hot plate (HP) tests and learning was measured with the Morris water maze (MWM). Tolerance or cross-tolerance was induced by daily injection (i.p.) of morphine sulfate (10 mg/kg for 7 days) or sodium salicylate (300 mg/kg for 6 days). The injection of sodium salicylate increased both TF and HP latencies. This anti-nociceptive effect was progressively decreased across six injections and tolerance to sodium salicylate was developed. When M was injected to sodium salicylate-tolerant rats, a weakened anti-nociceptive effect was seen, indicating cross-tolerance to M. Acute treatment with M also increased TF latency. This anti-nociceptive effect was successively decreased across seven injections and tolerance to M was developed. When sodium salicylate was injected to M-tolerant rats, a diminished anti-nociceptive effect was seen, indicating cross-tolerance to sodium salicylate. Acute M impaired water maze performance, while chronic M and sodium salicylate had no effects on MWM performance. However, when M was injected to rats that had received sodium salicylate after each training trial for 7 days, these rats spent less time in target quadrant as compared to M and saline groups. It is concluded that chronic sodium salicylate induces tolerance to anti-nociceptive effects of M and vice versa. Also chronic salicylate may produce lasting metaplastic changes in brain mechanisms behind spatial learning and memory, which can be visualized in cross-sensitization to morphine. Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
Keywords: Cross-tolerance Morphine Morris water maze Salicylate Spatial memory
1. Introduction The combination of opioids and non-opioids analgesics (NSAIDs) is commonly used to control severe pain. This combination therapy can raise analgesic effects while avoiding adverse side effects (Deciga-Campos et al., 2003). However, tolerance to anti-nociceptive effect of non-opiate analgesics and crosstolerance to morphine have been reported (Pernia-Andrade et al., 2004; Trujillo, 2000). It has been reported that NSAIDs (e.g. metamizol and lysine-acetylsalicylate, LASA) influence periaqueductal gray matter (PAG) and descending pain-control system, so that at least a part of their anti-nociceptive effects is induced through endogenous opioidergic structures (Tsagareli et al., 2011; Vanegas and Tortorici, 2002). Drug tolerance as a result of adaptation in the brain mechanisms appears with a decrease in responsiveness and an increase in demand for drug (Trujillo, 2002). In some cases, drug tolerance and physical dependence are considered as an aberrant form of synaptic plasticity, cause stable change in synaptic strength, and thereby act on learning and memory (Trujillo, 2000, 2002). There
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Corresponding author. Tel.: þ98 2188011001; fax: þ 98 2188013030. E-mail address: [email protected] (Y. Fathollahi).
are controversial reports about the effects of chronic morphine on learning ability. Some of them indicated learning impairment (Farahmandfar et al., 2010; Lu et al., 2010) and some others demonstrated no deficit (Miladi Gorji et al., 2008; Wang et al., 2006). The problem of tolerance and cross-tolerance for the effects of morphine and sodium salicylate on nociception and learning has not yet been clearly established. Likewise very few reports had put to the test so far the related actions of these two drugs. Herein, therefore, the effects of acute and chronic sodium salicylate and morphine on analgesia and spatial learning were examined. Furthermore, cross-tolerance effects of the drugs for one another were also explored.
2. Materials and methods 2.1. Animals and tolerance induction Subjects were 66 Wistar male rats that were born and reared at the colony room of Pasteur Institute (Alborz providence, Karaj, Iran). Rats were 9–10 weeks of age during the experiments and maintained on a 12 h light/dark cycle with a free access to water and food except during practical tests. Experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (National Academy
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Press, Washington D.C., 2010) and every effort was made to reduce animal suffering and the number of animals used. Morphine (M) tolerance was induced by daily injection of morphine sulfate (10 mg/kg, i.p.) for 7 days. Tolerance to sodium salicylate was induced by daily injection of freshly prepared sodium salicylate solution (1 injection/day for 6 consecutive days, 300 mg/kg, i.p.). The anti-nociceptive effects of the drugs were quantified using classic tail flick (TF, Harvard apparatus 52-9495, US) or hot plate (HP, Heidolph MR-3001K, Germany) 20–30 min after each daily injection. One day before the starting drug injection, response latency were measured as a baseline (BL). For TF, a light beam was focused on the dorsal surface of the tail and the latency to TF was measured. Cut-off time was 10 s and intensity was set at 80% maximum intensity. For HP, the animal was placed on a metal plate at 50 72 1C and the latency of first hind paw lick or to jump was measured while cut-off time was 20 s. Both tests were conducted in triplicate and the averaged values were used for analysis. To study cross-tolerance between the drugs, a day after tolerance development, morphine sulfate (5 mg/kg, i.p.) or sodium salicylate (300 mg/kg, i.p.) was applied as cross in each group and the response latency was measured. Time-matched saline injected rats were used as control group. 2.2. Morris water maze The Morris water maze (MWM) consisted of a dark circular water tank (140 cm diameter, 40 cm deep) filled with water at 21–23 1C to a depth of 25 cm (Vorhees and Williams, 2006). An invisible colorless Plexiglass platform (10 cm diameter) was located 1.5 cm below the water surface in a fixed location in the center of a quadrant. The maze was located in a quiet test room and contained distinctive visual marks which were visible from within the pool and served as distal cues. Locations of the cues were constant throughout the testing period. A day before training, a 1-min free swim trial without the platform was run for habituation. The training session consisted of 4 or 7 days, 4 trials per day each from different starting points. After climbing onto the platform, the animal was allowed to stay on the platform for 30 s. In the cases where the animal was unable to find the platform in 90 s, it was gently guided to the target by the experimenter and was allowed to remain there for 30 s. After 30 s on the platform, animals were placed into a heated cage until next trial (4–8 min later). The probe test was performed 1 day after last training session; a 90-s trial was initiated whereby the platform was removed. This was used to evaluate whether rats would localize the platform to the spatial location by calculating of the exploring time in each quadrant (quadrant occupancy) and the number of cross over the platform site (crossing index). After the probe trial, the platform was elevated above the water surface (visible test) to evaluate visio-motor coordination toward a visible platform. The visible test was performed for four 90 s. Swimming paths for training session and probe test was monitored using an automatic tracking system (Ethovision XT8; Noldus Instruments). This system was used to record the swimming trace and to calculate the mean latency to find the platform, the time spent in each quadrant and swimming speed. 2.3. Experimental protocol 2.3.1. Experiment 1 To study tolerance to sodium salicylate and its cross-tolerance with M, two different methods for pain rating called TF and HP were used. For this, a day after test for baseline in TF or HP, animals randomly were assigned to sodium salicylate-TF (n¼5), sodium salicylate-HP (n¼4), saline-TF (n ¼4) and saline-HP (n ¼4) groups. On first day, animals received sodium salicylate (300 mg/
kg, i.p.) or saline and TF or HP latencies were measured after 20– 30 min. The same was done on the following days until tolerance to sodium salicylate was developed. A day after tolerance development, all animals received a single dose of morphine sulfate (5 mg/kg, i.p.) and TF or HP latencies were measured 30 min thereafter. Since the former practical tests reproduced the results of Pernia-Andrade et al. (2004), to study M cross-tolerance with sodium salicylate, only TF test was used. For this, a day after test for TF baseline, animals randomly were divided in M-TF (n¼ 5) and saline-TF (n ¼4) groups. On first day, animals received saline or morphine sulfate (10 mg/kg, i.p.) and TF latencies were measured 30 min thereafter. The same was done on the following days until tolerance to M was developed. A day after tolerance development, all animals received a single dose of sodium salicylate (300 mg/kg, i.p.) and TF latencies were measured 30 min thereafter. 2.3.2. Experiment 2 To quantify the effects of acute and chronic M and sodium salicylate on water maze performance, three different groups of animals were selected as saline, M and sodium salicylate (n¼4 for each group). In acute application, MWM task were done 30 min after each injection. This protocol was continued for 4 days. In chronic application, again three different groups of animals (n¼4 for each group) were selected and received the drugs or saline for 6–7 consecutive days. Then MWM task was done as mentioned above. 2.3.3. Experiment 3 To assess the effects of M and sodium salicylate on memory consolidation, same as two former experiments, groups were arranged as saline, M and sodium salicylate (n¼4 for each group), but the drugs or saline were administrated immediately after each trial of MWM task. This was continued for 7 days. To take measure of tolerance development to M, on 8th day all animals were received a single dose of morphine sulfate (10 mg/kg, i.p.) and 30 min thereafter a probe test was done. 2.4. Data analysis The data were expressed as mean7S.E.M. After data were tested for normal distribution (Kolmogorov–Smirnov), a one-way or two-way ANOVA followed by Bonferroni post-hoc test were performed. Two independent groups were compared by unpaired t-test. Statistically significant differences were acknowledged when Po0.05.
3. Results 3.1. Experiment 1 3.1.1. Chronic sodium salicylate induces tolerance to antinociceptive effect of M The latencies to TF or HP were increased within initial days of sodium salicylate injections as compared to its baseline and saline group (TF: F7, 56 ¼21.81, P o0.0001 and HP: F7, 48 ¼46.29, Po0.001). But, in the subsequent days (6th day for TF, 4th or 5th day for HP) this anti-nociceptive effect was progressively decreased so that during 5th and 6th days of the injections, the latencies to TF or HP was same as saline and the baseline (P4 0.05), indication of the development of tolerance to sodium salicylate. On the other hand, M injection on the 7th day increased the latencies to TF or HP in saline group but not in sodium salicylate group (Po0.001, Fig. 1A, B), indication of the development of cross-tolerance to M.
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Fig. 2. Chronic M leads to tolerance to the anti-nociceptive effect of sodium salicylate. The injection of morphine sulfate (10 mg/kg, i.p.) increased TF (n ¼5) latency, which was successively reduced by morphine sulfate injections. Sodium salicylate injection (300 mg/kg, i.p.) on 8th day to M-tolerant rats was noneffective to produce the anti-nociceptive effect, which indicates the occurrence of cross-tolerance to sodium salicylate. Latency was measured 20–30 after each injection. Data presented as mean 7S.E.M. nP o0.05, Bonferroni post-hoc test following two-way ANOVA. BL, Baseline latency; M, morphine.
(F2, 11 ¼6.02, Fig. 3B, Po 0.05). When retention of previously learned spatial memory (platform location recalling) was tested, statistical analysis revealed that the crossing index (F2, 23 ¼7.90, Po0.05,), but not the quadrant occupancy (F9, 18 ¼0.36, P¼0.94) was significantly decreased (Fig. 3C).
Fig. 1. Chronic sodium salicylate induces tolerance to anti-nociceptive effect of M. The injection of sodium salicylate increased both TF (n¼ 5) and HP (n¼ 4) latencies, which were successively reduced by sodium salicylate injections. Morphine sulfate injection (5 mg/kg, i.p.) on 7th day to sodium salicylate-tolerant rats was non-effective to produce the anti-nociceptive effect, which indicates the occurrence of cross-tolerance. Latency was measured 20–30 after each injection. Data presented as mean 7S.E.M. nnP o 0.01, Bonferroni post-hoc test following two-way ANOVA. BL, Baseline latency; M, morphine.
3.1.2. Chronic M leads to tolerance to anti-nociceptive effect of sodium salicylate As expected, the latency to TF was increased by daily injection of morphine sulfate (10 mg/kg, i.p.) and followed by a tolerance to its anti-nociceptive effects during 7 days of morphine sulfate injections (TF: P o0.0001, F8, 56 ¼10.62). Interestingly, at the 8th day, the TF latency was not increased due to a single dose of sodium salicylate (300 mg/kg, i.p.) indicating the occurrence of cross-tolerance to sodium salicylate (Po0.05, Fig. 2). 3.2. Experiment 2 3.2.1. Acute M impairs water maze performance Learning ability of animals was tested in MWM 30 min after drug injection. As demonstrated in Fig. 3, sodium salicylate had no effect on water maze performance as compared to saline group. However, M-injected animals took significantly more time to find the platform. Statistical analysis using two-way ANOVA followed by the Bonferroni post-hoc tests revealed that M impaired water maze performance (distance: F2, 135 ¼17.98; Po0.0001, Fig. 3 A; latency: F2, 135 ¼22.79; Po0.0001). Thirty minutes after M injection but not sodium salicylate, swimming speed was significantly increased as compared to saline group
3.2.2. Chronic M or sodium salicylate has no effect on water maze performance when injected pre-training To measure water maze performance of M- and sodium salicylate-tolerant rats, spatial learning ability of chronically Mand sodium salicylate-injected rats were measured in MWM. Chronic M and sodium salicylate were without effects on water maze performance across trials (Fig. 4C). Statistical analysis using two-way ANOVA followed by the Bonferroni post-hoc tests revealed that the differences in the mean values of time spent and distance moved to find the platform were not significant between three groups (distance: F2, 159 ¼0.05; P¼0.94, Fig. 4A; latency: F2,159 ¼0.77; P¼ 0.46). As seen in Fig. 4B neither M nor sodium salicylate had no effect on swimming speed (F2, 11 ¼0.22, P¼0.80). There were no significant changes in the crossing index and the quadrant occupancy (F2, 22 ¼0.84, P¼0.45, Fig. 4C) during probe tests. 3.3. Experiment 3 3.3.1. M disrupts water maze performance of sodium salicylateinjected rats when injected post-training In these series of practical tests the drugs or saline administrated immediately after each trial during MWM task learning. There were no significant differences in the water maze performance of M- and sodium salicylate-injected groups as compared to saline injected group (distance: F2, 270 ¼2.64; P¼0.08, Fig. 5A; latency: F2, 270 ¼1.87; P ¼0.16), except on 2nd day of sodium salicylate group (t48 ¼2.87, Po0.05). Swimming speed did not show significant differences between groups (F2, 18 ¼2.11, Fig. 5B, P¼0.15). On 8th day, a single dose of morphine sulfate (10 mg/kg, i.p.) was injected to three groups of animals and 30 min after injection, a probe test was done. Results (Fig. 5C, D) showed that the time spent in the target quadrant for chronically sodium salicylate-treated group was less than those of chronically
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Fig. 3. Acute M but not sodium salicylate impairs acquisition and retention of spatial reference memory. (A) The mean values of latency to find hidden platform vs. training days. Sodium salicylate (n ¼4) or morphine sulfate (n¼ 4) was injected 30 min before each training trial for 4 days. Sodium salicylate (300 mg/kg, i.p.) had no significant effect on water maze performance. But, morphine sulfate (10 mg/kg, i.p.) significantly influenced reference memory acquisition. Data presented as mean 7S.E.M. (B) Averaged swim speeds across the training days (each bar shows the mean þ S.E.M.). Notice a significant increase in velocity due to M. (C) In the probe test (5th day) the crossing index (each bar shows the mean þS.E.M.) significantly decreased due to M. nP o 0.05, M vs. saline, nnnPo 0.001, M vs. sodium salicylate or saline, Bonferroni post-hoc test following one-way ANOVA. M, morphine.
Fig. 4. Chronic sodium salicylate or M has no effects on acquisition and retention of spatial reference memory. (A) The mean values of latency ( 7 S.E.M) to find hidden platform across 4 training days in water maze (4 trials/day for 4 days; 5 min inter-trial interval) for three groups. Sodium salicylate (300 mg/kg for 6 days, i.p., n¼ 4) or morphine sulfate (10 mg/kg for 7 days, i.p., n ¼4) was chronically injected. The drugs had no significant effect on water maze performance. (B) Averaged swim speeds across the training days (each bar shows the mean þ S.E.M.). Notice no significant differences between groups. (C) In the probe test (5th day), the crossing index (each bar shows the mean þS.E.M.) did not show significant differences between groups. M, morphine.
saline- or M-injected rats (t15 ¼ 2.75, Po0.05) due to a single dose of M. However there were no significant differences for the crossing index between groups.
4. Discussion The effects of acute and chronic M and sodium salicylate on analgesia and spatial learning were examined. Furthermore, cross-tolerance effects of the drugs for one another were also explored. Our results show: (1) that chronic sodium salicylate induces tolerance to anti-nociceptive effects of M and vice versa, (2) that acute but not chronic M impairs spatial memory with a tolerance to the effects of morphine. To our knowledge, there are very few reports concerning the related actions of M and sodium salicylate and this study in fact confirms and extends our previously published results (Hosseinmardi et al., 2011). In the present study we found tolerance to anti-nociceptive effect of sodium salicylate and cross-tolerance to anti-nociceptive effect of M due to chronic sodium salicylate. Consistent with our results, tolerance to anti-nociceptive effect of other NSAIDs such as LASA and metamizol and their cross-tolerance to M (PerniaAndrade et al., 2004; Tortorici et al., 2009; Tortorici and Vanegas, 2000) have been reported. Tolerance to anti-nociceptive effect of LASA appeared after three or four systemic injections of this drug at therapeutic dose with 12 h interinjection interval. After five consecutive injections, cross-tolerance to anti-nociceptive effect of M was induced. In the present study, we used another
derivative of salicylic acid, sodium salicylate in a dose same as Pernia-Andrade et al. (2004), but our injection protocol was different. The injection protocol consisted of one injection per day for six consecutive days. We discovered that this scheduled sodium salicylate injection can induce tolerance to antinociceptive effect of the drug and cross-tolerance to M. In our preliminary experiments, we used same protocol that had been used by Pernia-Andrade et al. (2004) in which two injections per day was scheduled, but the mortality rate of animals was high (nearly 50%) after 2nd or 3rd injection. So, the injection protocol was changed as mentioned to reduce the mortality rate of animals and two animals died during the study. Perhaps this high mortality rate might be related to salicylic derivatives, which were used in these studies or may be related to subjects. We used Wistar rat (9–10 weeks age, 180–200 g), whereas Pernia-Andrade et al. (2004) used Sprague-Dawley rats (250–350 g). To remedy the mortality issues, we suggest using lower doses for testing to survive experimental animals in the future. To our knowledge, this is the first time that chronic M was shown to not only induced tolerance to M but also induced a cross-tolerance to the anti-nociceptive effect of sodium salicylate. It has been shown that PAG and downstream pain-control structures have an important role to the analgesic effect of LASA and metamizol involving opioidergic mechanisms (Christie et al., 2000; Tsagareli et al., 2011; Vorhees and Williams, 2006). Chronic exposure to an exogenous opiate such as M may act through this pathway at downstream pain-control structures, causing tolerance to other opiates and cross-tolerance to NSAIDs.
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Fig. 5. M disrupts spatial memory in the sodium salicylate-injected rats. (A) The mean values of latency ( 7 S.E.M) to find hidden platform vs. training days in water maze (4 trials/day; 5 min inter-trial interval) for three groups of animals. Sodium salicylate (300 mg/kg, i.p. n¼ 4) or morphine sulfate (10 mg/kg, i.p. n¼ 4) was daily injected immediately after each trail in water maze for 7 days. Notice a significant effect on 2nd day for sodium salicylate group, but not in M group. (B) Averaged swim speeds across training days (each bar shows the mean þS.E.M.). Notice no significant differences between groups. (C, D) In the probe test (5th day), the effect of a single dose of morphine sulfate (10 mg/kg) on the retention was examined. Notice no significant differences in the crossing index between groups (D) but quadrant occupancy (C) for the target quadrant showed a significance decrease for sodium salicylate group due to M. Each bar shows the mean þ S.E.M. nP o 0.05, Bonferroni post-hoc test following oneway ANOVA. M, morphine.
We demonstrated an impairment of water maze performance 30 min after M injection. An impairment of water maze learning ability 20 min after M injection (5 and 10 mg/kg) in mice has been shown (Li et al., 2001). They explained that inhibition of brain cholinergic system by M caused this memory impairment. It has also been shown that 30 min after M injection acquisition of spatial memory and also retention of reference memory were impaired (Farahmandfar et al., 2010). However, neither of these studies reported an increase of swimming speed during the training tests, but our result showed considerable increase of swimming speed and animal motivation due to acute M. This increase of swimming speed may be attributed to experimental procedures used in their experiments. Li et al. used mice as experimental subjects and Farahmandfar et al. used 8 trials in one as training protocol. In addition, acute sodium salicylate did not act on learning ability in the present study. It has been suggested a protective effect for a combination of ibuprofen and glutathione as a potential therapeutic agent for treating Alzheimer’s disease (Pinnen et al., 2011). However, the present study is the first to report on the cognitive effects of NSAIDs (with antinociceptive dose) in the water maze task. We found that chronic exposure to M or sodium salicylate was without effect on water maze performance. But, as shown in Fig. 5, sodium salicylate can act on memory consolidation on second day when it was injected immediately after each training trial, while M had no effect during this experiment. In addition, a single M injection during the probe test reduced time spent in target quadrant in sodium salicylate group but not in saline or M groups (Fig. 5D), may be related to disruption of memory consolidation. In addition, as the probe test revealed, the effect
of acute M (Fig. 3) was not seen when M was applied after each training trial. A learning deficit and impaired memory formation following the chronic M (Lu et al., 2010; Miladi Gorji et al., 2008) have been described. However, some others (Miladi Gorji et al., 2008; Wang et al., 2006) demonstrated no effect of chronic M on learning ability and retrieval. An impairment of spatial memory in water maze by chronic M was demonstrated (Pu et al., 2002). They used a different M injection protocol, which consisted of 2 injections per day for 15 days which induced M dependence, instead of 1 injection per day for 7 days, which made up tolerance to M. They showed a deficit in learning that was restored if task were done 1 h after M injection. Furthermore in the above mentioned study, learning deficit was observed during 2 h before the second daily injection, which might be related to withdrawal signs, not M effects. Also, there are contrary reports using similar protocol of dependence development, in which no effect of M on acquisition of water maze task or even working memory was found (Miladi Gorji et al., 2008; Lu et al., 2010). But, a significant effect on retention test as previously reported (Lu et al., 2010) was found in the present study. Herein, for the first time we provided experimental evidence on the effects of chronic sodium salicylate on spatial memory. In addition, acute effects of M on spatial reference memory were not observed if animals received M immediately after each training trail during task learning. These findings indicated that when the drugs were injected daily for 7 days immediately after each training trial of water maze task leaning, tolerance to M was developed in M-injected rats, so that a single dose of M was without effect on water maze performance of M-injected rats. But, a single dose of M impaired water maze performance of sodium salicylate-injected rats, which indicated
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that M was effective and these rats did not show cross-tolerance to M. More experimentation would clarify the related actions of M and sodium salicylate by quantifying of spatial learning and memory in the water maze.
5. Conclusions It is concluded that chronic sodium salicylate induces tolerance to anti-nociceptive effects of M and vice versa. Also acute but not chronic M impairs spatial memory with a tolerance to the effects of morphine. Furthermore, cross-tolerance effects of the drugs for one another on learning and memory are not seen. Chronic salicylate may produce lasting metaplastic changes in brain mechanisms behind spatial learning and memory, which can be visualized in cross-sensitization to morphine.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Morphine deteriorates spatial memory in sodium salicylate treated rats Mehdi Sadegh a, Yaghoub Fathollahi a,n, Nasser Naghdi b, Saeed Semnanian a a b
Department of Physiology, School of Medical Sciences, Tarbiat Modares University, PO Box 14115-111, Tehran, Iran Department of Physiology & Pharmacology, Pasteur Institute, Tehran, Iran
a r t i c l e i n f o
abstract
Article history: Received 15 November 2012 Received in revised form 5 February 2013 Accepted 7 February 2013 Available online 24 February 2013
Tolerance and cross-tolerance for the effects of morphine (M) and sodium salicylate on nociception and learning were examined. The anti-nociceptive effects were measured by using the classic tail flick (TF) and hot plate (HP) tests and learning was measured with the Morris water maze (MWM). Tolerance or cross-tolerance was induced by daily injection (i.p.) of morphine sulfate (10 mg/kg for 7 days) or sodium salicylate (300 mg/kg for 6 days). The injection of sodium salicylate increased both TF and HP latencies. This anti-nociceptive effect was progressively decreased across six injections and tolerance to sodium salicylate was developed. When M was injected to sodium salicylate-tolerant rats, a weakened anti-nociceptive effect was seen, indicating cross-tolerance to M. Acute treatment with M also increased TF latency. This anti-nociceptive effect was successively decreased across seven injections and tolerance to M was developed. When sodium salicylate was injected to M-tolerant rats, a diminished anti-nociceptive effect was seen, indicating cross-tolerance to sodium salicylate. Acute M impaired water maze performance, while chronic M and sodium salicylate had no effects on MWM performance. However, when M was injected to rats that had received sodium salicylate after each training trial for 7 days, these rats spent less time in target quadrant as compared to M and saline groups. It is concluded that chronic sodium salicylate induces tolerance to anti-nociceptive effects of M and vice versa. Also chronic salicylate may produce lasting metaplastic changes in brain mechanisms behind spatial learning and memory, which can be visualized in cross-sensitization to morphine. Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
Keywords: Cross-tolerance Morphine Morris water maze Salicylate Spatial memory
1. Introduction The combination of opioids and non-opioids analgesics (NSAIDs) is commonly used to control severe pain. This combination therapy can raise analgesic effects while avoiding adverse side effects (Deciga-Campos et al., 2003). However, tolerance to anti-nociceptive effect of non-opiate analgesics and crosstolerance to morphine have been reported (Pernia-Andrade et al., 2004; Trujillo, 2000). It has been reported that NSAIDs (e.g. metamizol and lysine-acetylsalicylate, LASA) influence periaqueductal gray matter (PAG) and descending pain-control system, so that at least a part of their anti-nociceptive effects is induced through endogenous opioidergic structures (Tsagareli et al., 2011; Vanegas and Tortorici, 2002). Drug tolerance as a result of adaptation in the brain mechanisms appears with a decrease in responsiveness and an increase in demand for drug (Trujillo, 2002). In some cases, drug tolerance and physical dependence are considered as an aberrant form of synaptic plasticity, cause stable change in synaptic strength, and thereby act on learning and memory (Trujillo, 2000, 2002). There
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Corresponding author. Tel.: þ98 2188011001; fax: þ 98 2188013030. E-mail address: [email protected] (Y. Fathollahi).
are controversial reports about the effects of chronic morphine on learning ability. Some of them indicated learning impairment (Farahmandfar et al., 2010; Lu et al., 2010) and some others demonstrated no deficit (Miladi Gorji et al., 2008; Wang et al., 2006). The problem of tolerance and cross-tolerance for the effects of morphine and sodium salicylate on nociception and learning has not yet been clearly established. Likewise very few reports had put to the test so far the related actions of these two drugs. Herein, therefore, the effects of acute and chronic sodium salicylate and morphine on analgesia and spatial learning were examined. Furthermore, cross-tolerance effects of the drugs for one another were also explored.
2. Materials and methods 2.1. Animals and tolerance induction Subjects were 66 Wistar male rats that were born and reared at the colony room of Pasteur Institute (Alborz providence, Karaj, Iran). Rats were 9–10 weeks of age during the experiments and maintained on a 12 h light/dark cycle with a free access to water and food except during practical tests. Experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (National Academy
0014-2999/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.02.017
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Press, Washington D.C., 2010) and every effort was made to reduce animal suffering and the number of animals used. Morphine (M) tolerance was induced by daily injection of morphine sulfate (10 mg/kg, i.p.) for 7 days. Tolerance to sodium salicylate was induced by daily injection of freshly prepared sodium salicylate solution (1 injection/day for 6 consecutive days, 300 mg/kg, i.p.). The anti-nociceptive effects of the drugs were quantified using classic tail flick (TF, Harvard apparatus 52-9495, US) or hot plate (HP, Heidolph MR-3001K, Germany) 20–30 min after each daily injection. One day before the starting drug injection, response latency were measured as a baseline (BL). For TF, a light beam was focused on the dorsal surface of the tail and the latency to TF was measured. Cut-off time was 10 s and intensity was set at 80% maximum intensity. For HP, the animal was placed on a metal plate at 50 72 1C and the latency of first hind paw lick or to jump was measured while cut-off time was 20 s. Both tests were conducted in triplicate and the averaged values were used for analysis. To study cross-tolerance between the drugs, a day after tolerance development, morphine sulfate (5 mg/kg, i.p.) or sodium salicylate (300 mg/kg, i.p.) was applied as cross in each group and the response latency was measured. Time-matched saline injected rats were used as control group. 2.2. Morris water maze The Morris water maze (MWM) consisted of a dark circular water tank (140 cm diameter, 40 cm deep) filled with water at 21–23 1C to a depth of 25 cm (Vorhees and Williams, 2006). An invisible colorless Plexiglass platform (10 cm diameter) was located 1.5 cm below the water surface in a fixed location in the center of a quadrant. The maze was located in a quiet test room and contained distinctive visual marks which were visible from within the pool and served as distal cues. Locations of the cues were constant throughout the testing period. A day before training, a 1-min free swim trial without the platform was run for habituation. The training session consisted of 4 or 7 days, 4 trials per day each from different starting points. After climbing onto the platform, the animal was allowed to stay on the platform for 30 s. In the cases where the animal was unable to find the platform in 90 s, it was gently guided to the target by the experimenter and was allowed to remain there for 30 s. After 30 s on the platform, animals were placed into a heated cage until next trial (4–8 min later). The probe test was performed 1 day after last training session; a 90-s trial was initiated whereby the platform was removed. This was used to evaluate whether rats would localize the platform to the spatial location by calculating of the exploring time in each quadrant (quadrant occupancy) and the number of cross over the platform site (crossing index). After the probe trial, the platform was elevated above the water surface (visible test) to evaluate visio-motor coordination toward a visible platform. The visible test was performed for four 90 s. Swimming paths for training session and probe test was monitored using an automatic tracking system (Ethovision XT8; Noldus Instruments). This system was used to record the swimming trace and to calculate the mean latency to find the platform, the time spent in each quadrant and swimming speed. 2.3. Experimental protocol 2.3.1. Experiment 1 To study tolerance to sodium salicylate and its cross-tolerance with M, two different methods for pain rating called TF and HP were used. For this, a day after test for baseline in TF or HP, animals randomly were assigned to sodium salicylate-TF (n¼5), sodium salicylate-HP (n¼4), saline-TF (n ¼4) and saline-HP (n ¼4) groups. On first day, animals received sodium salicylate (300 mg/
kg, i.p.) or saline and TF or HP latencies were measured after 20– 30 min. The same was done on the following days until tolerance to sodium salicylate was developed. A day after tolerance development, all animals received a single dose of morphine sulfate (5 mg/kg, i.p.) and TF or HP latencies were measured 30 min thereafter. Since the former practical tests reproduced the results of Pernia-Andrade et al. (2004), to study M cross-tolerance with sodium salicylate, only TF test was used. For this, a day after test for TF baseline, animals randomly were divided in M-TF (n¼ 5) and saline-TF (n ¼4) groups. On first day, animals received saline or morphine sulfate (10 mg/kg, i.p.) and TF latencies were measured 30 min thereafter. The same was done on the following days until tolerance to M was developed. A day after tolerance development, all animals received a single dose of sodium salicylate (300 mg/kg, i.p.) and TF latencies were measured 30 min thereafter. 2.3.2. Experiment 2 To quantify the effects of acute and chronic M and sodium salicylate on water maze performance, three different groups of animals were selected as saline, M and sodium salicylate (n¼4 for each group). In acute application, MWM task were done 30 min after each injection. This protocol was continued for 4 days. In chronic application, again three different groups of animals (n¼4 for each group) were selected and received the drugs or saline for 6–7 consecutive days. Then MWM task was done as mentioned above. 2.3.3. Experiment 3 To assess the effects of M and sodium salicylate on memory consolidation, same as two former experiments, groups were arranged as saline, M and sodium salicylate (n¼4 for each group), but the drugs or saline were administrated immediately after each trial of MWM task. This was continued for 7 days. To take measure of tolerance development to M, on 8th day all animals were received a single dose of morphine sulfate (10 mg/kg, i.p.) and 30 min thereafter a probe test was done. 2.4. Data analysis The data were expressed as mean7S.E.M. After data were tested for normal distribution (Kolmogorov–Smirnov), a one-way or two-way ANOVA followed by Bonferroni post-hoc test were performed. Two independent groups were compared by unpaired t-test. Statistically significant differences were acknowledged when Po0.05.
3. Results 3.1. Experiment 1 3.1.1. Chronic sodium salicylate induces tolerance to antinociceptive effect of M The latencies to TF or HP were increased within initial days of sodium salicylate injections as compared to its baseline and saline group (TF: F7, 56 ¼21.81, P o0.0001 and HP: F7, 48 ¼46.29, Po0.001). But, in the subsequent days (6th day for TF, 4th or 5th day for HP) this anti-nociceptive effect was progressively decreased so that during 5th and 6th days of the injections, the latencies to TF or HP was same as saline and the baseline (P4 0.05), indication of the development of tolerance to sodium salicylate. On the other hand, M injection on the 7th day increased the latencies to TF or HP in saline group but not in sodium salicylate group (Po0.001, Fig. 1A, B), indication of the development of cross-tolerance to M.
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Fig. 2. Chronic M leads to tolerance to the anti-nociceptive effect of sodium salicylate. The injection of morphine sulfate (10 mg/kg, i.p.) increased TF (n ¼5) latency, which was successively reduced by morphine sulfate injections. Sodium salicylate injection (300 mg/kg, i.p.) on 8th day to M-tolerant rats was noneffective to produce the anti-nociceptive effect, which indicates the occurrence of cross-tolerance to sodium salicylate. Latency was measured 20–30 after each injection. Data presented as mean 7S.E.M. nP o0.05, Bonferroni post-hoc test following two-way ANOVA. BL, Baseline latency; M, morphine.
(F2, 11 ¼6.02, Fig. 3B, Po 0.05). When retention of previously learned spatial memory (platform location recalling) was tested, statistical analysis revealed that the crossing index (F2, 23 ¼7.90, Po0.05,), but not the quadrant occupancy (F9, 18 ¼0.36, P¼0.94) was significantly decreased (Fig. 3C).
Fig. 1. Chronic sodium salicylate induces tolerance to anti-nociceptive effect of M. The injection of sodium salicylate increased both TF (n¼ 5) and HP (n¼ 4) latencies, which were successively reduced by sodium salicylate injections. Morphine sulfate injection (5 mg/kg, i.p.) on 7th day to sodium salicylate-tolerant rats was non-effective to produce the anti-nociceptive effect, which indicates the occurrence of cross-tolerance. Latency was measured 20–30 after each injection. Data presented as mean 7S.E.M. nnP o 0.01, Bonferroni post-hoc test following two-way ANOVA. BL, Baseline latency; M, morphine.
3.1.2. Chronic M leads to tolerance to anti-nociceptive effect of sodium salicylate As expected, the latency to TF was increased by daily injection of morphine sulfate (10 mg/kg, i.p.) and followed by a tolerance to its anti-nociceptive effects during 7 days of morphine sulfate injections (TF: P o0.0001, F8, 56 ¼10.62). Interestingly, at the 8th day, the TF latency was not increased due to a single dose of sodium salicylate (300 mg/kg, i.p.) indicating the occurrence of cross-tolerance to sodium salicylate (Po0.05, Fig. 2). 3.2. Experiment 2 3.2.1. Acute M impairs water maze performance Learning ability of animals was tested in MWM 30 min after drug injection. As demonstrated in Fig. 3, sodium salicylate had no effect on water maze performance as compared to saline group. However, M-injected animals took significantly more time to find the platform. Statistical analysis using two-way ANOVA followed by the Bonferroni post-hoc tests revealed that M impaired water maze performance (distance: F2, 135 ¼17.98; Po0.0001, Fig. 3 A; latency: F2, 135 ¼22.79; Po0.0001). Thirty minutes after M injection but not sodium salicylate, swimming speed was significantly increased as compared to saline group
3.2.2. Chronic M or sodium salicylate has no effect on water maze performance when injected pre-training To measure water maze performance of M- and sodium salicylate-tolerant rats, spatial learning ability of chronically Mand sodium salicylate-injected rats were measured in MWM. Chronic M and sodium salicylate were without effects on water maze performance across trials (Fig. 4C). Statistical analysis using two-way ANOVA followed by the Bonferroni post-hoc tests revealed that the differences in the mean values of time spent and distance moved to find the platform were not significant between three groups (distance: F2, 159 ¼0.05; P¼0.94, Fig. 4A; latency: F2,159 ¼0.77; P¼ 0.46). As seen in Fig. 4B neither M nor sodium salicylate had no effect on swimming speed (F2, 11 ¼0.22, P¼0.80). There were no significant changes in the crossing index and the quadrant occupancy (F2, 22 ¼0.84, P¼0.45, Fig. 4C) during probe tests. 3.3. Experiment 3 3.3.1. M disrupts water maze performance of sodium salicylateinjected rats when injected post-training In these series of practical tests the drugs or saline administrated immediately after each trial during MWM task learning. There were no significant differences in the water maze performance of M- and sodium salicylate-injected groups as compared to saline injected group (distance: F2, 270 ¼2.64; P¼0.08, Fig. 5A; latency: F2, 270 ¼1.87; P ¼0.16), except on 2nd day of sodium salicylate group (t48 ¼2.87, Po0.05). Swimming speed did not show significant differences between groups (F2, 18 ¼2.11, Fig. 5B, P¼0.15). On 8th day, a single dose of morphine sulfate (10 mg/kg, i.p.) was injected to three groups of animals and 30 min after injection, a probe test was done. Results (Fig. 5C, D) showed that the time spent in the target quadrant for chronically sodium salicylate-treated group was less than those of chronically
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Fig. 3. Acute M but not sodium salicylate impairs acquisition and retention of spatial reference memory. (A) The mean values of latency to find hidden platform vs. training days. Sodium salicylate (n ¼4) or morphine sulfate (n¼ 4) was injected 30 min before each training trial for 4 days. Sodium salicylate (300 mg/kg, i.p.) had no significant effect on water maze performance. But, morphine sulfate (10 mg/kg, i.p.) significantly influenced reference memory acquisition. Data presented as mean 7S.E.M. (B) Averaged swim speeds across the training days (each bar shows the mean þ S.E.M.). Notice a significant increase in velocity due to M. (C) In the probe test (5th day) the crossing index (each bar shows the mean þS.E.M.) significantly decreased due to M. nP o 0.05, M vs. saline, nnnPo 0.001, M vs. sodium salicylate or saline, Bonferroni post-hoc test following one-way ANOVA. M, morphine.
Fig. 4. Chronic sodium salicylate or M has no effects on acquisition and retention of spatial reference memory. (A) The mean values of latency ( 7 S.E.M) to find hidden platform across 4 training days in water maze (4 trials/day for 4 days; 5 min inter-trial interval) for three groups. Sodium salicylate (300 mg/kg for 6 days, i.p., n¼ 4) or morphine sulfate (10 mg/kg for 7 days, i.p., n ¼4) was chronically injected. The drugs had no significant effect on water maze performance. (B) Averaged swim speeds across the training days (each bar shows the mean þ S.E.M.). Notice no significant differences between groups. (C) In the probe test (5th day), the crossing index (each bar shows the mean þS.E.M.) did not show significant differences between groups. M, morphine.
saline- or M-injected rats (t15 ¼ 2.75, Po0.05) due to a single dose of M. However there were no significant differences for the crossing index between groups.
4. Discussion The effects of acute and chronic M and sodium salicylate on analgesia and spatial learning were examined. Furthermore, cross-tolerance effects of the drugs for one another were also explored. Our results show: (1) that chronic sodium salicylate induces tolerance to anti-nociceptive effects of M and vice versa, (2) that acute but not chronic M impairs spatial memory with a tolerance to the effects of morphine. To our knowledge, there are very few reports concerning the related actions of M and sodium salicylate and this study in fact confirms and extends our previously published results (Hosseinmardi et al., 2011). In the present study we found tolerance to anti-nociceptive effect of sodium salicylate and cross-tolerance to anti-nociceptive effect of M due to chronic sodium salicylate. Consistent with our results, tolerance to anti-nociceptive effect of other NSAIDs such as LASA and metamizol and their cross-tolerance to M (PerniaAndrade et al., 2004; Tortorici et al., 2009; Tortorici and Vanegas, 2000) have been reported. Tolerance to anti-nociceptive effect of LASA appeared after three or four systemic injections of this drug at therapeutic dose with 12 h interinjection interval. After five consecutive injections, cross-tolerance to anti-nociceptive effect of M was induced. In the present study, we used another
derivative of salicylic acid, sodium salicylate in a dose same as Pernia-Andrade et al. (2004), but our injection protocol was different. The injection protocol consisted of one injection per day for six consecutive days. We discovered that this scheduled sodium salicylate injection can induce tolerance to antinociceptive effect of the drug and cross-tolerance to M. In our preliminary experiments, we used same protocol that had been used by Pernia-Andrade et al. (2004) in which two injections per day was scheduled, but the mortality rate of animals was high (nearly 50%) after 2nd or 3rd injection. So, the injection protocol was changed as mentioned to reduce the mortality rate of animals and two animals died during the study. Perhaps this high mortality rate might be related to salicylic derivatives, which were used in these studies or may be related to subjects. We used Wistar rat (9–10 weeks age, 180–200 g), whereas Pernia-Andrade et al. (2004) used Sprague-Dawley rats (250–350 g). To remedy the mortality issues, we suggest using lower doses for testing to survive experimental animals in the future. To our knowledge, this is the first time that chronic M was shown to not only induced tolerance to M but also induced a cross-tolerance to the anti-nociceptive effect of sodium salicylate. It has been shown that PAG and downstream pain-control structures have an important role to the analgesic effect of LASA and metamizol involving opioidergic mechanisms (Christie et al., 2000; Tsagareli et al., 2011; Vorhees and Williams, 2006). Chronic exposure to an exogenous opiate such as M may act through this pathway at downstream pain-control structures, causing tolerance to other opiates and cross-tolerance to NSAIDs.
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Fig. 5. M disrupts spatial memory in the sodium salicylate-injected rats. (A) The mean values of latency ( 7 S.E.M) to find hidden platform vs. training days in water maze (4 trials/day; 5 min inter-trial interval) for three groups of animals. Sodium salicylate (300 mg/kg, i.p. n¼ 4) or morphine sulfate (10 mg/kg, i.p. n¼ 4) was daily injected immediately after each trail in water maze for 7 days. Notice a significant effect on 2nd day for sodium salicylate group, but not in M group. (B) Averaged swim speeds across training days (each bar shows the mean þS.E.M.). Notice no significant differences between groups. (C, D) In the probe test (5th day), the effect of a single dose of morphine sulfate (10 mg/kg) on the retention was examined. Notice no significant differences in the crossing index between groups (D) but quadrant occupancy (C) for the target quadrant showed a significance decrease for sodium salicylate group due to M. Each bar shows the mean þ S.E.M. nP o 0.05, Bonferroni post-hoc test following oneway ANOVA. M, morphine.
We demonstrated an impairment of water maze performance 30 min after M injection. An impairment of water maze learning ability 20 min after M injection (5 and 10 mg/kg) in mice has been shown (Li et al., 2001). They explained that inhibition of brain cholinergic system by M caused this memory impairment. It has also been shown that 30 min after M injection acquisition of spatial memory and also retention of reference memory were impaired (Farahmandfar et al., 2010). However, neither of these studies reported an increase of swimming speed during the training tests, but our result showed considerable increase of swimming speed and animal motivation due to acute M. This increase of swimming speed may be attributed to experimental procedures used in their experiments. Li et al. used mice as experimental subjects and Farahmandfar et al. used 8 trials in one as training protocol. In addition, acute sodium salicylate did not act on learning ability in the present study. It has been suggested a protective effect for a combination of ibuprofen and glutathione as a potential therapeutic agent for treating Alzheimer’s disease (Pinnen et al., 2011). However, the present study is the first to report on the cognitive effects of NSAIDs (with antinociceptive dose) in the water maze task. We found that chronic exposure to M or sodium salicylate was without effect on water maze performance. But, as shown in Fig. 5, sodium salicylate can act on memory consolidation on second day when it was injected immediately after each training trial, while M had no effect during this experiment. In addition, a single M injection during the probe test reduced time spent in target quadrant in sodium salicylate group but not in saline or M groups (Fig. 5D), may be related to disruption of memory consolidation. In addition, as the probe test revealed, the effect
of acute M (Fig. 3) was not seen when M was applied after each training trial. A learning deficit and impaired memory formation following the chronic M (Lu et al., 2010; Miladi Gorji et al., 2008) have been described. However, some others (Miladi Gorji et al., 2008; Wang et al., 2006) demonstrated no effect of chronic M on learning ability and retrieval. An impairment of spatial memory in water maze by chronic M was demonstrated (Pu et al., 2002). They used a different M injection protocol, which consisted of 2 injections per day for 15 days which induced M dependence, instead of 1 injection per day for 7 days, which made up tolerance to M. They showed a deficit in learning that was restored if task were done 1 h after M injection. Furthermore in the above mentioned study, learning deficit was observed during 2 h before the second daily injection, which might be related to withdrawal signs, not M effects. Also, there are contrary reports using similar protocol of dependence development, in which no effect of M on acquisition of water maze task or even working memory was found (Miladi Gorji et al., 2008; Lu et al., 2010). But, a significant effect on retention test as previously reported (Lu et al., 2010) was found in the present study. Herein, for the first time we provided experimental evidence on the effects of chronic sodium salicylate on spatial memory. In addition, acute effects of M on spatial reference memory were not observed if animals received M immediately after each training trail during task learning. These findings indicated that when the drugs were injected daily for 7 days immediately after each training trial of water maze task leaning, tolerance to M was developed in M-injected rats, so that a single dose of M was without effect on water maze performance of M-injected rats. But, a single dose of M impaired water maze performance of sodium salicylate-injected rats, which indicated
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that M was effective and these rats did not show cross-tolerance to M. More experimentation would clarify the related actions of M and sodium salicylate by quantifying of spatial learning and memory in the water maze.
5. Conclusions It is concluded that chronic sodium salicylate induces tolerance to anti-nociceptive effects of M and vice versa. Also acute but not chronic M impairs spatial memory with a tolerance to the effects of morphine. Furthermore, cross-tolerance effects of the drugs for one another on learning and memory are not seen. Chronic salicylate may produce lasting metaplastic changes in brain mechanisms behind spatial learning and memory, which can be visualized in cross-sensitization to morphine.
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