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Changes in hippocampal orexin 1 receptor expression involved in tooth pain-induced learning and memory impairment in rats
Neuropeptides 50 (2015) 9–16
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Neuropeptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n p e p
Changes in hippocampal orexin 1 receptor expression involved in tooth pain-induced learning and memory impairment in rats Ramin Raoof a,b, Saeed Esmaeili-Mahani a, Mehdi Abbasnejad a, Maryam Raoof b,c,*, Vahid Sheibani b, Razieh Kooshki a, Ladan Amirkhosravi a, Foroozan Rafie d a
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Laboratory of Molecular Neuroscience, Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran c Department of Endodontics, School of Dentistry, Kerman University of Medical Sciences, Kerman, Iran d Department of Motor Behaviour, Facaulty of Physical Education, Tehran University, Tehran, Iran b
A R T I C L E
I N F O
Article history: Received 4 July 2014 Accepted 2 March 2015 Available online 12 March 2015 Keywords: Tooth inflammatory pulpal pain Orexin 1 receptor Learning and memory Capsaicin Gene expression
A B S T R A C T
Orexin 1 receptor signaling plays a significant role in pain as well as learning and memory processes. This study was conducted to assess the changes in orexin 1 receptor expression levels in hippocampus following learning and memory impairment induced by tooth inflammatory pulpal pain. Adult male Wistar rats received intradental injection of 100 μg capsaicin to induce pulpal pain. After recording the pain scores, spatial learning and memory were assessed using Morris Water Maze test. The hippocampal levels of orexin 1 receptor mRNA and protein were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblotting respectively. The data showed that capsaicininduced tooth inflammatory pulpal pain was correlated with learning and memory impairment. Intrahippocampal injection of orexin A inhibited pain-induced learning and memory impairment. However, orexin 1 receptor antagonist, SB-334867, had no effect on learning and memory impairment. Moreover, capsaicin-induced pain significantly decreased hippocampal orexin 1 receptor mRNA and protein levels. Meanwhile, reversed changes took place in the ibuprofen-pretreated group (p < 0.05). It seems that decrease in orexin 1 receptor density and signaling could be involved in tooth pain-induced learning and memory impairment. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Orofacial pain is one of the most prevalent types of pain and odontalgia is the most commonly experienced one (Moure-Leite et al., 2011). Numerous studies have demonstrated the relationship between pain and changes in brain anatomy such as cortical thickness and gray matter density (Seminowicz et al., 2009). Moreover, neural systems involved in cognition and pain processing are closely linked, they may modulate one another reciprocally (Moriarty et al., 2011). So, cognitive function is thought to be affected in patients suffering pain. However, pain of trigeminal origin shows specific processing pathways and relay sites that make it different from pain originating from the anterolateral system (Upadhyay et al., 2008). Moreover, the episodes of pain, the thickness and pattern of the nerves, the size of terminal varicosities, and the length of intervaricose segments
* Corresponding author. Neuroscience Research Center, Jahad St., Kerman, Iran. E-mail address: [email protected] (M. Raoof). http://dx.doi.org/10.1016/j.npep.2015.03.002 0143-4179/© 2015 Elsevier Ltd. All rights reserved.
in dental pulp are quite different from that of other tissues (Zhang et al., 1998). Another clear difference is that the pulp vessels present rather rich calcitonin gene related peptide (CGRP) expressing nerve fibers but much less abundant substance P and neurokinin expressing nerve fibers, no vasoactive intestinal peptide (VIP) nerves, and few or no neuropeptide Y (NPY) expressing nerves (Kerezoudis et al., 1995; Uddman et al., 1980, 1984). Another unique feature of dental pulp is the direct innervation of both microvasculature and larger vessels by free nerve endings, suggesting that the local regulation of blood flow may take place not only at larger vessels but also at the level of the microvasculature in this tissue (Tabata et al., 1998). Learning and memory deficits have been reported in painful conditions (Hu et al., 2010; Yang et al., 2014). However, the investigation of the underlying mechanisms remains to be clarified. Orexin (A and B) and its receptors are widely distributed in the central nervous system. There are numerous reports indicating the antinociceptive effects of orexins in various animal models of pain, including trigeminovascular pain (Chiou et al., 2010). Orexins are antinociceptive at both spinal and supraspinal levels (Mobarakeh et al., 2005). Surprisingly, the antinociceptive effect of orexin-A is comparable to opioids (Yamamoto et al., 2002). This effect is
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opioid-independent and mainly mediated through orexin 1 receptors. Some animal studies suggest that endogenous orexins may be released during inflammatory pain states, or during some stress conditions, which may contribute to stress-induced analgesia (Chiou et al., 2010). Damage to the hippocampal structures has been also associated with learning and memory impairments (Kuhajda et al., 2002; Squire, 1992; Sun et al., 2013). Furthermore, hippocampal formation (CA1, CA2 and dentate gyrus) expresses orexin 1 receptors and receives orexinergic terminals. It has been shown that hippocampal orexin 1 receptor signaling has an important role in acquisition, consolidation and retrieval of spatial memory in Morris water maze (MWM) task (Akbari et al., 2007). Recently, Zhao et al. (2014) reported that orexin-A can attenuate the impairment of spatial learning and memory in PTZ-kindled rats through orexin 1 receptor-mediated signaling (Zhao et al., 2014). Since orexin 1 receptor signaling has a noticeable role in pain as well as learning and memory processes and the exact mechanisms of pain-induced learning and memory deficit have not yet been clarified, the present study was conducted to assess the role of hippocampal orexin 1 receptors and determine their expression levels in learning and memory impairment induced by tooth inflammatory pulpal pain.
2. Material and methods 2.1. Animals Adult male Wistar rats weighing 250–300 grams, purchased from the Neuroscience Research Center (Kerman University of Medical Sciences, Iran), were used in this study. The rats were housed (12-h light/dark cycle) one per cage in a room with a temperature of 23 ± 2 °C with unlimited access to standard rat chow and water before and during the study. Rats were randomly allocated into experimental groups, each comprising 6–7 animals. Animals that were used for the behavioral studies (n = 7) were different from animals that were used for molecular experiments (n = 6). All experimental procedures were approved by the Animal Research Ethics Committee of Kerman University of Medical Sciences, Kerman, Iran (Code: K/90/258).
2.2. Dental procedure Inflammatory pulpal pain induction was constructed as our modified model, representing a modification to the Chidiac study (Chidiac et al., 2002) described in a previous article (Raoof et al., 2012). In brief, the distal 2 mm of the rats’ mandibular incisors were cut off and special polyethylene crowns were fixed on the teeth using a flow composite resin (Tetric Flow, IvoclarVivadent). A small space remained between the tooth structure and the internal surface of the crown.
2.3. Drugs Capsaicin (Sigma-Aldrich, USA) was dissolved in Tween 80 – ethanol solution (Merck, Germany) (10% ethanol, 10% Tween 80, 80% distilled water, w/w) at a concentration of 10 mg/ml. Ten μl of capsaicin solution which contains 100 μg of drug was administrated intradentally (i.d.). Orexin A and SB-334867 (Tocris, London, UK) were dissolved in distilled water. Ibuprofen (Rouzdaru, Iran) powder was dissolved in a vehicle (2% Tween 80/distilled water) and given intragastrically (oral gavage) at a dose of 120 mg/kg (Raoof et al., 2012).
2.4. Experimental design The animals were randomly divided into five experimental groups (n = 7) as follows: 1: Control group, included intact animals. 2: Sham-operated group, which took the crown but received no injection. 3: Sham-vehicle group received i.d. injection of capsaicin vehicle for five days. 4: Capsaicin-treated group received capsaicin (100 μg, i.d.) for five consecutive days. 5: Ibuprofen-treated group received 120 mg/kg ibuprofen 20 min before capsaicin injection for five consecutive days. 6: Orexin A-treated group received orexin A (40 pM) 20 min before capsaicin injection for five consecutive days. 7: Orexin 1 receptor antagonist-treated group received 80 nM SB-334867 (Azhdari-Zarmehri et al., 2013) 20 min before capsaicin injection for five consecutive days. 2.5. Nociceptive behavior Test sessions were carried out during the light phase, between 09:00 a.m. and 13:00 p.m., in a quiet room maintained automatically at 23 ± 2 °C. Before drug injection, each animal was placed in the test box for a 30 min habituation period to minimize additional stress. The rats did not have access to food or water during the test. Immediately following the injection, each rat was placed back in the transparent Plexi glass box (25 × 35 × 35) with a transparent floor positioned over a mirror at an angle of 45° to allow for observation of nociceptive behavior. The rats’ behavior was observed for 21 minutes, divided into 7 blocks of 3 minutes. The person investigating the behavioral test was blinded to the group assignment. A pain score was determined for each block by measuring the number of seconds that the animal presented each of the following responses which represents the same scoring criteria as described previously (Chidiac et al., 2002): 0 – Calm, normal behavior such as grooming; 1– Abnormal head movements such as mild head shaking or continuous placement of the jaw on the floor or the wall of the cage; 2– Abnormal continuous shaking of the lower jaw; 3– Excessive rubbing of the mouth with foreleg movements, such as head grooming, but concentrated consistently and mainly on the lower jaw. A video camera was used to record the behavioral response (Raoof et al., 2012). 2.6. Morris water maze test The water maze test was used (Morris et al., 1982). Briefly, it was a black circular pool with a diameter of 136 cm and a height of 60 cm, filled with 20 ± 1 °C water to a depth of 25 cm. The maze was divided geographically into four equal quadrants and release points that were designed at each quadrant as N, E, S and W. A hidden circular platform (10 cm in diameter), made of Plexiglas, was located in the center of the southwest (target) quadrant, submerged 1.5 cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze consisting of geometric shapes on the walls, shelves, computer, a window, a door and posters. These were kept in fixed positions with respect to the swimming pool to allow the rat to locate the escape platform hidden below the water surface. After completion of training, the rats were returned to their cages and the retention test (probe trial) was performed 2 h later. In probe test, animals had 60 s free swim period without a platform and the time spent in the target quadrant was recorded. A video
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camera was mounted directly above the water maze to record the rats’ swim path. A tracking system was used to measure the distance each rat traveled, the percent of distance, and the time in each quadrant. 2.7. Tissue extraction and preparation The rats were anesthetized (exposed to a CO2 atmosphere) and decapitated and the brains were removed immediately. Brains were dissected along the sagittal midline, followed by bilateral removal of the hippocampus (Duric and McCarson, 2007). The hippocampus was immediately placed on ice in a glass petri dish. The dissected hippocampi from each rat were randomly distributed for further western blot and RT-PCR assays. Tissue samples were weighed and immediately frozen in liquid nitrogen and stored at −70 °C until assay (Kaeidi et al., 2011). 2.8. mRNA analysis Total cellular RNAs were isolated from the hippocampus by a modification of the guanidine isothiocyanate–phenol–chloroform method using RNX+ reagent (Ausubel et al., 2002). A semiquantitative RT-PCR method was used (Marone et al., 2001). Briefly, the RTPCR reaction was performed using Oligo-dT primer and M-MuLV reverse transcriptase, based on the manufacturer’s protocol (Fermentas GMBH, Germany). The reactions were incubated at 42 °C for 60 min and then inactivated at 70 °C for 10 min. Three separate PCR reactions were used for studying gene expression in the samples obtained from each rat. Each PCR reaction was carried out using selective forward and reverse primers for β-actin (as an internal standard) and orexin 1 receptor proteins. The sequence of the primers used was: orexin 1 receptor forward: 5′-AGG TGG ATG GAA GCG TGA AG-3′, orexin 1 receptor reverse: 5′-AGA GAT AAT CGC GCC ACA GG-3′, β-actin forward: 5′-CCC AGA GCA AGA GAG GCA TC-3′, β-actin reverse: 5′-CTC AGG AGG AGC AAT GAT CT-3′. Taq DNA polymerase (Cinaclon, Iran) used for DNA amplification and reactions were set up according to the manufacturer’s protocol. The PCR reactions were incubated at 94 °C for 5 min, followed by 25 cycles of thermal cycling (45 s at 94 °C, 45 s at 55 °C and 45 s at 72 °C). The final cycle was followed by a 5 min extension step at 72 °C. The reaction parameters were adjusted to obtain a condition with a linear relation between the number of PCR cycles and PCR products and with linear relation between the initial amount of cDNA template and PCR product. Based on the results obtained from these experiments, 25 cycles of PCR amplification were used for analyzing all samples. PCR products were subsequently analyzed on 1.5% agarose LMMP (Roche, Germany) gel and bands were quantified by densitometry using Lab Works analyzing software (UVP, UK). The possibility of the presence of contaminating genomic DNA was ruled out by using the yield of reverse transcriptase-minus (RT−) reaction, instead of cDNA template, which caused no DNA amplification (data not shown). Semiquantitative PCR technique was used to estimate orexin 1 receptor mRNA levels in tissue samples, normalized to an internal standard (Hajializadeh et al., 2010). 2.9. Protein analysis The dissected hippocampal tissues were homogenized using RIPA buffer, containing 10 mM trisaminomethane–HCL (Tris–HCl) (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl sulfate (SDS), 0.1% Na-deoxycholate, 1% NP-40 with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2.5 μg/ml of leupeptin, 10 μg/ml of aprotinin) and 1 mM sodium orthovanadate. The homogenate was centrifuged at 14,000 rpm at 4 °C for 15 min. The resulting supernatant was retained as the whole cell fraction. Protein
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concentrations were measured using the Bradford method (BioRad Laboratories, Muenchen, Germany). Equal amounts of proteins of the sample were separated according to molecular weight using SDS–PAGE gel electrophoresis (Cinaclone, Iran) and transferred to PVDF membrane. After blocking with 5% non-fat dried milk in Trisbuffered saline with Tween 20 (blocking buffer, TBS-T, 150 mM NaCl, 20 mM Tris–HCl, pH 7.5, 0.1% Tween 20) the membrane was probed for the protein of interest with specific antibodies. All antibodies were diluted in blocking buffer. A dilute solution of primary antibody to orexin 1 receptor (sc8073; Santa Cruz biotechnology, USA, 1:1000) was incubated with the membrane under gentle agitation (overnight at 4°C for). After washing in TBS-T (three times, 5 min), the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (1:15,000, Santa Cruz biotechnology, USA). The incubation time for orexin 1 receptor antibody was 3 hours at room temperature. The antibody–antigen complexes were detected using the Electrochemiluminescence-Advance system (ECL-Advance system) and exposed to Lumi-Film chemiluminescent detection film (Roch, Germany) (Kaeidi et al., 2011). For ensuring the loading of proteins, the amount of β-actin protein was evaluated. After digitizing the films, the images of bands were evaluated using Lab Work analyzing software (UVP, UK) (Kaeidi et al., 2011). 2.10. Statistical analysis All statistical analyses were carried out by an observer blinded to the experimental groups. Data are presented as mean ± standard error of mean (S.E.M.). Differences between groups regarding pain scores and learning and memory indices were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. The differences in the amount of orexin 1 receptor mRNA and protein levels between groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. P < 0.05 was considered significant. 3. Results 3.1. Assessment of recorded pain scores in the control (intact, sham-operated and sham-vehicle), capsaicin- and ibuprofen-treated groups There were no significant differences in baseline nociceptive behaviors [F(2,18) = 2.949, P = 0.0780] between intact, sham-operated (took crown but received no injection) and sham-vehicle (received i.d. injection of capsaicin vehicle) animals (Fig. 1). In addition, intradental application of capsaicin (100 μg/rat) significantly increased pain scores [F(4,30) = 29.761, P = 0.0001]. In ibuprofenpretreated rats (Caps + Ibup), capsaicin-induced nociception was decreased (Fig. 1). 3.2. Assessment of spatial learning and memory in experimental groups 3.2.1. Hidden platform trials There was no significant difference between intact, sham and sham-vehicle animals as control groups in time to find the platform [F(2,18) = 1.658, P = 0.2183]. Capsaicin injection significantly increased time to find the platform [F(6,42) = 12.119, P = 0.0001] which was decreased by 40 pM orexin A or ibuprofen (120 mg/kg, i.g.) pretreatment (Fig. 2). As shown in Fig. 3, there was no significant difference between control, sham-operated and sham-vehicle groups in traveled distance to reach the platform [F(2,18) = 3.239, P = 0.0629]. Traveled distance was significantly increased in capsaicin-treated (Caps) animals which was reversed by orexin A (Caps + Orex) or ibuprofen
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Fig. 1. Comparison of recorded dental pain score in different experimental groups. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. Each value in the graph represents the mean ± SEM. ***P < 0.001 versus control (intact, sham and sham-vehicle) groups. +++P < 0.001 versus capsaicintreated (Caps) animals.
(Caps + Ibup) pretreatment [F(6,42) = 6.610, P = 0.0001]. In addition, the increase in traveled distance (P < 0.05) was also observed in animals that received capsaicin plus 80 nM SB-334867 (Caps + SB). Fig. 4 depicts typical swimming tracks of the rats in the Morris water maze test. The animals in the capsaicin-treated group often searched for the platform in an inappropriate way resulting in the longer latency to locate the platform. The effect was attenuated by orexin or ibuprofen treatment. Furthermore, one-way ANOVA showed that there were no significant differences [F(6,42) = 2.178, P = 0.0001] between swim speeds in different experimental groups (Fig. 5). 3.2.2. Probe trials Fig. 6 shows the retention performance on the probe test trial for experimental groups. The mean of time spent in the target quadrant during the 60 s probe test was significantly decreased in capsaicin-treated rats as compared to the control groups
[F(3,27) = 8.422, P = 0.0006]. Orexin A or ibuprofen pretreatment significantly prevented the effect of capsaicin on time spent in the target quadrant [F(6,42) = 6.718, P = 0.0001]. However in SB-334867pretreated rats the mean value was closed to those observed in control rats (P > 0.05). 3.3. Effect of tooth inflammatory pulpal pain on hippocampal orexin 1 receptor mRNA and protein levels The expression of orexin 1 receptor mRNA and protein in the control (intact, sham-operated and sham vehicle-treated) groups were the same (data not shown). The levels of orexin 1 receptor mRNA (Fig. 7) and protein (Fig. 8) in hippocampus of capsaicintreated rats were significantly lower than those observed in control (vehicle-treated sham) rats (P < 0.01 and P < 0.001 respectively). However, in ibuprofen-pretreated rats, orexin 1 receptor mRNA and protein levels were closed to the control levels (p > 0.05).
Fig. 2. Comparison of time between study groups. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. Each value in the graph represents mean ± SEM. ***p < 0.001 versus intact and sham-operated (Sham) groups. ##p < 0.01 and ###p < 0.001 versus sham-vehicle group. ++P < 0.01 and +++P < 0.001 versus capsaicin-treated (Caps) animals.
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Fig. 3. Comparison of traveled distance between study groups. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. Each value in the graph represents mean ± SEM. *P < 0.05 versus control (intact, sham and sham-veh) groups. + P < 0.05 and ++P < 0.01 versus capsaicin-treated animals.
Fig. 4. Typical swimming tracks of the rats in the Morris water maze test. The animals in capsaicin-treated group often searched for the platform in an inappropriate way. The effect was attenuated by orexin or ibuprofen treatment.
Fig. 5. Comparison of speed between study groups. One-way analysis of variance showed there were no significant differences. Each value in the graph represents mean ± SEM (n = 7).
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Fig. 6. Comparison of the time spent in the target quadrant during the 60 s probe test. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. The columns represent the mean ± S.E.M. *p < 0.05 versus intact and sham-operated (Sham) and vehicle-treated sham (Sham-Veh) groups. ++P < 0.01 and +++P < 0.001 versus capsaicin-treated (Caps) animals.
4. Discussion In the present study, intradental injection of capsaicin produced a significant pain response score, which was inhibited by ibuprofen. It has been demonstrated that activation of capsaicin-sensitive fibers in
Fig. 7. The levels of orexin 1 receptor mRNA in control (Cont), capsaicin-treated (Caps) and ibuprofen-pretreated (Caps + Ibup) rats. One-way analysis of variance was used. Values represent mean ± SEM (n = 6). ** P < 0.01 versus control group. + P < 0.05 versus capsaicin-treated group.
Fig. 8. The levels of orexin 1 receptor protein in control (Cont), capsaicin-treated (Caps) and ibuprofen-pretreated (Caps + Ibup) rats. One-way analysis of variance was used. Values represent mean ± SEM (n = 6). *** P < 0.001 versus control group. +++ P < 0.001 versus capsaicin-treated group.
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dental pulp increases pulpal blood flow and produces changes in escape behavior, suggesting a thermal and mechanical hyperalgesia (Kupers et al., 1997). The capsaicin-induced pain affected the neural processing in diencephalon and telencephalon; for example, the spontaneous firing of neurons in the primary somatosensory cortex and the ventral posterior medial nucleus of the thalamus was increased by orofacial capsaicin injection (Katz et al., 1999). Surprisingly, intradental capsaicin-treated rats spent more time and distance to find the hidden platform than control animals in the MWM test, which was attenuated by ibuprofen pretreatment. When the platform was removed for the probe trial, capsaicintreated animals spent significantly less time in the training quadrant than control ones. On the other hand, capsaicin had no significant effect on motor performance as measured by swim speed. Furthermore, visual platform performance did not differ between different groups. It seems likely that capsaicin-related spatial learning impairment is not related to the changes in motivation and/or sensory functions. Learning and memory impairment induced by intradental injection of capsaicin showed a positive correlation with pain score. It has been also reported that chronic pain stress significantly impairs spatial learning and memory function in neonatal rats (Li et al., 2005). Pais-Vieira et al. have shown that chronic inflammatory pain and monoarthritis causes attention impairment that could not be reversed by subcutaneous injection of carprofen (Pais-Vieira et al., 2009). So, there is sufficient evidence from preclinical and clinical investigations to support the theory that pain is associated with impaired cognitive function. This impairment may have a noticeable impact on patients’ quality of life. There is also some evidence for a mechanistic, neuropathological basis for pain-related cognitive impairment (Reneman et al., 2014). There is a considerable overlap between the neuroanatomical and neurochemical substrates implicated in both pain and cognition (Moriarty et al., 2011). It has been reported that orexin receptors, which have antinociceptive signaling at both spinal and supraspinal levels, are significantly expressed in the hippocampus (Chiou et al., 2010). Orexin is distributed in the periaqueductal gray matter (PAG), raphe nuclei, nucleus locus coeruleus and superficial and profound layers of the spinal dorsal horn; all of these areas are involved in nociceptive pathways (Trivedi et al., 1998; van den Pol, 1999). This study showed that the expression level of orexin 1 receptor is reduced in the hippocampus of rats that had pulpal pain and impaired learning and memory. The expression pattern of orexin receptors in rat brain has been previously reported (Cluderay et al., 2002; Marcus et al., 2001). Orexin 1 receptor immunoreactivity showed that the cerebral neocortex, basal ganglia, hippocampal formation, and many other regions in the hypothalamus, thalamus, midbrain and reticular formation express orexin 1 receptors (Marcus et al., 2001). However, since immunohistochemistry is also helpful to estimate the tissue protein expression levels further studies are needed to confirm the different expression levels of orexin 1 receptor in the hippocampus of capsaicin-treated animals. It has been demonstrated that the orexinergic system has an important role in learning and memory processes and induces structural changes in the hippocampus and related structures. Orexin neurons play an important role in the consolidation of social recognition memory through enhancements of hippocampal synaptic plasticity (Yang et al., 2013). Our results (Figs. 2–5) indicate that intrahyppocampal injection of orexin attenuates learning and memory impairment in capsaicin-treated rats. However, the orexin-1 receptor antagonist, SB-334867, has been shown to reverse cognitive deficits. The effects appear to be mediated by the orexin-A receptor. Despite orexin-B, Orexin-A induces a state-dependent longterm potentiation of Schaffer collateral-CA1 synapses in the adult mouse hippocampal slices (Selbach et al., 2010). Akbari et al. also demonstrated that functional inactivation of orexin 1 receptors in
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CA1 region impairs Morris water maze performance (Akbari et al., 2006). Surprisingly, orexin-A improved memory retention in both young and old SAMP8 mice through cholinergic, GABAergic and serotonergic pathways (Jaeger et al., 2002). There are numerous pieces of evidence that there is an interaction between orexin-A and neuropeptide Y that may contribute to appetite regulation. If orexin-A is able to interact with NPY to regulate feeding behaviors, then this interaction may also be responsible for the regulation of other physiological processes, such as memory consolidation (Jaeger et al., 2002). In addition, the nucleus locus coeruleus, which plays a significant role in pain, consciousness, learning and memory, receives orexinergic neurons. It seems dysregulation of the orexin system may contribute to the etiology of cognitive disorders (Sears et al., 2013). As mentioned above, orexin receptors are involved in nociceptive pathways as well as learning and memory. Our data showed that orexin 1 receptor expression significantly decreased in the hippocampus as one of the most important regions in learning and memory. So maybe orexin 1 receptor down-regulation could be one reason for learning and memory impairment caused by tooth inflammatory pulpal pain. However, the underlying mechanisms need to be clarified by further investigations. Acknowledgments This work was supported by funds from Neuroscience Research Center and Kerman University of Medical Sciences (grant no: 93-26). References Akbari, E., Naghdi, N., Motamedi, F., 2006. Functional inactivation of orexin 1 receptors in CA1 region impairs acquisition, consolidation and retrieval in Morris water maze task. Behav. Brain Res. 173, 47–52. Akbari, E., Naghdi, N., Motamedi, F., 2007. The selective orexin 1 receptor antagonist SB-334867-A impairs acquisition and consolidation but not retrieval of spatial memory in Morris water maze. Peptides 28, 650–656. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., et al., 2002. Short Protocols in Molecular Biology. Wiley, USA. Azhdari-Zarmehri, H., Esmaeili, M.H., Sofiabadi, M., Haghdoost-Yazdi, H., 2013. Orexin receptor type-1 antagonist SB-334867 decreases morphine-induced antinociceptive effect in formalin test. Pharmacol. Biochem. Behav. 112, 64–70. Chidiac, J.J., Rifai, K., Hawwa, N.N., Massaad, C.A., Jurjus, A.R., Jabbur, S.J., et al., 2002. Nociceptive behaviour induced by dental application of irritants to rat incisors: a new model for tooth inflammatory pain. Eur. J. Pain 6, 55–67. Chiou, L.C., Lee, H.J., Ho, Y.C., Chen, S.P., Liao, Y.Y., Ma, C.H., et al., 2010. Orexins/ hypocretins: pain regulation and cellular actions. Curr. Pharm. Des. 16, 3089– 3100. Cluderay, J.E., Harrison, D.C., Hervieu, G.J., 2002. Protein distribution of the orexin-2 receptor in the rat central nervous system. Regul. Pept. 104, 131–144. Duric, V., McCarson, K.E., 2007. Neurokinin-1 (NK-1) receptor and brain-derived neurotrophic factor (BDNF) gene expression is differentially modulated in the rat spinal dorsal horn and hippocampus during inflammatory pain. Mol. Pain 3, 32–40. Hajializadeh, Z., Esmaeili-Mahani, S., Sheibani, V., Kaeidi, A., Atapour, M., Abbasnejad, M., 2010. Changes in the gene expression of specific G-protein subunits correlate with morphine insensitivity in streptozotocin-induced diabetic rats. Neuropeptides 44, 299–304. Hu, Y., Yang, J., Hu, Y., Wang, Y., Li, W., 2010. Amitriptyline rather than lornoxicam ameliorates neuropathic pain-induced deficits in abilities of spatial learning and memory. Eur. J. Anaesthesiol. 27, 162–168. Jaeger, L.B., Farr, S.A., Banks, W.A., Morley, J.E., 2002. Effects of orexin-A on memory processing. Peptides 23, 1683–1688. Kaeidi, A., Esmaeili-Mahani, S., Sheibani, V., Abbasnejad, M., Rasoulian, B., Hajializadeh, Z., et al., 2011. Olive (Olea europaea L.) leaf extract attenuates early diabetic neuropathic pain through prevention of high glucose-induced apoptosis: in vitro and in vivo studies. J. Ethnopharmacol. 136, 188–196. Katz, D.B., Simon, S., Moody, A., Nicolelis, M.A., 1999. Simultaneous reorganization in thalamocortical ensembles evolves over several hours after perioral capsaicin injections. J. Neurophysiol. 82, 963–977. Kerezoudis, N.P., Fried, K., Olgart, L., 1995. Haemodynamic and immunohistochemical studies of rat incisor pulp after denervation and subsequent re-innervation. Arch. Oral Biol. 40, 815–823. Kuhajda, M.C., Thorn, B.E., Klinger, M.R., Rubin, N.J., 2002. The effect of headache pain on attention (encoding) and memory (recognition). Pain 97, 213–221.
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Kupers, R.C., Chen, C.C., Bushnell, M.C., 1997. A model of transient hyperalgesia in the behaving monkey induced by topical application of capsaicin. Pain 72, 269–275. Li, Y., Peng, S., Wan, C., Cao, L., Li, Y., 2005. Chronic pain impairs spatial learning and memory ability and down-regulates Bcl-2 and BDNF mRNA expression in hippocampus of neonatal rats. Zhonghua Er Ke Za Zhi. 43, 444–448. Marcus, J.N., Aschkenasi, C.J., Lee, C.E., Chemelli, R.M., Saper, C.B., Yanagisawa, M., et al., 2001. Differential expression of orexin receptors 1 and 2 in the rat brain. J. Comp. Neurol. 435, 6–25. Marone, M., Mozzetti, S., Ritis, D.D., Pierelli, L., Scambia, G., 2001. Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. Biol. Proced. Online 3, 19–25. Mobarakeh, J.I., Takahashi, K., Sakurada, S., Nishino, S., Watanabe, H., Kato, M., et al., 2005. Enhanced antinociception by intracerebroventricularly and intrathecallyadministered orexin A and B (hypocretin-1 and -2) in mice. Peptides 26, 767–777. Moriarty, O., McGuire, B.E., Finn, D.P., 2011. The effect of pain on cognitive function: a review of clinical and preclinical research. Prog. Neurobiol. 93, 385–404. Morris, R.G.M., Rawlins, J., O’Keeffe, J., 1982. Place navigation impaired in rats with hippocampal lesions. Nature 297, 681–683. Moure-Leite, F.R., Ramos-Jorge, J., Ramos-Jorge, M.L., Paiva, S.M., Vale, M.P., Pordeus, I.A., 2011. Impact of dental pain on daily living of five-year-old Brazilian preschool children: prevalence and associated factors. Eur. Arch. Paediatr. Dent. 12, 293–297. Pais-Vieira, M., Lima, D., Galhardo, V., 2009. Sustained attention deficits in rats with chronic inflammatory pain. Neurosci. Lett. 463, 98–102. Raoof, M., Abbasnejad, M., Amirkhosravi, L., Ebrahimnejad, H., Raoof, R., 2012. A modification of a previous model for inflammatory tooth pain: effects of different capsaicin and formalin concentrations and ibuprofen. J. Oral Health Oral Epidemiol. 1, 7–15. Reneman, M.F., Versteegen, G.J., Huitema, R.B., 2014. The evidence for pain-related cognitive impairment. Pain 155, 645–646. Sears, R.M., Fink, A.E., Wigestrand, M.B., Farb, C.R., de Lecea, L., Ledoux, J.E., 2013. Orexin/hypocretin system modulates amygdala-dependent threat learning through the locus coeruleus. Proc. Natl. Acad. Sci. U.S.A. 110, 20260–20265. Selbach, O., Bohla, C., Barbara, A., Doreulee, N., Eriksson, K.S., Sergeeva, O.A., et al., 2010. Orexins/hypocretins control bistability of hippocampal long-term synaptic plasticity through co-activation of multiple kinases. Acta Physiol. (Oxf.) 198, 277–285. Seminowicz, D.A., Laferriere, A.L., Millecamps, M., Yu, J.S., Coderre, T.J., Bushnell, M.C., 2009. MRI structural brain changes associated with sensory and emotional
function in a rat model of long-term neuropathic pain. Neuroimage 47, 1007– 1014. Squire, L.R., 1992. Memory and the hippocampus: a synthesis from findings with rats, monkeys, and humans. Psychol. Rev. 99, 195–231. Sun, A.M., Li, C.G., Han, Y.Q., Liu, Q.L., Xia, Q., Yuan, Y.W., 2013. X-ray irradiation promotes apoptosis of hippocampal neurons through up-regulation of Cdk5 and p25. Cancer Cell Int. 13, 13–47. Tabata, S., Ozaki, H.S., Nakashima, M., Uemura, M., Iwamoto, H., 1998. Innervation of blood vessels in the rat incisor pulp: a scanning electron microscopic and immunoelectron microscopic study. Anat. Rec. 251, 384–391. Trivedi, P., Yu, H., MacNeil, D.J., Van der Ploeg, L.H., Guan, X.M., 1998. Distribution of orexin receptor mRNA in the rat brain. FEBS Lett. 438, 71–75. Erratum in: 1999 FEBS. Lett. 442, 122. Uddman, R., Björlin, G., Möller, B., Sundler, F., 1980. Occurrence of VIP nerves in mammalian dental pulps. Acta Odontol. Scand. 38, 325–328. Uddman, R., Grunditz, J., Sundler, F., 1984. Neuropeptide Y: occurrence and distribution in dental pulps. Acta Odontol. Scand. 42, 361–365. Upadhyay, J., Knudsen, J., Anderson, J., Becerra, L., Borsook, D., 2008. Noninvasive mapping of human trigeminal brainstem pathways. Mag. Reson. Med. 60, 1037–1046. van den Pol, A.N., 1999. Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J. Neurosci. 19, 3171–3182. Yamamoto, T., Nozaki-Taguchi, N., Chiba, T., 2002. Analgesic effect of intrathecally administered orexin-A in the rat formalin test and in the rat hot plate test. Br. J. Pharmacol. 137, 170–176. Yang, L., Zou, B., Xiong, X., Pascual, C., Xie, J., Malik, A., et al., 2013. Hypocretin/orexin neurons contribute to hippocampus-dependent social memory and synaptic plasticity in mice. J. Neurosci. 33, 5275–5284. Yang, L., Xin, X., Zhang, J., Zhang, L., Dong, Y., Zhang, Y., et al., 2014. Inflammatory pain may induce cognitive impairment through an interleukin-6-dependent and postsynaptic density-95-associated mechanism. Anesth. Analg. 119, 471– 480. Zhang, J.Q., Nagata, K., Iijima, T., 1998. Scanning electron microscopy and immunohistochemical observations of the vascular nerve plexuses in the dental pulp of rat incisor. Anat. Rec. 251, 214–220. Zhao, X., Zhang, R.X., Tang, S., Ren, Y.Y., Yang, W.X., Liu, X.M., et al., 2014. Orexin-Ainduced ERK1/2 activation reverses impaired spatial learning and memory in pentylenetetrazol-kindled rats via OX1R-mediated hippocampal neurogenesis. Peptides 54, 140–147.
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Neuropeptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n p e p
Changes in hippocampal orexin 1 receptor expression involved in tooth pain-induced learning and memory impairment in rats Ramin Raoof a,b, Saeed Esmaeili-Mahani a, Mehdi Abbasnejad a, Maryam Raoof b,c,*, Vahid Sheibani b, Razieh Kooshki a, Ladan Amirkhosravi a, Foroozan Rafie d a
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Laboratory of Molecular Neuroscience, Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran c Department of Endodontics, School of Dentistry, Kerman University of Medical Sciences, Kerman, Iran d Department of Motor Behaviour, Facaulty of Physical Education, Tehran University, Tehran, Iran b
A R T I C L E
I N F O
Article history: Received 4 July 2014 Accepted 2 March 2015 Available online 12 March 2015 Keywords: Tooth inflammatory pulpal pain Orexin 1 receptor Learning and memory Capsaicin Gene expression
A B S T R A C T
Orexin 1 receptor signaling plays a significant role in pain as well as learning and memory processes. This study was conducted to assess the changes in orexin 1 receptor expression levels in hippocampus following learning and memory impairment induced by tooth inflammatory pulpal pain. Adult male Wistar rats received intradental injection of 100 μg capsaicin to induce pulpal pain. After recording the pain scores, spatial learning and memory were assessed using Morris Water Maze test. The hippocampal levels of orexin 1 receptor mRNA and protein were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblotting respectively. The data showed that capsaicininduced tooth inflammatory pulpal pain was correlated with learning and memory impairment. Intrahippocampal injection of orexin A inhibited pain-induced learning and memory impairment. However, orexin 1 receptor antagonist, SB-334867, had no effect on learning and memory impairment. Moreover, capsaicin-induced pain significantly decreased hippocampal orexin 1 receptor mRNA and protein levels. Meanwhile, reversed changes took place in the ibuprofen-pretreated group (p < 0.05). It seems that decrease in orexin 1 receptor density and signaling could be involved in tooth pain-induced learning and memory impairment. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Orofacial pain is one of the most prevalent types of pain and odontalgia is the most commonly experienced one (Moure-Leite et al., 2011). Numerous studies have demonstrated the relationship between pain and changes in brain anatomy such as cortical thickness and gray matter density (Seminowicz et al., 2009). Moreover, neural systems involved in cognition and pain processing are closely linked, they may modulate one another reciprocally (Moriarty et al., 2011). So, cognitive function is thought to be affected in patients suffering pain. However, pain of trigeminal origin shows specific processing pathways and relay sites that make it different from pain originating from the anterolateral system (Upadhyay et al., 2008). Moreover, the episodes of pain, the thickness and pattern of the nerves, the size of terminal varicosities, and the length of intervaricose segments
* Corresponding author. Neuroscience Research Center, Jahad St., Kerman, Iran. E-mail address: [email protected] (M. Raoof). http://dx.doi.org/10.1016/j.npep.2015.03.002 0143-4179/© 2015 Elsevier Ltd. All rights reserved.
in dental pulp are quite different from that of other tissues (Zhang et al., 1998). Another clear difference is that the pulp vessels present rather rich calcitonin gene related peptide (CGRP) expressing nerve fibers but much less abundant substance P and neurokinin expressing nerve fibers, no vasoactive intestinal peptide (VIP) nerves, and few or no neuropeptide Y (NPY) expressing nerves (Kerezoudis et al., 1995; Uddman et al., 1980, 1984). Another unique feature of dental pulp is the direct innervation of both microvasculature and larger vessels by free nerve endings, suggesting that the local regulation of blood flow may take place not only at larger vessels but also at the level of the microvasculature in this tissue (Tabata et al., 1998). Learning and memory deficits have been reported in painful conditions (Hu et al., 2010; Yang et al., 2014). However, the investigation of the underlying mechanisms remains to be clarified. Orexin (A and B) and its receptors are widely distributed in the central nervous system. There are numerous reports indicating the antinociceptive effects of orexins in various animal models of pain, including trigeminovascular pain (Chiou et al., 2010). Orexins are antinociceptive at both spinal and supraspinal levels (Mobarakeh et al., 2005). Surprisingly, the antinociceptive effect of orexin-A is comparable to opioids (Yamamoto et al., 2002). This effect is
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opioid-independent and mainly mediated through orexin 1 receptors. Some animal studies suggest that endogenous orexins may be released during inflammatory pain states, or during some stress conditions, which may contribute to stress-induced analgesia (Chiou et al., 2010). Damage to the hippocampal structures has been also associated with learning and memory impairments (Kuhajda et al., 2002; Squire, 1992; Sun et al., 2013). Furthermore, hippocampal formation (CA1, CA2 and dentate gyrus) expresses orexin 1 receptors and receives orexinergic terminals. It has been shown that hippocampal orexin 1 receptor signaling has an important role in acquisition, consolidation and retrieval of spatial memory in Morris water maze (MWM) task (Akbari et al., 2007). Recently, Zhao et al. (2014) reported that orexin-A can attenuate the impairment of spatial learning and memory in PTZ-kindled rats through orexin 1 receptor-mediated signaling (Zhao et al., 2014). Since orexin 1 receptor signaling has a noticeable role in pain as well as learning and memory processes and the exact mechanisms of pain-induced learning and memory deficit have not yet been clarified, the present study was conducted to assess the role of hippocampal orexin 1 receptors and determine their expression levels in learning and memory impairment induced by tooth inflammatory pulpal pain.
2. Material and methods 2.1. Animals Adult male Wistar rats weighing 250–300 grams, purchased from the Neuroscience Research Center (Kerman University of Medical Sciences, Iran), were used in this study. The rats were housed (12-h light/dark cycle) one per cage in a room with a temperature of 23 ± 2 °C with unlimited access to standard rat chow and water before and during the study. Rats were randomly allocated into experimental groups, each comprising 6–7 animals. Animals that were used for the behavioral studies (n = 7) were different from animals that were used for molecular experiments (n = 6). All experimental procedures were approved by the Animal Research Ethics Committee of Kerman University of Medical Sciences, Kerman, Iran (Code: K/90/258).
2.2. Dental procedure Inflammatory pulpal pain induction was constructed as our modified model, representing a modification to the Chidiac study (Chidiac et al., 2002) described in a previous article (Raoof et al., 2012). In brief, the distal 2 mm of the rats’ mandibular incisors were cut off and special polyethylene crowns were fixed on the teeth using a flow composite resin (Tetric Flow, IvoclarVivadent). A small space remained between the tooth structure and the internal surface of the crown.
2.3. Drugs Capsaicin (Sigma-Aldrich, USA) was dissolved in Tween 80 – ethanol solution (Merck, Germany) (10% ethanol, 10% Tween 80, 80% distilled water, w/w) at a concentration of 10 mg/ml. Ten μl of capsaicin solution which contains 100 μg of drug was administrated intradentally (i.d.). Orexin A and SB-334867 (Tocris, London, UK) were dissolved in distilled water. Ibuprofen (Rouzdaru, Iran) powder was dissolved in a vehicle (2% Tween 80/distilled water) and given intragastrically (oral gavage) at a dose of 120 mg/kg (Raoof et al., 2012).
2.4. Experimental design The animals were randomly divided into five experimental groups (n = 7) as follows: 1: Control group, included intact animals. 2: Sham-operated group, which took the crown but received no injection. 3: Sham-vehicle group received i.d. injection of capsaicin vehicle for five days. 4: Capsaicin-treated group received capsaicin (100 μg, i.d.) for five consecutive days. 5: Ibuprofen-treated group received 120 mg/kg ibuprofen 20 min before capsaicin injection for five consecutive days. 6: Orexin A-treated group received orexin A (40 pM) 20 min before capsaicin injection for five consecutive days. 7: Orexin 1 receptor antagonist-treated group received 80 nM SB-334867 (Azhdari-Zarmehri et al., 2013) 20 min before capsaicin injection for five consecutive days. 2.5. Nociceptive behavior Test sessions were carried out during the light phase, between 09:00 a.m. and 13:00 p.m., in a quiet room maintained automatically at 23 ± 2 °C. Before drug injection, each animal was placed in the test box for a 30 min habituation period to minimize additional stress. The rats did not have access to food or water during the test. Immediately following the injection, each rat was placed back in the transparent Plexi glass box (25 × 35 × 35) with a transparent floor positioned over a mirror at an angle of 45° to allow for observation of nociceptive behavior. The rats’ behavior was observed for 21 minutes, divided into 7 blocks of 3 minutes. The person investigating the behavioral test was blinded to the group assignment. A pain score was determined for each block by measuring the number of seconds that the animal presented each of the following responses which represents the same scoring criteria as described previously (Chidiac et al., 2002): 0 – Calm, normal behavior such as grooming; 1– Abnormal head movements such as mild head shaking or continuous placement of the jaw on the floor or the wall of the cage; 2– Abnormal continuous shaking of the lower jaw; 3– Excessive rubbing of the mouth with foreleg movements, such as head grooming, but concentrated consistently and mainly on the lower jaw. A video camera was used to record the behavioral response (Raoof et al., 2012). 2.6. Morris water maze test The water maze test was used (Morris et al., 1982). Briefly, it was a black circular pool with a diameter of 136 cm and a height of 60 cm, filled with 20 ± 1 °C water to a depth of 25 cm. The maze was divided geographically into four equal quadrants and release points that were designed at each quadrant as N, E, S and W. A hidden circular platform (10 cm in diameter), made of Plexiglas, was located in the center of the southwest (target) quadrant, submerged 1.5 cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze consisting of geometric shapes on the walls, shelves, computer, a window, a door and posters. These were kept in fixed positions with respect to the swimming pool to allow the rat to locate the escape platform hidden below the water surface. After completion of training, the rats were returned to their cages and the retention test (probe trial) was performed 2 h later. In probe test, animals had 60 s free swim period without a platform and the time spent in the target quadrant was recorded. A video
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camera was mounted directly above the water maze to record the rats’ swim path. A tracking system was used to measure the distance each rat traveled, the percent of distance, and the time in each quadrant. 2.7. Tissue extraction and preparation The rats were anesthetized (exposed to a CO2 atmosphere) and decapitated and the brains were removed immediately. Brains were dissected along the sagittal midline, followed by bilateral removal of the hippocampus (Duric and McCarson, 2007). The hippocampus was immediately placed on ice in a glass petri dish. The dissected hippocampi from each rat were randomly distributed for further western blot and RT-PCR assays. Tissue samples were weighed and immediately frozen in liquid nitrogen and stored at −70 °C until assay (Kaeidi et al., 2011). 2.8. mRNA analysis Total cellular RNAs were isolated from the hippocampus by a modification of the guanidine isothiocyanate–phenol–chloroform method using RNX+ reagent (Ausubel et al., 2002). A semiquantitative RT-PCR method was used (Marone et al., 2001). Briefly, the RTPCR reaction was performed using Oligo-dT primer and M-MuLV reverse transcriptase, based on the manufacturer’s protocol (Fermentas GMBH, Germany). The reactions were incubated at 42 °C for 60 min and then inactivated at 70 °C for 10 min. Three separate PCR reactions were used for studying gene expression in the samples obtained from each rat. Each PCR reaction was carried out using selective forward and reverse primers for β-actin (as an internal standard) and orexin 1 receptor proteins. The sequence of the primers used was: orexin 1 receptor forward: 5′-AGG TGG ATG GAA GCG TGA AG-3′, orexin 1 receptor reverse: 5′-AGA GAT AAT CGC GCC ACA GG-3′, β-actin forward: 5′-CCC AGA GCA AGA GAG GCA TC-3′, β-actin reverse: 5′-CTC AGG AGG AGC AAT GAT CT-3′. Taq DNA polymerase (Cinaclon, Iran) used for DNA amplification and reactions were set up according to the manufacturer’s protocol. The PCR reactions were incubated at 94 °C for 5 min, followed by 25 cycles of thermal cycling (45 s at 94 °C, 45 s at 55 °C and 45 s at 72 °C). The final cycle was followed by a 5 min extension step at 72 °C. The reaction parameters were adjusted to obtain a condition with a linear relation between the number of PCR cycles and PCR products and with linear relation between the initial amount of cDNA template and PCR product. Based on the results obtained from these experiments, 25 cycles of PCR amplification were used for analyzing all samples. PCR products were subsequently analyzed on 1.5% agarose LMMP (Roche, Germany) gel and bands were quantified by densitometry using Lab Works analyzing software (UVP, UK). The possibility of the presence of contaminating genomic DNA was ruled out by using the yield of reverse transcriptase-minus (RT−) reaction, instead of cDNA template, which caused no DNA amplification (data not shown). Semiquantitative PCR technique was used to estimate orexin 1 receptor mRNA levels in tissue samples, normalized to an internal standard (Hajializadeh et al., 2010). 2.9. Protein analysis The dissected hippocampal tissues were homogenized using RIPA buffer, containing 10 mM trisaminomethane–HCL (Tris–HCl) (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl sulfate (SDS), 0.1% Na-deoxycholate, 1% NP-40 with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2.5 μg/ml of leupeptin, 10 μg/ml of aprotinin) and 1 mM sodium orthovanadate. The homogenate was centrifuged at 14,000 rpm at 4 °C for 15 min. The resulting supernatant was retained as the whole cell fraction. Protein
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concentrations were measured using the Bradford method (BioRad Laboratories, Muenchen, Germany). Equal amounts of proteins of the sample were separated according to molecular weight using SDS–PAGE gel electrophoresis (Cinaclone, Iran) and transferred to PVDF membrane. After blocking with 5% non-fat dried milk in Trisbuffered saline with Tween 20 (blocking buffer, TBS-T, 150 mM NaCl, 20 mM Tris–HCl, pH 7.5, 0.1% Tween 20) the membrane was probed for the protein of interest with specific antibodies. All antibodies were diluted in blocking buffer. A dilute solution of primary antibody to orexin 1 receptor (sc8073; Santa Cruz biotechnology, USA, 1:1000) was incubated with the membrane under gentle agitation (overnight at 4°C for). After washing in TBS-T (three times, 5 min), the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (1:15,000, Santa Cruz biotechnology, USA). The incubation time for orexin 1 receptor antibody was 3 hours at room temperature. The antibody–antigen complexes were detected using the Electrochemiluminescence-Advance system (ECL-Advance system) and exposed to Lumi-Film chemiluminescent detection film (Roch, Germany) (Kaeidi et al., 2011). For ensuring the loading of proteins, the amount of β-actin protein was evaluated. After digitizing the films, the images of bands were evaluated using Lab Work analyzing software (UVP, UK) (Kaeidi et al., 2011). 2.10. Statistical analysis All statistical analyses were carried out by an observer blinded to the experimental groups. Data are presented as mean ± standard error of mean (S.E.M.). Differences between groups regarding pain scores and learning and memory indices were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. The differences in the amount of orexin 1 receptor mRNA and protein levels between groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. P < 0.05 was considered significant. 3. Results 3.1. Assessment of recorded pain scores in the control (intact, sham-operated and sham-vehicle), capsaicin- and ibuprofen-treated groups There were no significant differences in baseline nociceptive behaviors [F(2,18) = 2.949, P = 0.0780] between intact, sham-operated (took crown but received no injection) and sham-vehicle (received i.d. injection of capsaicin vehicle) animals (Fig. 1). In addition, intradental application of capsaicin (100 μg/rat) significantly increased pain scores [F(4,30) = 29.761, P = 0.0001]. In ibuprofenpretreated rats (Caps + Ibup), capsaicin-induced nociception was decreased (Fig. 1). 3.2. Assessment of spatial learning and memory in experimental groups 3.2.1. Hidden platform trials There was no significant difference between intact, sham and sham-vehicle animals as control groups in time to find the platform [F(2,18) = 1.658, P = 0.2183]. Capsaicin injection significantly increased time to find the platform [F(6,42) = 12.119, P = 0.0001] which was decreased by 40 pM orexin A or ibuprofen (120 mg/kg, i.g.) pretreatment (Fig. 2). As shown in Fig. 3, there was no significant difference between control, sham-operated and sham-vehicle groups in traveled distance to reach the platform [F(2,18) = 3.239, P = 0.0629]. Traveled distance was significantly increased in capsaicin-treated (Caps) animals which was reversed by orexin A (Caps + Orex) or ibuprofen
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Fig. 1. Comparison of recorded dental pain score in different experimental groups. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. Each value in the graph represents the mean ± SEM. ***P < 0.001 versus control (intact, sham and sham-vehicle) groups. +++P < 0.001 versus capsaicintreated (Caps) animals.
(Caps + Ibup) pretreatment [F(6,42) = 6.610, P = 0.0001]. In addition, the increase in traveled distance (P < 0.05) was also observed in animals that received capsaicin plus 80 nM SB-334867 (Caps + SB). Fig. 4 depicts typical swimming tracks of the rats in the Morris water maze test. The animals in the capsaicin-treated group often searched for the platform in an inappropriate way resulting in the longer latency to locate the platform. The effect was attenuated by orexin or ibuprofen treatment. Furthermore, one-way ANOVA showed that there were no significant differences [F(6,42) = 2.178, P = 0.0001] between swim speeds in different experimental groups (Fig. 5). 3.2.2. Probe trials Fig. 6 shows the retention performance on the probe test trial for experimental groups. The mean of time spent in the target quadrant during the 60 s probe test was significantly decreased in capsaicin-treated rats as compared to the control groups
[F(3,27) = 8.422, P = 0.0006]. Orexin A or ibuprofen pretreatment significantly prevented the effect of capsaicin on time spent in the target quadrant [F(6,42) = 6.718, P = 0.0001]. However in SB-334867pretreated rats the mean value was closed to those observed in control rats (P > 0.05). 3.3. Effect of tooth inflammatory pulpal pain on hippocampal orexin 1 receptor mRNA and protein levels The expression of orexin 1 receptor mRNA and protein in the control (intact, sham-operated and sham vehicle-treated) groups were the same (data not shown). The levels of orexin 1 receptor mRNA (Fig. 7) and protein (Fig. 8) in hippocampus of capsaicintreated rats were significantly lower than those observed in control (vehicle-treated sham) rats (P < 0.01 and P < 0.001 respectively). However, in ibuprofen-pretreated rats, orexin 1 receptor mRNA and protein levels were closed to the control levels (p > 0.05).
Fig. 2. Comparison of time between study groups. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. Each value in the graph represents mean ± SEM. ***p < 0.001 versus intact and sham-operated (Sham) groups. ##p < 0.01 and ###p < 0.001 versus sham-vehicle group. ++P < 0.01 and +++P < 0.001 versus capsaicin-treated (Caps) animals.
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Fig. 3. Comparison of traveled distance between study groups. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. Each value in the graph represents mean ± SEM. *P < 0.05 versus control (intact, sham and sham-veh) groups. + P < 0.05 and ++P < 0.01 versus capsaicin-treated animals.
Fig. 4. Typical swimming tracks of the rats in the Morris water maze test. The animals in capsaicin-treated group often searched for the platform in an inappropriate way. The effect was attenuated by orexin or ibuprofen treatment.
Fig. 5. Comparison of speed between study groups. One-way analysis of variance showed there were no significant differences. Each value in the graph represents mean ± SEM (n = 7).
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Fig. 6. Comparison of the time spent in the target quadrant during the 60 s probe test. One-way analysis of variance was used to compare the multiple group means followed by Tukey’s test. The columns represent the mean ± S.E.M. *p < 0.05 versus intact and sham-operated (Sham) and vehicle-treated sham (Sham-Veh) groups. ++P < 0.01 and +++P < 0.001 versus capsaicin-treated (Caps) animals.
4. Discussion In the present study, intradental injection of capsaicin produced a significant pain response score, which was inhibited by ibuprofen. It has been demonstrated that activation of capsaicin-sensitive fibers in
Fig. 7. The levels of orexin 1 receptor mRNA in control (Cont), capsaicin-treated (Caps) and ibuprofen-pretreated (Caps + Ibup) rats. One-way analysis of variance was used. Values represent mean ± SEM (n = 6). ** P < 0.01 versus control group. + P < 0.05 versus capsaicin-treated group.
Fig. 8. The levels of orexin 1 receptor protein in control (Cont), capsaicin-treated (Caps) and ibuprofen-pretreated (Caps + Ibup) rats. One-way analysis of variance was used. Values represent mean ± SEM (n = 6). *** P < 0.001 versus control group. +++ P < 0.001 versus capsaicin-treated group.
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dental pulp increases pulpal blood flow and produces changes in escape behavior, suggesting a thermal and mechanical hyperalgesia (Kupers et al., 1997). The capsaicin-induced pain affected the neural processing in diencephalon and telencephalon; for example, the spontaneous firing of neurons in the primary somatosensory cortex and the ventral posterior medial nucleus of the thalamus was increased by orofacial capsaicin injection (Katz et al., 1999). Surprisingly, intradental capsaicin-treated rats spent more time and distance to find the hidden platform than control animals in the MWM test, which was attenuated by ibuprofen pretreatment. When the platform was removed for the probe trial, capsaicintreated animals spent significantly less time in the training quadrant than control ones. On the other hand, capsaicin had no significant effect on motor performance as measured by swim speed. Furthermore, visual platform performance did not differ between different groups. It seems likely that capsaicin-related spatial learning impairment is not related to the changes in motivation and/or sensory functions. Learning and memory impairment induced by intradental injection of capsaicin showed a positive correlation with pain score. It has been also reported that chronic pain stress significantly impairs spatial learning and memory function in neonatal rats (Li et al., 2005). Pais-Vieira et al. have shown that chronic inflammatory pain and monoarthritis causes attention impairment that could not be reversed by subcutaneous injection of carprofen (Pais-Vieira et al., 2009). So, there is sufficient evidence from preclinical and clinical investigations to support the theory that pain is associated with impaired cognitive function. This impairment may have a noticeable impact on patients’ quality of life. There is also some evidence for a mechanistic, neuropathological basis for pain-related cognitive impairment (Reneman et al., 2014). There is a considerable overlap between the neuroanatomical and neurochemical substrates implicated in both pain and cognition (Moriarty et al., 2011). It has been reported that orexin receptors, which have antinociceptive signaling at both spinal and supraspinal levels, are significantly expressed in the hippocampus (Chiou et al., 2010). Orexin is distributed in the periaqueductal gray matter (PAG), raphe nuclei, nucleus locus coeruleus and superficial and profound layers of the spinal dorsal horn; all of these areas are involved in nociceptive pathways (Trivedi et al., 1998; van den Pol, 1999). This study showed that the expression level of orexin 1 receptor is reduced in the hippocampus of rats that had pulpal pain and impaired learning and memory. The expression pattern of orexin receptors in rat brain has been previously reported (Cluderay et al., 2002; Marcus et al., 2001). Orexin 1 receptor immunoreactivity showed that the cerebral neocortex, basal ganglia, hippocampal formation, and many other regions in the hypothalamus, thalamus, midbrain and reticular formation express orexin 1 receptors (Marcus et al., 2001). However, since immunohistochemistry is also helpful to estimate the tissue protein expression levels further studies are needed to confirm the different expression levels of orexin 1 receptor in the hippocampus of capsaicin-treated animals. It has been demonstrated that the orexinergic system has an important role in learning and memory processes and induces structural changes in the hippocampus and related structures. Orexin neurons play an important role in the consolidation of social recognition memory through enhancements of hippocampal synaptic plasticity (Yang et al., 2013). Our results (Figs. 2–5) indicate that intrahyppocampal injection of orexin attenuates learning and memory impairment in capsaicin-treated rats. However, the orexin-1 receptor antagonist, SB-334867, has been shown to reverse cognitive deficits. The effects appear to be mediated by the orexin-A receptor. Despite orexin-B, Orexin-A induces a state-dependent longterm potentiation of Schaffer collateral-CA1 synapses in the adult mouse hippocampal slices (Selbach et al., 2010). Akbari et al. also demonstrated that functional inactivation of orexin 1 receptors in
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CA1 region impairs Morris water maze performance (Akbari et al., 2006). Surprisingly, orexin-A improved memory retention in both young and old SAMP8 mice through cholinergic, GABAergic and serotonergic pathways (Jaeger et al., 2002). There are numerous pieces of evidence that there is an interaction between orexin-A and neuropeptide Y that may contribute to appetite regulation. If orexin-A is able to interact with NPY to regulate feeding behaviors, then this interaction may also be responsible for the regulation of other physiological processes, such as memory consolidation (Jaeger et al., 2002). In addition, the nucleus locus coeruleus, which plays a significant role in pain, consciousness, learning and memory, receives orexinergic neurons. It seems dysregulation of the orexin system may contribute to the etiology of cognitive disorders (Sears et al., 2013). As mentioned above, orexin receptors are involved in nociceptive pathways as well as learning and memory. Our data showed that orexin 1 receptor expression significantly decreased in the hippocampus as one of the most important regions in learning and memory. So maybe orexin 1 receptor down-regulation could be one reason for learning and memory impairment caused by tooth inflammatory pulpal pain. However, the underlying mechanisms need to be clarified by further investigations. Acknowledgments This work was supported by funds from Neuroscience Research Center and Kerman University of Medical Sciences (grant no: 93-26). References Akbari, E., Naghdi, N., Motamedi, F., 2006. Functional inactivation of orexin 1 receptors in CA1 region impairs acquisition, consolidation and retrieval in Morris water maze task. Behav. Brain Res. 173, 47–52. Akbari, E., Naghdi, N., Motamedi, F., 2007. The selective orexin 1 receptor antagonist SB-334867-A impairs acquisition and consolidation but not retrieval of spatial memory in Morris water maze. Peptides 28, 650–656. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., et al., 2002. Short Protocols in Molecular Biology. Wiley, USA. Azhdari-Zarmehri, H., Esmaeili, M.H., Sofiabadi, M., Haghdoost-Yazdi, H., 2013. Orexin receptor type-1 antagonist SB-334867 decreases morphine-induced antinociceptive effect in formalin test. Pharmacol. Biochem. Behav. 112, 64–70. Chidiac, J.J., Rifai, K., Hawwa, N.N., Massaad, C.A., Jurjus, A.R., Jabbur, S.J., et al., 2002. Nociceptive behaviour induced by dental application of irritants to rat incisors: a new model for tooth inflammatory pain. Eur. J. Pain 6, 55–67. Chiou, L.C., Lee, H.J., Ho, Y.C., Chen, S.P., Liao, Y.Y., Ma, C.H., et al., 2010. Orexins/ hypocretins: pain regulation and cellular actions. Curr. Pharm. Des. 16, 3089– 3100. Cluderay, J.E., Harrison, D.C., Hervieu, G.J., 2002. Protein distribution of the orexin-2 receptor in the rat central nervous system. Regul. Pept. 104, 131–144. Duric, V., McCarson, K.E., 2007. Neurokinin-1 (NK-1) receptor and brain-derived neurotrophic factor (BDNF) gene expression is differentially modulated in the rat spinal dorsal horn and hippocampus during inflammatory pain. Mol. Pain 3, 32–40. Hajializadeh, Z., Esmaeili-Mahani, S., Sheibani, V., Kaeidi, A., Atapour, M., Abbasnejad, M., 2010. Changes in the gene expression of specific G-protein subunits correlate with morphine insensitivity in streptozotocin-induced diabetic rats. Neuropeptides 44, 299–304. Hu, Y., Yang, J., Hu, Y., Wang, Y., Li, W., 2010. Amitriptyline rather than lornoxicam ameliorates neuropathic pain-induced deficits in abilities of spatial learning and memory. Eur. J. Anaesthesiol. 27, 162–168. Jaeger, L.B., Farr, S.A., Banks, W.A., Morley, J.E., 2002. Effects of orexin-A on memory processing. Peptides 23, 1683–1688. Kaeidi, A., Esmaeili-Mahani, S., Sheibani, V., Abbasnejad, M., Rasoulian, B., Hajializadeh, Z., et al., 2011. Olive (Olea europaea L.) leaf extract attenuates early diabetic neuropathic pain through prevention of high glucose-induced apoptosis: in vitro and in vivo studies. J. Ethnopharmacol. 136, 188–196. Katz, D.B., Simon, S., Moody, A., Nicolelis, M.A., 1999. Simultaneous reorganization in thalamocortical ensembles evolves over several hours after perioral capsaicin injections. J. Neurophysiol. 82, 963–977. Kerezoudis, N.P., Fried, K., Olgart, L., 1995. Haemodynamic and immunohistochemical studies of rat incisor pulp after denervation and subsequent re-innervation. Arch. Oral Biol. 40, 815–823. Kuhajda, M.C., Thorn, B.E., Klinger, M.R., Rubin, N.J., 2002. The effect of headache pain on attention (encoding) and memory (recognition). Pain 97, 213–221.
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