Pharmacology, Biochemistry and Behavior 122 (2014) 286–290
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Orexin-A microinjection into the rostral ventromedial medulla causes antinociception on formalin test☆,☆☆ Hassan Azhdari-Zarmehri a,b, Saeed Semnanian b,⁎, Yaghoub Fathollahi b a b
Department of Basic Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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Article history: Received 18 May 2013 Received in revised form 19 March 2014 Accepted 20 March 2014 Available online 29 March 2014 Keywords: Orexin-A Rostral ventromedial medulla Orexin receptor 1 Formalin test
a b s t r a c t Orexins are produced from neurons which are restricted to a few regions of the lateral hypothalamus (LH), where they are important in pain modulation. The orexin receptors and orexinergic projections are localized in regions previously shown to play a role in pain modulation such as rostral ventromedial medulla (RVM). The effect of orexin-A (ORXA) microinjection into the RVM on nociceptive behaviors was examined using the formalin test. Microinjection of ORXA into the RVM, but not adjacent reticularis gigantocellularis (Gi) nucleus, decreased formalin induced nociceptive behaviors. Pretreatment with a selective OX1R antagonist, SB-334867 inhibited the antinociception produced by ORXA, while the administration of SB-334867 alone had no effect. These data demonstrate that ORXA-induced antinociception in the formalin test is mediated in part through orexin1 receptors in the RVM. © 2014 Published by Elsevier Inc.
1. Introduction Orexin-A (ORXA, hypocretin-1) and orexin-B (ORXB, or hypocretin2) are produced from the precursor protein prepro-orexin in neurons which are restricted to a few regions of the lateral hypothalamus (LH) (Peyron et al., 1998; Sakurai et al., 1998). ORXA and ORXB activate two G-protein coupled receptors, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) (Sakurai et al., 1998). The orexin receptors and orexinergic projections are localized in regions previously shown to play a role in pain modulation such as periaqueductal gray (PAG), locus coeruleus, and rostral ventromedial medulla (RVM) (Mondal et al., 1999; Peyron et al., 1998). Furthermore, electrical or chemical stimulation of the LH has been shown to induce excitation of PAG neurons and this stimulation produced an increase in tail flick latency. The lateral hypothalamus (LH) appears to modify nociception, in part, through the PAG and rostral ventromedial medulla (RVM) (Behbehani et al., 1988; Holden and Pizzi, 2008). It has been found that orexins have a wide variety of functions such as the regulation of appetite (Sakurai et al., 1998; Yamamoto et al., 1999), involvement in arousal (Chemelli et al., 1999; Lin et al., 1999),
and central control of autonomic activity, stress and addiction (Erami et al., 2012b; Harris et al., 2005; Heidari-Oranjaghi et al., 2012; Narita et al., 2006). Several lines of evidence imply that ORX also is involved in nociceptive sensory processes (Azhdari-Zarmehri et al., 2011; Bingham et al., 2001; Cheng et al., 2003; Erami et al., 2012a; Mobarakeh et al., 2005; Sadeghi et al., 2013; Yamamoto et al., 2002). In addition, the administration of ORXA has been shown to induce naloxone-insensitive antinociception effects as a result of spinal and/or supraspinal OX1R binding (Bingham et al., 2001; Yamamoto et al., 2002). However, the role of ORXA within the RVM has not been identified despite previous research showing that the antinociception induced by activating neurons in the LH is mediated in part by activation of spinally-projecting neurons from the RVM (Holden et al., 2005; Holden and Pizzi, 2008; Vaccarino and Chorney, 1994). Therefore, in this study, the effect of ORXA microinjection into RVM and adjacent reticularis gigantocellularis (Gi) on formalin induced nociceptive behaviors was investigated. Also, in order to investigate the role of OX1R in the RVM, the effect of the selective OX1R antagonist on antinociceptive effect of ORXA was also examined. 2. Materials and methods
☆ This research was supported by a grant from the INSF, Tehran, Iran. ☆☆ We have no financial or other conflicts of interest. ⁎ Corresponding author at: Department of Physiology, School of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-116, Tehran, Iran. E-mail address: [email protected] (S. Semnanian).
http://dx.doi.org/10.1016/j.pbb.2014.03.017 0091-3057/© 2014 Published by Elsevier Inc.
2.1. Subjects All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996) and were approved by the
H. Azhdari-Zarmehri et al. / Pharmacology, Biochemistry and Behavior 122 (2014) 286–290
Ethics Committee of the School of Medical Sciences, Tarbiat Modares University (TMU), Tehran, Iran. Adult male Sprague–Dawley rats (n = 52, 220–300 g) were purchased from Razi Institute (Karaj, Iran). Animals were housed in groups of three rats per cage in a temperature-controlled room, under a 12 h light–dark cycle with lights on from 7:00 to 19:00. Food and water were provided ad libitum. In all experiments, strict attention was paid to the regulations of local authorities for handling of laboratory animals. 2.2. Surgical preparation for intra-nucleus microinjections To perform direct intra-nucleus administrations of drugs or respective vehicle, an intracranial guide cannula was implanted 7 days before the experiments. Rats were anesthetized with ketamine (100 mg/kg)/ xylazine (10 mg/kg) and a 23-gauge, 8.5 mm-long stainless steel guide cannula (Narishige, Japan) was stereotaxically lowered until its tip was 2 mm above the RVM or Gi according to coordinates from the atlas of Paxinos and Watson (2005) [Gi; A, − 11. 2 to − 11. 8 mm from bregma; L, ± 1 mm; V, 9 mm below the bregma and RVM; A, −11. 2 to −11. 8 from bregma; L, midline; V, 10. 5 mm below the bregma]. The cannula was anchored with dental cement to two stainless steel screws (2 pieces) in the skull. Immediately after waking from surgery, rats were returned to their home cages to recover for 7 days. Direct intra-nucleus administration of drugs or the respective vehicle was done using a stainless steel injection cannula (30 G, 0. 3 mm outer diameter) connected by a polyethylene tube to a Hamilton syringe. The injection cannula was extended 2 mm beyond the tip of the guide cannula to reach the nucleus. Drug solutions and vehicles were injected manually in a volume of 0.5 μl over a period of 60 s. The injection cannula was gently removed 1.5 min later. During microinjections, animals were free to move around the cage with the cannula in place. The microinjection was verified by monitoring the movement of an air bubble along the polyethylene tube. After 5 min, formalin (2%) was injected into the plantar surface of the right hind paw using a disposable syringe with a 30 G needle.
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of animals injected with drug (post-drug) or saline (post-saline). The following formula was used:
%MPE ¼
post‐saline‐post‐drug 100: post‐saline
2.4. Histology At the end of each test session, rats were given an overdose of Ketamine or urethane (N 1.2 g/kg) followed by microinjection of 0.5 μl of pontamine sky blue (0.2%) into the RVM or Gi (10–20 min before sacrificing the animals). Rats were perfused intracardially with saline and then 4% formalin solution and the brain was removed and sectioned. The cerebellum was removed, and the brain stem block was fixed by cyanoacrylate glue to a slicing tray. Coronal slices were prepared with 300–400 μm thickness until the darkness of the pontamine sky blue injection could be seen, then subsequent sections were cut with a thickness of 50–60 μm. Slices were cut through the brain stem from the trapezoid body to the inferior olivary nuclei using a vibrating microtome (Vibatome 1000 plus, US). Injection sites in the RVM were visualized in the triangular midline region dorsal to the pyramidal tracts and visually identified under a microscope. Only those rats with microinjection site and diffusion areas located within the nucleus were included in the results (Fig. 1, data from 100 nM ORXA are shown). 2.5. Data analysis Data were presented as mean ± SEM. The formalin pain scores in all groups were subjected to one-way ANOVA followed by protected Dunnett/Tukey's tests for multiple comparisons as needed. Each of the three phases of the formalin test (phase 1: 0 to 7 min), interphase (8 to 14 min), and phase 2 (15 to 60 min). The defined level for statistical significance was P b 0. 05.
2.3. Formalin test Seven days after surgery, rats were moved to the test room at least 1 h before the commencement of the experiment. The formalin test was performed in clear plastic boxes (30 × 30 × 30 cm) with a mirror placed underneath at a 45° angle to allow an unimpeded view of the animals' paws. Rats were acclimatized to this chamber for 30 min before intranucleus microinjection of ORXA or vehicle followed 5 min later by subcutaneous injection of 50 μl of 2% formalin into the plantar surface of the right hind paw. Formalin was administered via a 30 gauge needle inserted 5 mm under the skin. Each rat was immediately returned to the observation box for behavioral recording of nociception. Pain behaviors were scored as follows: 0, the injected paw was not favored,; 1, the injected paw had little or no weight placed on it with no toe splaying; 2, the injected paw was elevated and not in contact with any surface; and 3, the injected paw was licked or bitten. Recording of the nociceptive behaviors was started immediately after formalin injection (time 0) and continued for 60 min. The scores of nociceptive behaviors for each 3-minute interval were calculated as the weighted average of the number of seconds engaged in each behavior. The behavioral responses of each rat were evaluated separately for the first phase (1–7 min), inter-phase (8–14 min) and the second phase (15–60 min). The same procedure was followed in Experiment 2 except that the OX1R antagonist, SB-334867, was administered 5 min before ORXA. SB-334867 was dissolved in DMSO on the day of the experiment and then diluted in saline before use. The percentage of maximum possible effect (%MPE) in the formalin test was calculated as the percentage difference between the measured nociceptive behaviors during the late phase (15–60 min) for the group
Fig. 1. Location of injections in the RVM and Gi plotted as percentage of maximum possible effect (MPE%): Black squares for 0–10%, black circles for 10–25%, white circles for 25–50%, Black triangles for 50–75%, and white triangle for an effect higher than 75%. The plane is modified to a series of three sections for the brain areas from the atlas of Paxinos and Watson (2005). The approximate coordinate is indicated in millimeters posterior to bregma (data from 100 nM ORXA shown). A number of injections were lateral in the Gi-alpha, which is a part of the RVM.
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3. Results 3.1. Effects of ORXA injection into the RVM nucleus and the Gi region on formalin-induced nociceptive behaviors Intra-RVM microinjection of ORXA (100 nM) caused a decrease in formalin induced nociceptive behaviors in phases 1 and 2 compared to vehicle treated rats (phase 1 [T (1, 17) = 4.195, P = 0.001], interphase [T (1, 17) = 2.210, P = 0.041], and phase 2 [T (1, 17) = 8.009, P = 0.000]; (Fig. 2B1)). Intra-Gi microinjection of ORXA (100 nM, 0.5 μl) failed to show any significant change in formalin induced nociceptive behaviors in phase 1 [T (1, 20) = 0.761, P = 0.455], interphase [T (1, 20) = 1.393, P = 0.179], or phase 2 [T (1, 20) = 0.431, P = 0.671] (Fig. 2B2). 3.2. Effects of SB-334867 (OX1R antagonist) microinjection into the RVM regions on ORXA-induced attenuation of nociceptive behaviors in formalin test Pre-treatment with SB-334867 (100 μM/0. 5 μl) partially antagonized the effect of ORXA induced antinociceptive behaviors in the RVM ((for phase 1 [F (2, 20) = 16.042, P = 0.000]; for interphase: [F (2, 20) = 3.304, P = 0.058]; for phase 2 [F (2, 20) = 41.446, P = 0.000]); Fig. 3). Intra RVM microinjection of SB-334867 had no effect on phase 1, inter-phase, and phase 2 in the formalin test compared with the vehicle injected rats in RVM (for phase 1: 1.80 ± 0.14: 1.73 ± 0.06, P N 0.05; for interphase: 0.93 ± 0.18: 0.95 ± 0.16, P N 0.05; for phase 2: 1.81 ±0.08: 1.87 ± 0.05, P N 0.05). 4. Discussion In this study, we showed that the microinjection of ORXA into RVM has an antinociceptive effect on the formalin test, but no effect when microinjected into Gi. The lack of antinociception following ORXA
injection into Gi suggests that the antinociception obtained by microinjection into the RVM cannot be due to the diffusion of ORX into Gi areas. However, our previous study showed that areas immediately adjacent to the RVM may support ORXA antinociception (Erami et al., 2012a). We should emphasize that drug diffusion is a minor limitation for this type of experimental protocol. Injection spread is affected by the molecular weight, concentration (Myers and Hoch, 1978), and water solubility of the solution (Sakai et al., 1979). The fact that microinjections in the Gi did not produce antinociception is evidence that diffusion does not go very far from the microinjection site because the RVM in only 2 mm away. Pretreatment with a selective OX1R antagonist (SB-334867) partially antagonized the ORXA induced antinociception produced by intraRVM injection of ORXA, indicating that the antinociceptive effect of ORXA is mediated, at least in part, through OX1R. The formalin test is a widely used animal model of unrelieved pain (Dubuisson and Dennis, 1977). Formalin as a chemical noxious stimulus induces distinct nociceptive behaviors that have two phases which possibly reflect different types of pain (Abbott et al., 1995; Hunskaar et al., 1986; Hunskaar and Hole, 1987; Tjolsen et al., 1992) and sensitive to two different pain modulating mechanisms (Vaccarino and Chorney, 1994). In this study, the selective OX1R antagonist SB-334867 antagonized the antinociceptive effect of ORXA during phase 2 of the formalin test and partially antagonized the antinociceptive effect during the first phase. The difference between SB-334867 administration on the antinociceptive effect of ORXA during the first and second phases of the formalin test may well be due to the mechanisms by which formalin induces nociceptive behaviors in both phases. Previous studies have shown that (a) ORXA projections and OX1Rs are found in the brainstem (Ciriello et al., 2003; Marcus et al., 2001; Mondal et al., 1999; Peyron et al., 1998; Trivedi et al., 1998), (b) ORXA produces excitatory effects on neurons in the RVM (Azhdari Zarmehri et al., 2010; Dun et al., 2000; Vaccarino and Chorney, 1994); and (c) some evidence support that the antinociception produced by activating neurons in the lateral hypothalamus is mediated in part by the subsequent activation of spinally-projecting neurons in the RVM (Holden
Fig. 2. Time course for formalin induced nociceptive behavior score (±S. E. M.) following intra-RVM (n = 9) and Gi (n = 10) infusion of ORXA (100 nM, 0.5 μl,) measured every 3 min for 60 min (A). The columns represent the mean nociceptive scores in each phase: phase 1 (1–7 min), inter-phase (8–14 min), and phase 2 (15–60 min) (B). All microinjections in the midline raphe magnus and lateral in the Gi-alpha are included in the RVM and those outside of RVM are included in Gi. **P b 0.01; ***P b 0.001 compared with saline group.
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Fig. 3. Time course of formalin induced nociceptive behavior score (±S. E. M.) following infusions of the vehicle (0.5 μl) + ORXA (100 nM / 0.5 μl) (n = 9), SB-334867 (100 μM / 0.5 μl) + vehicle (0.5 μl) (n = 6) and SB-334867 (100 μM / 0.5 μl) + ORXA (100 nM / 0.5 μl) (n = 7) into the RVM (A) measured every 3 min for 60 min. The columns represent the mean nociceptive scores in each phase: phase 1 (1–7 min), inter-phase (8–14 min), and phase 2 (15–60 min) (B). *P b 0.05; ***P b 0.001 compared to vehicle group.
et al., 2005; Holden and Pizzi, 2008; Vaccarino and Chorney, 1994), a region known to play a role in pain modulation (Fields and Heinricher, 1985; Heinricher et al., 1989). These findings support our observations showing the involvement of ORXA in modulating pain in the RVM. Ciriello et al. found that the distribution of ORXA-labeled axons varies within the specific regions of the magnocellular reticular area (Ciriello et al., 2003). ORXA axon labeling was observed mainly within the GiA and to a lesser extent within the Gi and RVM (Ciriello et al., 2003). However, there are significant differences among the sites that mediated the ORXA induced analgesia on the formalin test. As previously reported, ORXA injections directly into the GiA elicit a decrease in muscle tone and cardioacceleration (Ciriello et al., 2003; Mileykovskiy et al., 2002). The present findings show that ORXA microinjection into the RVM caused antinociception on the formalin test while considering the possibility that the origin of the orexinergic inputs to these structures might be involved in top-down pain modulation. The RVM may influence neurons in the dorsal horn of the spinal cord which are involved in the neurotransmission of noxious stimuli (Vanegas and Schaible, 2004; Zhuo and Gebhart, 1997). Because the LH is the exclusive area to express ORX and implicated as part of a descending system associated with the modulation of nociceptive transmission in the first synapse in dorsal horn neuron of the spinal cord, the antinociception mediated by the LH appears to occur via activation of brainstem nuclei (Behbehani et al., 1988; Dafny et al., 1996). We suggest that ORX might act as a mediator between LH and RVM in modulating pain. Consistent with other reports using the formalin and hot plate tests (Yamamoto et al., 2002), pretreatment with selective OX1R (SB334867) antagonized the antinociceptive behaviors of ORXA in the RVM, demonstrating likely, the potential importance of OX1R in the modulation of pain. Our data corroborate the previous studies in which SB-334867, a selective antagonist of OX1R, was demonstrated to restore the antinociception effect of ORXA after ICV administration in mice and rats (Bingham et al., 2001; Yamamoto et al., 2002). It has been suggested that the antinociceptive effects of ORXA are mediated both via the spinal and supraspinal mechanisms. In conclusion, ORXA-induced antinociception in the formalin test is mediated in part through OX1R in the RVM, although other structures may be involved. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was supported by a grant from INSF, Tehran, Iran. We thank Dr. Ali A. Pahlevan (Iran) and Professor Morgan (Washington State University Vancouver, Vancouver) and Mary M. Heinricher,
(Oregon Health & Science University) for their meticulous work on revision of the final English version of the manuscript.
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Orexin-A microinjection into the rostral ventromedial medulla causes antinociception on formalin test☆,☆☆ Hassan Azhdari-Zarmehri a,b, Saeed Semnanian b,⁎, Yaghoub Fathollahi b a b
Department of Basic Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 18 May 2013 Received in revised form 19 March 2014 Accepted 20 March 2014 Available online 29 March 2014 Keywords: Orexin-A Rostral ventromedial medulla Orexin receptor 1 Formalin test
a b s t r a c t Orexins are produced from neurons which are restricted to a few regions of the lateral hypothalamus (LH), where they are important in pain modulation. The orexin receptors and orexinergic projections are localized in regions previously shown to play a role in pain modulation such as rostral ventromedial medulla (RVM). The effect of orexin-A (ORXA) microinjection into the RVM on nociceptive behaviors was examined using the formalin test. Microinjection of ORXA into the RVM, but not adjacent reticularis gigantocellularis (Gi) nucleus, decreased formalin induced nociceptive behaviors. Pretreatment with a selective OX1R antagonist, SB-334867 inhibited the antinociception produced by ORXA, while the administration of SB-334867 alone had no effect. These data demonstrate that ORXA-induced antinociception in the formalin test is mediated in part through orexin1 receptors in the RVM. © 2014 Published by Elsevier Inc.
1. Introduction Orexin-A (ORXA, hypocretin-1) and orexin-B (ORXB, or hypocretin2) are produced from the precursor protein prepro-orexin in neurons which are restricted to a few regions of the lateral hypothalamus (LH) (Peyron et al., 1998; Sakurai et al., 1998). ORXA and ORXB activate two G-protein coupled receptors, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) (Sakurai et al., 1998). The orexin receptors and orexinergic projections are localized in regions previously shown to play a role in pain modulation such as periaqueductal gray (PAG), locus coeruleus, and rostral ventromedial medulla (RVM) (Mondal et al., 1999; Peyron et al., 1998). Furthermore, electrical or chemical stimulation of the LH has been shown to induce excitation of PAG neurons and this stimulation produced an increase in tail flick latency. The lateral hypothalamus (LH) appears to modify nociception, in part, through the PAG and rostral ventromedial medulla (RVM) (Behbehani et al., 1988; Holden and Pizzi, 2008). It has been found that orexins have a wide variety of functions such as the regulation of appetite (Sakurai et al., 1998; Yamamoto et al., 1999), involvement in arousal (Chemelli et al., 1999; Lin et al., 1999),
and central control of autonomic activity, stress and addiction (Erami et al., 2012b; Harris et al., 2005; Heidari-Oranjaghi et al., 2012; Narita et al., 2006). Several lines of evidence imply that ORX also is involved in nociceptive sensory processes (Azhdari-Zarmehri et al., 2011; Bingham et al., 2001; Cheng et al., 2003; Erami et al., 2012a; Mobarakeh et al., 2005; Sadeghi et al., 2013; Yamamoto et al., 2002). In addition, the administration of ORXA has been shown to induce naloxone-insensitive antinociception effects as a result of spinal and/or supraspinal OX1R binding (Bingham et al., 2001; Yamamoto et al., 2002). However, the role of ORXA within the RVM has not been identified despite previous research showing that the antinociception induced by activating neurons in the LH is mediated in part by activation of spinally-projecting neurons from the RVM (Holden et al., 2005; Holden and Pizzi, 2008; Vaccarino and Chorney, 1994). Therefore, in this study, the effect of ORXA microinjection into RVM and adjacent reticularis gigantocellularis (Gi) on formalin induced nociceptive behaviors was investigated. Also, in order to investigate the role of OX1R in the RVM, the effect of the selective OX1R antagonist on antinociceptive effect of ORXA was also examined. 2. Materials and methods
☆ This research was supported by a grant from the INSF, Tehran, Iran. ☆☆ We have no financial or other conflicts of interest. ⁎ Corresponding author at: Department of Physiology, School of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-116, Tehran, Iran. E-mail address: [email protected] (S. Semnanian).
http://dx.doi.org/10.1016/j.pbb.2014.03.017 0091-3057/© 2014 Published by Elsevier Inc.
2.1. Subjects All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996) and were approved by the
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Ethics Committee of the School of Medical Sciences, Tarbiat Modares University (TMU), Tehran, Iran. Adult male Sprague–Dawley rats (n = 52, 220–300 g) were purchased from Razi Institute (Karaj, Iran). Animals were housed in groups of three rats per cage in a temperature-controlled room, under a 12 h light–dark cycle with lights on from 7:00 to 19:00. Food and water were provided ad libitum. In all experiments, strict attention was paid to the regulations of local authorities for handling of laboratory animals. 2.2. Surgical preparation for intra-nucleus microinjections To perform direct intra-nucleus administrations of drugs or respective vehicle, an intracranial guide cannula was implanted 7 days before the experiments. Rats were anesthetized with ketamine (100 mg/kg)/ xylazine (10 mg/kg) and a 23-gauge, 8.5 mm-long stainless steel guide cannula (Narishige, Japan) was stereotaxically lowered until its tip was 2 mm above the RVM or Gi according to coordinates from the atlas of Paxinos and Watson (2005) [Gi; A, − 11. 2 to − 11. 8 mm from bregma; L, ± 1 mm; V, 9 mm below the bregma and RVM; A, −11. 2 to −11. 8 from bregma; L, midline; V, 10. 5 mm below the bregma]. The cannula was anchored with dental cement to two stainless steel screws (2 pieces) in the skull. Immediately after waking from surgery, rats were returned to their home cages to recover for 7 days. Direct intra-nucleus administration of drugs or the respective vehicle was done using a stainless steel injection cannula (30 G, 0. 3 mm outer diameter) connected by a polyethylene tube to a Hamilton syringe. The injection cannula was extended 2 mm beyond the tip of the guide cannula to reach the nucleus. Drug solutions and vehicles were injected manually in a volume of 0.5 μl over a period of 60 s. The injection cannula was gently removed 1.5 min later. During microinjections, animals were free to move around the cage with the cannula in place. The microinjection was verified by monitoring the movement of an air bubble along the polyethylene tube. After 5 min, formalin (2%) was injected into the plantar surface of the right hind paw using a disposable syringe with a 30 G needle.
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of animals injected with drug (post-drug) or saline (post-saline). The following formula was used:
%MPE ¼
post‐saline‐post‐drug 100: post‐saline
2.4. Histology At the end of each test session, rats were given an overdose of Ketamine or urethane (N 1.2 g/kg) followed by microinjection of 0.5 μl of pontamine sky blue (0.2%) into the RVM or Gi (10–20 min before sacrificing the animals). Rats were perfused intracardially with saline and then 4% formalin solution and the brain was removed and sectioned. The cerebellum was removed, and the brain stem block was fixed by cyanoacrylate glue to a slicing tray. Coronal slices were prepared with 300–400 μm thickness until the darkness of the pontamine sky blue injection could be seen, then subsequent sections were cut with a thickness of 50–60 μm. Slices were cut through the brain stem from the trapezoid body to the inferior olivary nuclei using a vibrating microtome (Vibatome 1000 plus, US). Injection sites in the RVM were visualized in the triangular midline region dorsal to the pyramidal tracts and visually identified under a microscope. Only those rats with microinjection site and diffusion areas located within the nucleus were included in the results (Fig. 1, data from 100 nM ORXA are shown). 2.5. Data analysis Data were presented as mean ± SEM. The formalin pain scores in all groups were subjected to one-way ANOVA followed by protected Dunnett/Tukey's tests for multiple comparisons as needed. Each of the three phases of the formalin test (phase 1: 0 to 7 min), interphase (8 to 14 min), and phase 2 (15 to 60 min). The defined level for statistical significance was P b 0. 05.
2.3. Formalin test Seven days after surgery, rats were moved to the test room at least 1 h before the commencement of the experiment. The formalin test was performed in clear plastic boxes (30 × 30 × 30 cm) with a mirror placed underneath at a 45° angle to allow an unimpeded view of the animals' paws. Rats were acclimatized to this chamber for 30 min before intranucleus microinjection of ORXA or vehicle followed 5 min later by subcutaneous injection of 50 μl of 2% formalin into the plantar surface of the right hind paw. Formalin was administered via a 30 gauge needle inserted 5 mm under the skin. Each rat was immediately returned to the observation box for behavioral recording of nociception. Pain behaviors were scored as follows: 0, the injected paw was not favored,; 1, the injected paw had little or no weight placed on it with no toe splaying; 2, the injected paw was elevated and not in contact with any surface; and 3, the injected paw was licked or bitten. Recording of the nociceptive behaviors was started immediately after formalin injection (time 0) and continued for 60 min. The scores of nociceptive behaviors for each 3-minute interval were calculated as the weighted average of the number of seconds engaged in each behavior. The behavioral responses of each rat were evaluated separately for the first phase (1–7 min), inter-phase (8–14 min) and the second phase (15–60 min). The same procedure was followed in Experiment 2 except that the OX1R antagonist, SB-334867, was administered 5 min before ORXA. SB-334867 was dissolved in DMSO on the day of the experiment and then diluted in saline before use. The percentage of maximum possible effect (%MPE) in the formalin test was calculated as the percentage difference between the measured nociceptive behaviors during the late phase (15–60 min) for the group
Fig. 1. Location of injections in the RVM and Gi plotted as percentage of maximum possible effect (MPE%): Black squares for 0–10%, black circles for 10–25%, white circles for 25–50%, Black triangles for 50–75%, and white triangle for an effect higher than 75%. The plane is modified to a series of three sections for the brain areas from the atlas of Paxinos and Watson (2005). The approximate coordinate is indicated in millimeters posterior to bregma (data from 100 nM ORXA shown). A number of injections were lateral in the Gi-alpha, which is a part of the RVM.
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3. Results 3.1. Effects of ORXA injection into the RVM nucleus and the Gi region on formalin-induced nociceptive behaviors Intra-RVM microinjection of ORXA (100 nM) caused a decrease in formalin induced nociceptive behaviors in phases 1 and 2 compared to vehicle treated rats (phase 1 [T (1, 17) = 4.195, P = 0.001], interphase [T (1, 17) = 2.210, P = 0.041], and phase 2 [T (1, 17) = 8.009, P = 0.000]; (Fig. 2B1)). Intra-Gi microinjection of ORXA (100 nM, 0.5 μl) failed to show any significant change in formalin induced nociceptive behaviors in phase 1 [T (1, 20) = 0.761, P = 0.455], interphase [T (1, 20) = 1.393, P = 0.179], or phase 2 [T (1, 20) = 0.431, P = 0.671] (Fig. 2B2). 3.2. Effects of SB-334867 (OX1R antagonist) microinjection into the RVM regions on ORXA-induced attenuation of nociceptive behaviors in formalin test Pre-treatment with SB-334867 (100 μM/0. 5 μl) partially antagonized the effect of ORXA induced antinociceptive behaviors in the RVM ((for phase 1 [F (2, 20) = 16.042, P = 0.000]; for interphase: [F (2, 20) = 3.304, P = 0.058]; for phase 2 [F (2, 20) = 41.446, P = 0.000]); Fig. 3). Intra RVM microinjection of SB-334867 had no effect on phase 1, inter-phase, and phase 2 in the formalin test compared with the vehicle injected rats in RVM (for phase 1: 1.80 ± 0.14: 1.73 ± 0.06, P N 0.05; for interphase: 0.93 ± 0.18: 0.95 ± 0.16, P N 0.05; for phase 2: 1.81 ±0.08: 1.87 ± 0.05, P N 0.05). 4. Discussion In this study, we showed that the microinjection of ORXA into RVM has an antinociceptive effect on the formalin test, but no effect when microinjected into Gi. The lack of antinociception following ORXA
injection into Gi suggests that the antinociception obtained by microinjection into the RVM cannot be due to the diffusion of ORX into Gi areas. However, our previous study showed that areas immediately adjacent to the RVM may support ORXA antinociception (Erami et al., 2012a). We should emphasize that drug diffusion is a minor limitation for this type of experimental protocol. Injection spread is affected by the molecular weight, concentration (Myers and Hoch, 1978), and water solubility of the solution (Sakai et al., 1979). The fact that microinjections in the Gi did not produce antinociception is evidence that diffusion does not go very far from the microinjection site because the RVM in only 2 mm away. Pretreatment with a selective OX1R antagonist (SB-334867) partially antagonized the ORXA induced antinociception produced by intraRVM injection of ORXA, indicating that the antinociceptive effect of ORXA is mediated, at least in part, through OX1R. The formalin test is a widely used animal model of unrelieved pain (Dubuisson and Dennis, 1977). Formalin as a chemical noxious stimulus induces distinct nociceptive behaviors that have two phases which possibly reflect different types of pain (Abbott et al., 1995; Hunskaar et al., 1986; Hunskaar and Hole, 1987; Tjolsen et al., 1992) and sensitive to two different pain modulating mechanisms (Vaccarino and Chorney, 1994). In this study, the selective OX1R antagonist SB-334867 antagonized the antinociceptive effect of ORXA during phase 2 of the formalin test and partially antagonized the antinociceptive effect during the first phase. The difference between SB-334867 administration on the antinociceptive effect of ORXA during the first and second phases of the formalin test may well be due to the mechanisms by which formalin induces nociceptive behaviors in both phases. Previous studies have shown that (a) ORXA projections and OX1Rs are found in the brainstem (Ciriello et al., 2003; Marcus et al., 2001; Mondal et al., 1999; Peyron et al., 1998; Trivedi et al., 1998), (b) ORXA produces excitatory effects on neurons in the RVM (Azhdari Zarmehri et al., 2010; Dun et al., 2000; Vaccarino and Chorney, 1994); and (c) some evidence support that the antinociception produced by activating neurons in the lateral hypothalamus is mediated in part by the subsequent activation of spinally-projecting neurons in the RVM (Holden
Fig. 2. Time course for formalin induced nociceptive behavior score (±S. E. M.) following intra-RVM (n = 9) and Gi (n = 10) infusion of ORXA (100 nM, 0.5 μl,) measured every 3 min for 60 min (A). The columns represent the mean nociceptive scores in each phase: phase 1 (1–7 min), inter-phase (8–14 min), and phase 2 (15–60 min) (B). All microinjections in the midline raphe magnus and lateral in the Gi-alpha are included in the RVM and those outside of RVM are included in Gi. **P b 0.01; ***P b 0.001 compared with saline group.
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Fig. 3. Time course of formalin induced nociceptive behavior score (±S. E. M.) following infusions of the vehicle (0.5 μl) + ORXA (100 nM / 0.5 μl) (n = 9), SB-334867 (100 μM / 0.5 μl) + vehicle (0.5 μl) (n = 6) and SB-334867 (100 μM / 0.5 μl) + ORXA (100 nM / 0.5 μl) (n = 7) into the RVM (A) measured every 3 min for 60 min. The columns represent the mean nociceptive scores in each phase: phase 1 (1–7 min), inter-phase (8–14 min), and phase 2 (15–60 min) (B). *P b 0.05; ***P b 0.001 compared to vehicle group.
et al., 2005; Holden and Pizzi, 2008; Vaccarino and Chorney, 1994), a region known to play a role in pain modulation (Fields and Heinricher, 1985; Heinricher et al., 1989). These findings support our observations showing the involvement of ORXA in modulating pain in the RVM. Ciriello et al. found that the distribution of ORXA-labeled axons varies within the specific regions of the magnocellular reticular area (Ciriello et al., 2003). ORXA axon labeling was observed mainly within the GiA and to a lesser extent within the Gi and RVM (Ciriello et al., 2003). However, there are significant differences among the sites that mediated the ORXA induced analgesia on the formalin test. As previously reported, ORXA injections directly into the GiA elicit a decrease in muscle tone and cardioacceleration (Ciriello et al., 2003; Mileykovskiy et al., 2002). The present findings show that ORXA microinjection into the RVM caused antinociception on the formalin test while considering the possibility that the origin of the orexinergic inputs to these structures might be involved in top-down pain modulation. The RVM may influence neurons in the dorsal horn of the spinal cord which are involved in the neurotransmission of noxious stimuli (Vanegas and Schaible, 2004; Zhuo and Gebhart, 1997). Because the LH is the exclusive area to express ORX and implicated as part of a descending system associated with the modulation of nociceptive transmission in the first synapse in dorsal horn neuron of the spinal cord, the antinociception mediated by the LH appears to occur via activation of brainstem nuclei (Behbehani et al., 1988; Dafny et al., 1996). We suggest that ORX might act as a mediator between LH and RVM in modulating pain. Consistent with other reports using the formalin and hot plate tests (Yamamoto et al., 2002), pretreatment with selective OX1R (SB334867) antagonized the antinociceptive behaviors of ORXA in the RVM, demonstrating likely, the potential importance of OX1R in the modulation of pain. Our data corroborate the previous studies in which SB-334867, a selective antagonist of OX1R, was demonstrated to restore the antinociception effect of ORXA after ICV administration in mice and rats (Bingham et al., 2001; Yamamoto et al., 2002). It has been suggested that the antinociceptive effects of ORXA are mediated both via the spinal and supraspinal mechanisms. In conclusion, ORXA-induced antinociception in the formalin test is mediated in part through OX1R in the RVM, although other structures may be involved. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was supported by a grant from INSF, Tehran, Iran. We thank Dr. Ali A. Pahlevan (Iran) and Professor Morgan (Washington State University Vancouver, Vancouver) and Mary M. Heinricher,
(Oregon Health & Science University) for their meticulous work on revision of the final English version of the manuscript.
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