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Intra-Periaqueductal Gray Matter Microinjection of Orexin-A Decreases Formalin-Induced Nociceptive Behaviors in Adult Male Rats
The Journal of Pain, Vol 12, No 2 (February), 2011: pp 280-287 Available online at www.sciencedirect.com
Intra-Periaqueductal Gray Matter Microinjection of Orexin-A Decreases Formalin-Induced Nociceptive Behaviors in Adult Male Rats Hassan Azhdari Zarmehri,*,y Saeed Semnanian,* Yaghoub Fathollahi,* Elaheh Erami,y Roghaieh Khakpay,* Hossein Azizi,* and Kambiz Rohampour* * Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran. y Department of Physiology, School of Medical Sciences, Qazvin University of Medical Science, Qazvin, Iran.
Abstract: Intracerebroventricular injection of orexin-A (hypocretin-1) has been shown to elicit the analgesic responses. However, the locations of central sites that may mediate these effects have not been clearly elucidated. This study was performed using male Sprague Dawley rats to investigate the antinociceptive effects of intra-periaqueductal gray matter (PAG) administration of orexin-A, 5 minutes prior to formalin (50 mL of 2%) injection. Orexin-A had no effect on tail-flick test as thermal and acute model. In the formalin test, intra-PAG injection of orexin-A (10 nM) decreased the formalin-induced nociceptive behaviors in the interphase and phase 2, but not in phase 1, indicating an antinociceptive role of exogenous orexin-A in the PAG. While Orexin-A failed to produce a dose-dependent decrease in formalin-evoked behaviors in phase 1, it may have induced a dose-dependent decrease in formalinevoked behaviors in early phase 2. Control injections of orexin-A into the sites near the PAG resulted in less or no reduction in pain, indicating that the analgesia observed is probably due to a site of action within the PAG rather than at surrounding neural structures. The antinociceptive effect of orexin-A was compared with intra-PAG administration of morphine (.5 mL of 20 mM, 5 minutes before the formalin injection). Morphine decreased the formalin-induced nociceptive behaviors in all phases. To investigate whether the orexin has a special action on the early part of the second phase, or its delayed effects are related to its pharmacokinetics, the orexin-A was injected into the PAG, 10 minutes before the formalin injection. No difference was observed between 5 and 10 minutes injection of orexin-A prior to formalin injection. The antinociceptive effect of orexin was blocked by intra-PAG injection of SB-334867, a putative type 1 orexin receptor antagonist, suggesting the involvement of orexin receptor type 1 in antinociception produced with intra-PAG injection of orexin-A. The results showed that the orexin-A plays an antinociceptive role in PAG in the interphase and the late phase of formalin test through type 1 orexin receptor dependent mechanism. Perspective: Orexin is produced exclusively in the lateral hypothalamus, where it is known to modulate the pain processing through PAG. The antinociceptive effect of orexin in PAG may provide a role for this neurotransmitter in the up-down modulating pain system and further support the development of orexin-1 agonists for pain treatment. ª 2011 by the American Pain Society Key words: Orexin-A, periaqueductal gray matter, orexin receptor type 1, SB-334867, formalin test.
Received March 9, 2010; Revised July 21, 2010; Accepted September 17, 2010. Supported by grants from the Neuroscience Research Center, Shahid Beheshti University, and Iran National Science Foundation (INSF), Tehran, Iran. Address reprint requests to Saeed Semnanian, Department of Physiology, School of Medical Sciences, Tarbiat Modares University, P.O. Box 14115116, Tehran, Iran. E-mail: [email protected] 1526-5900/$36.00 ª 2011 by the American Pain Society doi:10.1016/j.jpain.2010.09.006
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he neuropeptides orexin-A and -B (also called hypocretin-1 and -2) are expressed in neurons of the lateral hypothalamus (LH), with a few orexin cells extending into the dorsomedial hypothalamic nucleus (DMH).21 Orexin-A is a 33 amino acid peptide and orexinB a 28 amino acid peptide.21 Sakurai et al21 described 2 orexin receptors coupled to G proteins. Orexin-A binds equally to both orexin receptor type 1 and 2, while orexin-B has a preferential affinity for orexin receptor type 2. The broad projections of the orexinergic system have led to its implication in a variety of functions,
Azhdari Zarmehri et al including feeding, sleep-wake cycle, cardiovascular function, hormone secretion,8,20,22,23 and, more recently, the modulation of nociceptive processing.4,5,18,24,26,27 Initial support for this idea came from a study in which intracerebroventricular (ICV) Orexin-A (3–30 mg) produced a dose-related analgesia in the hotplate test in rats.4 Systemic (iv) administration of orexin-A also proved to be effective in this rat model and also in the equivalent mouse paradigm at doses between 3 and 30 mg/kg.4 In other mouse models, orexin-A inhibited the visceral nociception (abdominal constriction; 10 and 30 mg/kg) and thermal hyperalgesia (in intraplantar carrageenan model; 3–30 mg/kg), with an efficacy equivalent to the opioid analgesic morphine.4 Morphological studies have established that the orexin-containing neurons and fibers as well as orexin receptors (orexin receptor type 1 and 2) are distributed along all parts of pain circuitry, including the periaqueductal gray matter (PAG) region which is considered to be important for pain modulation.13,19,25 Stimulation of the lateral hypothalamus elicits antinociception via relays to the PAG and the rostral ventromedial medulla (RVM), which ultimately triggers the activation of descending noradrenergic pathways.3 Electrical stimulation applied in the PAG produced antinociception analogous to 10 mg/kg morphine.28 Like electrical stimulation, opioid administration into the ventrolateral parts of PAG also produces robust behavioral signs of antinociception.16 Although orexin-A exhibits analgesic effects when administered via ICV or intrathecal microinjection,4,17,26 the effect of orexin-A in the PAG is unclear. In the present study, the effect of orexin microinjected into the PAG region on the tail-flick test and formalin test was investigated. The tail-flick test was used to evaluate the acute nociceptive transmission, and the formalin test to reflect an inflammatory pain condition to measure its therapeutic potential. Portions of these data were presented as abstracts at Neuro2010.1
Methods Subjects All experiments involving the animals were conducted according to the policy of Iranian Convention for the Protection of Vertebrate Animals used for experiments and the protocol was approved by the Ethics Committee of the School of Medical Sciences, Tarbiat Modares University (TMU), Tehran, Iran. Efforts were made throughout the experiments to minimize the animal discomfort and to reduce the number of animals used. Adult male, Sprague-Dawley rats (220–300 g) purchased from Razi Institute (Hesarak Karj, Iran), were housed in groups of 3 in a temperature controlled room, under a 12 hour light/ dark cycle with lights on at 0700 to 1900. Food and water were provided ad libitum. During the experiments, attention was strictly paid to the regulations of local authorities for handling laboratory animals.
Surgical Preparation for Intra-PAG and ICV Microinjections To perform direct intra-PAG and ICV administrations of drugs or the respective vehicle (saline), rats were anaes-
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thetized with Ketamine (100 mg/kg)/Xylazine (10 mg/ kg). The rats were placed in a stereotaxic apparatus (Narishige, Japan), holes were drilled in the skull over the PAG, and the dura was removed to allow the placement of a guide cannula. 23-gauge, 5 mm-long stainless steel guide cannula was stereotaxically lowered until its tip was 2 mm above the PAG by applying coordinates from the atlas of Paxinos and Watson10 (for PAG: A, 7.8 mm from bregma; L, .5 mm; V, 5.7 mm below the bregma and for ICV A, .9 mm from bregma; L, .1.8 mm; V, 3.8 mm below the bregma). The cannula was anchored with dental cement to a stainless steel screw in the skull. The guide cannula for intra-PAG microinjection was implanted 7 days before the experiment for pain formalin or tail-flick tests in awake rats. Immediately after waking from surgery, rats were returned to their home cages to await the formalin test procedure. Direct intra-PAG administration of drugs, or respective vehicle, was conducted with a stainless steel cannula (30 gauge) connected via a polyethylene tube to a Hamilton syringe, inserted through the guide cannula, and extended 2 mm beyond the tip of the guide cannula to reach the PAG. At day 7, while the animals were habituated, volumes of .5 mL of drug solutions or vehicles were injected into the PAG over a period of 60 seconds or 5 mL orexin-A 10 nM or vehicles injected into the ICV over a period of 100 seconds and the injection cannula was gently removed later. After 5 minutes, formalin was injected into the plantar surface of the right hind paw using a disposable insulin syringe with a fixed 30-gauge needle. A separate group of animals were used for conducting tail-flick test.
Tail-Flick Test Latency to tail-flick against heat was used as a measure of nociceptive responsiveness. At day 7, rats were kept in a glass restrainer and the dorsal surface of the tail between 4 and 6 cm from the tip of the tail was exposed to a beam of light generated from an automated analgesia meter (Harvard Tail-flick Analgesia Meter, Holliston, MA). The timer stopped when the animal flicked its tail away from the beam of light. Tail-flick latency was measured at 5-minute intervals until a stable baseline was obtained over 3 consecutive trials. The latency was measured 10 minutes after intra-PAG orexin-A injection up to 60 minutes. Orexin-A (.01–10 nM) and saline were given intra-PAG, in experimental and vehicle groups, respectively. Latencies were determined twice at 10-minutes interval after the administration of orexin-A or vehicle, and the average of 2 frequent readings was taken as the predrug latency. To avoid tissue damage, a 10-second cut-off was used.
Formalin-Induced Nociceptive Behavior Formalin-induced nociceptive behavior is a widely used animal model of persistent pain.2,7 Formalininduced nociceptive behavior was used because it employs an adequate painful stimulus; the animals show a spontaneous response and it is sensitive to the commonly used analgesics. Moreover, the pain stimulus
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is a continuous rather than a transient one and thus may have resemblance to some kinds of clinical pain, and the observations were obtained from the animals which were under mild or no stress. Also, the test employs a chemical nociceptive stimulus that has 2 distinct phases which possibly indicate different types of pain modulations.14,15 Rats were moved to the test room at least 1 hour before the commencement of the experiment. In the present study, the rats were first acclimatized for 30
Orexin-A Decreased Formalin-Induced Nociceptive Behaviors minutes in an acrylic observation chamber (30 cm in diameter and in height) and then 50 mL of 2% formalin was injected subcutaneously into the plantar surface of the right hind paw with a 30-gauge needle. Each rat was then immediately returned to the observation box, and the behavioral recording commenced. A mirror, placed at a 45 angle beneath the box, permitted the observation of behaviors without moving the box. Pain behavior was scored as follows: 0, the injected paw was not favored; 1, the injected paw had little or no weight
Figure 1. Time scores of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 to 8 rats per group) following intraperiaqueductal gray matter (PAG) infusion of the saline (.5 mL, A1) or orexin-A and control site (A2-A6) measured every 3 minutes for 60 minutes (A) and bar chart for different doses of orexin-A 1, 5, 10, 20 nM/.5 mL) in the formalin test and control site (B1-B6). The columns represent the mean of nociceptive score in each phase: phase 1 (minutes 1–7), interphase (minutes 8–14), and phase 2 (minutes 15–60) (B1-B6). Rats received orexin-A into the PAG 5 minutes before formalin injection in the right hind paw. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. For clarity, each dose of orexin-A was plotted in separate graphs, with the same vehicle group in each. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the orexin-A groups, open circles correspond to the vehicle group. All microinjections were made on the right side of the PAG, and are separated here for clarity only. *P < .05; ***P < .001 in comparison with saline.
Azhdari Zarmehri et al placed on it; 2, the injected paw was elevated and not in contact with any surface; and 3, the injected paw was licked or bit. Scores were continuously observed for the duration of the experiment (60 minutes). The nociceptive behaviors score for each 3-minute interval was calculated as the weighted average of the number of seconds spent in each nociceptive behavioral, from the start of the experiment. The scores were recorded in normal rats as well as in those who received orexin-A. In each group, the behavioral response of each rat during the first phase (1–7 minutes), interphase (8–14), and the second phase (15–60) was separately evaluated.
Drugs Two percent formalin was prepared by diluting 37% formaldehyde (ie, the commercially available saturated aqueous solution of formaldehyde, (Temad, Iran) 2:100 with sterile physiological saline solution. Morphine sulfate (Temad, Iran) was dissolved in saline. Orexin-A (molecular weight = 3,561, Sigma) was dissolved in saline, an orexin-1 receptor agonist; SB 334867 (N-(2-methyl-6benzorexin-Azolyl)-N00 -1,5-naphthyridin-4-yl urea; (molecular weight = 356, Tocris), was dissolved in dimethyl sulfoxide (DMSO) and diluted in saline to reach the final concentration. All drugs were diluted in saline immediately before use on the morning of an experiment.
Histology At the end of the experiments, the animals were deeply anaesthetized with Ketamine overdose, a volume of .5 mL of pontamin sky blue (.2%) was also injected in the cannula 10 to 20 minutes before sacrificing the rat. Rats were then perfused intracardially with 100 mL of 4% formalin solution and the brain was removed and sectioned. Only those rats whose microinjection site and diffusion were located within the PAG were included in the results.10 In all experiments, the orexin-A was microinjected into the right PAG and the formalin injected into the plantar surface of the right hind paw.
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Data Analysis Data are presented as mean 6 S.E.M. and analyzed by 1-way analysis of variance and t-test between groups. The mean nociceptive score in each phase (I, interphase, II) of the formalin test for the effect of each dose of orexin-A was analyzed using 1-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Phase 1 (1–7 minutes), the interphase (8–14 minutes), and the phase 2 (15–60 minutes) of the formalin test were analyzed separately. The defined level for statistical significance was P < .05.
Results Effects of Different Doses of Orexin-A Injected into the PAG on FormalinInduced Nociceptive Behaviors Formalin produced typical biphasic pain responses. The first and second phases were separated by a brief interphase where little to no nociceptive behavior was observed in control group. Microinjections of saline (.5 mL), into the PAG, 5 minutes before the formalin, failed to change the pattern of the early and late hyperalgesic behaviors induced by formalin (Fig 1). While the intra-PAG microinjection of 5, 10, and 20 nM orexin-A decreased the formalin-induced nociceptive behaviors in phase 2 compared with saline-treated rats (Fig 1), the injection of 10 nM of orexin-A caused a decrease in formalin-induced nociceptive behaviors in the interphase (Fig 1). Control injections of orexin-A into the sites near the PAG resulted in less or no reduction of pain, indicating that the analgesia observed is probably due to a site of action within the PAG rather than the surrounding neural structures (Fig 1; A6, B6, and C6, n = 12). In this study, the selective effect of orexin-A on the early second phase response was intriguing and the effect of orexin on the second phase showed to be clearly time dependent. To investigate whether the orexin has a special action on the early part of the second phase, or its delayed
Figure 2. Time scores of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 to 8 rats per group) following intraperiaqueductal gray matter (PAG) infusion of the orexin-A (10 nM) measured every 3 minutes for 60 minutes (A) and bar chart (B). The columns represent the mean of nociceptive score in each phase: phase 1 (minutes 1–7), interphase (minutes 8–14), and phase 2 (minutes 15–60). Rats received orexin-A into the PAG 5 and 10 minutes before formalin injection in the right hind paw. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the orexin-A (5 minutes before formalin test) group, open circles correspond to the orexin-A (10 minutes before formalin test) group. All microinjections were made on the right side of the PAG, and are separated here for clarity only.
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effects are related to its pharmacokinetics, orexin-A was injected into the PAG, 10 minutes before the formalin injection. No difference between 5- and 10-minute injections of orexin-A prior to formalin injection was demonstrated (Fig 2, P > .05).
Dose-Response Relationships of Orexin-A Injected into the PAG on Formalin-Induced Nociceptive Behaviors The dose-response relationships of intra-PAG administered orexin-A on the first phase, interphase and the second phase of the formalin-induced nociceptive behaviors is shown in Fig 3.
Comparing the Effects of Morphine and Orexin-A Injected into the PAG on Formalin-Induced Nociceptive Behaviors Intra-PAG microinjection of morphine (.5 mL of 20 mM) vigorously decreased the formalin-induced nociceptive behaviors in phase 1, interphase as well as the phase 2 of the formalin test (Fig 4). As mentioned earlier, 10-nM orexin-A decreased the formalin-induced nociceptive behaviors in the interphase and phase 2, but not in phase 1.
Effects of SB-334867 (a Type 1 Orexin Receptor Antagonist) Injected into the PAG on Orexin-A-Induced Attenuation of Nociceptive Behaviors in Formalin Test Pretreatment with SB-334867 (100 nM/.5 mL) antagonized the effect of 10-nM orexin-A on the interphase and phase 2 nociceptive behaviors (Fig 5). Intra-PAG microinjection of SB-334867 produced no effect on phase 1, interphase, and the phase 2 in formalin test compared
Figure 3. Dose-response curves of formalin-induced nociceptive behaviors during phase 1 (minutes 1–7), interphase (minutes 8–14), and phase 2 (minutes 15–60) after microinjection of different dose of orexin-A. The ordinate scale shows percent 6 S.E.M. relative to the control group in formalin-induced nociceptive behaviors. Drugs were administered intraperiaqueductal gray matter (PAG) 5 minutes before formalin injection in the right hind paw. Orexin-A in 10 nM reduced formalin-induced nociceptive behaviors in the inter-phase (P < .001) and phase 2 (P < .05), and in 5 and 20 nM reduced formalin-induced nociceptive behaviors only phase 2 (P < .05). *P < .05; ***P < .001 in comparison with saline.
Orexin-A Decreased Formalin-Induced Nociceptive Behaviors with the saline-treated rats (Fig 5). The SB 334867 was microinjected 5 minutes before the orexin-A injection.
Effects Of Orexin-A Injected into the ICV on Formalin-Induced Nociceptive Behaviors and Orexin-A Injected into the PAG on Tail-Flick Withdrawal Test To determine the effect of ICV injection of orexin-A on formalin test, the orexin-A (10 nM, 5 mL) was injected into the right ventricle and the formalin-induced nociceptive behaviors were monitored. In this experiment, orexin-A failed to elicit significant changes in formalin-induced nociceptive behaviors (for phase 1: 1.89 6 .16, F > .05; for interphase: .98 6 .35, F > .05; for phase 2: 1.78 6 .11, F > .05; n = 10). Unilateral injections of saline (.5 mL) into the right PAG also caused no significant changes in latency of the tail-flick test. Orexin-A, injected unilaterally into the PAG at the concentrations up to 10 nM, demonstrated no significant effect on thermal tail-flick test (Table 1).
Discussion In this study we investigated the role of the midbrain PAG as a common substrate in orexin-A anti-nociception in formalin and tail-flick tests. The injection of orexin-A into the nuclei of the descending antinociceptive pathway may provide an opportunity to investigate the role of this neurotransmitter in the up-down modulating pain system. Orexin is produced exclusively in the dorsal and lateral hypothalamus,6,21 the regions known to modulate pain processing through the PAG.3 In addition, Orexin projections are widespread within the brain regions that play an important role in descending pain modulation such as PAG.13,19,25 We clearly demonstrated that the microinjection of orexin-A (5 nM) into the PAG reduced the formalin-evoked nociceptive behaviors in the interphase and the late phase but not the initial phase. Orexin-A had no effect on tail flick up to a dose of 10 nM that was effective in formalin test. Moreover, the SB-334867, a selective type 1 orexin receptor antagonist, antagonized the antinociceptive effect of orexin-A in the formalin test. Microinjection of morphine produced persistent and profound analgesia in all phases of the formalin test and the effect of orexin was less than the effect observed with morphine. In previous studies, the systemic administration of orexin-A produced a dose-related analgesia in hot plate analgesic test in rats4 and inhibited the visceral nociception (10 and 30 mg/kg) and thermal hyperalgesia induced with carrageenan (3–30 mg/kg), with an efficacy equivalent to the opioid analgesic morphine.4,26 Our findings on the antinociceptive effects of orexin-A are consistent with previous studies in which the intrathecal delivery of orexin-A was reported to have inhibited the pain perception.26 The site(s) of analgesic action of orexin-A following iv and ICV administration remains to be established. It has been suggested that the antinociceptive effects of
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Figure 4. Time scores of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 rats per group) following intra-periaqueductal
gray matter (PAG) infusion of the orexin-A (10 nM/.5 mL) and morphine (20 mM/.5 mL) measured every 3 minutes for 60 minutes (A) and bar chart (B). The columns represent the mean of nociceptive scores in each phase: phase 1 (minutes 1–7), inter-phase (minutes 8–14), and phase 2 (minutes 15–60). Rats received orexin-A 5 minutes before formalin injection. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the orexin-A group, open circles correspond to the vehicle group and tingle correspond morphine group. All microinjections were made on the right side of the PAG, and are separated here for clarity only. *P < .05; ** P< .01; ***P < .001 in comparison with saline.
orexin-A and B are mediated via spinal and supraspinal mechanisms.4,26 According to dense interaction between the LH and PAG,3 the orexinergic innervations and its receptors expression in PAG,19 it can be suggested that the orexinergic pathway may mediate the interaction between these 2 sites. The difference between their effects on the first and second phase of the test may well be due to the mechanisms by which the formalin induces nociceptive behaviors in 2 phases. Subcutaneous injection of formalin generates typical biphasic nociceptive response that is characterized by an early phase (minutes 0–7), a quiescent interphase, and a second prolonged phase (minutes 15–55).2,7 The early phase, the quiescent interval, and the second delayed phase are all active phases. The capacity of orexin-A to attenuate the second phase of the nociceptive behaviors suggests that it recruits the putative descending inhibitory control pathways and also influences the formalin-induced spinal nociceptive processing during the interphase and the late phase.
It seems that the effect of orexin-A in the PAG may not be related to control of acute nociception, as there was no effect on the tail-flick test and the first phase of the formalin test. On the other hand, it may play a critical role in the modulation of the active inhibition on the interphase as well as the neuronal process occurring during the late phase of the formalin test. The period between the 2 phases of nociceptive responses is largely overlooked, probably as it is generally considered to be a phase of inactivity. There is some evidence that this interphase in the formalin test may be an active process due to the action of endogenous pain-suppressing mechanisms.9,11,12 Franklin et al9 reported that pentobarbital, diazepam, and alcohol inhibit the ‘‘inter-phase diminution’’ and argued that these GABAA receptor agonists unmask the pain that is suppressed by some inhibitory mechanism. Henry et al12 showed that after 2 injections of formalin (with 20-minute interval), there was a second diminution of nociceptive scores after the second formalin injection, rather than an additive sum of the
Figure 5. Time course of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 to 8 rats per group) following intra-
periaqueductal gray matter (PAG) infusions of the vehicle 1 orexin-A (10 nM/.5 mL), SB-334867 (100 nM/.5 mL) 1 vehicle and orexin-A 1 SB-334867 measured every 3 minutes for 60 minutes (A) and bar chart for each phase (B). The columns represent the mean of nociceptive scores in each phase: phase 1 (minutes 1–7), interphase (minutes 8–14), and phase2 (minutes 15–60) (B). Rats received orexin-A 5 minutes before formalin injection. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the vehicle 1 orexin-A group, open circles correspond to the vehicle 1 vehicle group, square correspond SB334867 1 vehicle group and tingle correspond SB 334867 1 orexin-A group. All microinjections were made on the right side of the PAG. *P < .05; ***P < .001 in comparison with vehicle group.
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Mean Tail-Flick Latencies in Seconds (Mean 6 S.E.M. of 6 to 7 Rats per Group) of Test Given 2 Every 10 Minutes for 1 Hour Following Infusions of Orexin-A or Saline into the Periaqueductal Gray Matter (PAG)
Table 1.
REACTION TIME IN SECOND GROUPS
DOSE
PRE INJ.
10 MIN
20 MIN
30 MIN
40 MIN
50 MIN
60 MIN
Vehicle Orexin Orexin Orexin
0 .01 nM 1 nM 10 nM
6.3 6 .55 4.6 6 .20 5.7 6 .51 5.0 6 .43
5.8 6 .56 4.5 6 .32 5.1 6 .91 5.1 6 .46
6.2 6 .49 4.8 6 .38 5.2 6 .49 4.6 6 .31
5.9 6 .39 4.7 6 .07 5.1 6 .44 5.3 6 .84
5.5 6 .35 4.7 6 .4 5.3 6 .65 4.6 6 .47
5.9 6 .4 4.8 6 .26 5.6 6 1.06 5.4 6 .25
5.5 6 .32 4.9 6 .06 5.1 6 .69 5.5 6 .96
nociceptive responses to the 2 injections. Consistent with their suggestions that the diminution that normally occurs after the first phase of the second injection of formalin test is due to an active inhibitory process, the intra-PAG orexin-A injection increases the inhibitory period during this inter-phase.12 The active inhibitory phase may be considered as a form of nonopioid autoanalgesia brought about by the descending inhibitory controls arising above the PAG.9,12 The reduction in the nociceptive behaviors recorded during the first part of phase 2 may reflect a prolongation of the interphase in response to orexin microinjection or a short action on the phase 2 nociceptive mechanisms; even there are significant differences when analyzing the mean response for phase 2. In agreement with another report regarding the formalin and hot plate tests,4,26 the SB-334867, a selective type 1 orexin receptor antagonist, has no effect on formalin test when used alone, which suggests that a tonic orexin receptor type 1 mediated inhibitory system does not exist in the PAG during this test. It has been reported that the SB-334867 is acting as a pro-hyperalgesic in the mouse carrageenan assay (3 and 10 mg/kg IP), and this suggests a tonically activated type 1 orexin receptor mediated inhibitory system is present during a carrageenan-induced thermal hyperalgesia test.26 This may reflect the strain differences, different route of administration, differences between the models used (carrageenan hyperalgesia test and the formalin test), methodological differences (eg, lighting, noise, odors, handling stress, or anaesthesia prior to formalin injection, all of which are known to influence the test). Our
data corroborate the previous studies, all demonstrating that SB-334867 restores the analgesic effect of both ICV and intrathecal administration of orexin-A in mice and rats, respectively.4,26 It has been suggested that the antinociceptive effects of orexin-A are mediated via spinal and supraspinal mechanisms. Subsequently, we tested the antinociceptive activity of orexin-A and morphine in the formalin test. Microinjection of morphine produced persistent and profound analgesia in first, interphase, and late phase of formalin test. The present study has shown that the orexin causes a decrease in formalin induced nociceptive behaviors in interphase and late phase, although its antinociceptive effect is lower than morphine. In conclusion, the intra-PAG injection of orexin-A depresses the inter-phase and phase 2 in formalininduced nociceptive behaviors in rats without having any effect on tail-flick latency. These effects of orexin-A are mediated by the activation of type 1 orexin receptors expressed in the PAG. These data might suggest a potential therapeutic approach in treatment of pain.
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Acknowledgments The authors wish to express their appreciation to Drs Ali-Reza Mani and Mohammad Javan, both as the academic members at the Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran, for their assistance and support. Our thanks also go to Dr Ali A Pahlevan for his final revision of the English version of the manuscript.
6. de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG: The hypocretins: hypothalamusspecific peptides with neuroexcitatory activity. Proc Natl Acad Sci U S A 95:322-327, 1998 7. Dubuisson D, Dennis SG: The formalin test: A quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4:161-174, 1977
Azhdari Zarmehri et al 8. Ferguson AV, Samson WK: The orexin/hypocretin system: A critical regulator of neuroendocrine and autonomic function. Front Neuroendocrinol 24:141-150, 2003 9. Franklin KB, Abbott FV: Pentobarbital, diazepam, and ethanol abolish the interphase diminution of pain in the formalin test: Evidence for pain modulation by GABAA receptors. Pharmacol Biochem Behav 46:661-666, 1993 10. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates, 4th and 6th eds. New York, NY, Academic Press, 2005 11. Gaumond I, Arsenault P, Marchand S: The role of sex hormones on formalin-induced nociceptive responses. Brain Res 958:139-145, 2002 12. Henry JL, Yashpal K, Pitcher GM, Coderre TJ: Physiological evidence that the ‘interphase’ in the formalin test is due to active inhibition. Pain 82:57-63, 1999 13. Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, van den Pol AN: Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J Comp Neurol 415:145-159, 1999 14. Hunskaar S, Berge OG, Hole K: Dissociation between antinociceptive and anti-inflammatory effects of acetylsalicylic acid and indomethacin in the formalin test. Pain 25: 125-132, 1986 15. Hunskaar S, Hole K: The formalin test in mice: Dissociation between inflammatory and non-inflammatory pain. Pain 30:103-114, 1987 16. Lewis VA, Gebhart GF: Evaluation of the periaqueductal central gray (PAG) as a morphine-specific locus of action and examination of morphine-induced and stimulationproduced analgesia at coincident PAG loci. Brain Res 124: 283-303, 1977 17. Mobarakeh JI, Takahashi K, Sakurada S, Nishino S, Watanabe H, Kato M, Yanai K: Enhanced antinociception by intracerebroventricularly and intrathecally-administered orexin A and B (hypocretin-1 and -2) in mice. Peptides 26: 767-777, 2005 18. Mobarakeh JI, Takahashi K, Sakurada S, Nishino S, Watanabe H, Kato M, Naghdi N, Yanai K: Enhanced antinociception by intracerebroventricularly administered orexin A
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in histamine H1 or H2 receptor gene knockout mice. Pain 188:254-262, 2005 19. Peyron C, Tighe DK, van den Pol AN, de LL, Heller HC, Sutcliffe JG, Kilduff TS: Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996-10015, 1998 20. Sakurai T: Roles of orexin/hypocretin in regulation of sleep/wakefulness and energy homeostasis. Sleep Med Rev 9:231-241, 2005 21. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M: Orexins and orexin receptors: A family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92:573-585, 1998 22. Samson WK, Taylor MM, Ferguson AV: Non-sleep effects of hypocretin/orexin. Sleep Med Rev 9:243-252, 2005 23. Siegel JM: The narcoleptic borderland. Sleep Med 4:3-4, 2003 24. Suyama H, Kawamoto M, Shiraishi S, Gaus S, Kajiyama S, Yuge O: Analgesic effect of intrathecal administration of orexin on neuropathic pain in rats. In Vivo 18:119-123, 2004 25. Tao R, Ma Z, McKenna JT, Thakkar MM, Winston S, Strecker RE, McCarley RW: Differential effect of orexins (hypocretins) on serotonin release in the dorsal and median raphe nuclei of freely behaving rats. Neuroscience 141: 1101-1105, 2006 26. Yamamoto T, Nozaki-Taguchi N, Chiba T: 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, 2002 27. Yamamoto T, Saito O, Shono K, Hirasawa S: Activation of spinal orexin-1 receptor produces anti-allodynic effect in the rat carrageenan test. Eur J Pharmacol 481:175-180, 2003 28. Yeung JC, Yaksh TL, Rudy TA: Concurrent mapping of brain sites for sensitivity to the direct application of morphine and focal electrical stimulation in the production of antinociception in the rat. Pain 4:23-40, 1977
Intra-Periaqueductal Gray Matter Microinjection of Orexin-A Decreases Formalin-Induced Nociceptive Behaviors in Adult Male Rats Hassan Azhdari Zarmehri,*,y Saeed Semnanian,* Yaghoub Fathollahi,* Elaheh Erami,y Roghaieh Khakpay,* Hossein Azizi,* and Kambiz Rohampour* * Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran. y Department of Physiology, School of Medical Sciences, Qazvin University of Medical Science, Qazvin, Iran.
Abstract: Intracerebroventricular injection of orexin-A (hypocretin-1) has been shown to elicit the analgesic responses. However, the locations of central sites that may mediate these effects have not been clearly elucidated. This study was performed using male Sprague Dawley rats to investigate the antinociceptive effects of intra-periaqueductal gray matter (PAG) administration of orexin-A, 5 minutes prior to formalin (50 mL of 2%) injection. Orexin-A had no effect on tail-flick test as thermal and acute model. In the formalin test, intra-PAG injection of orexin-A (10 nM) decreased the formalin-induced nociceptive behaviors in the interphase and phase 2, but not in phase 1, indicating an antinociceptive role of exogenous orexin-A in the PAG. While Orexin-A failed to produce a dose-dependent decrease in formalin-evoked behaviors in phase 1, it may have induced a dose-dependent decrease in formalinevoked behaviors in early phase 2. Control injections of orexin-A into the sites near the PAG resulted in less or no reduction in pain, indicating that the analgesia observed is probably due to a site of action within the PAG rather than at surrounding neural structures. The antinociceptive effect of orexin-A was compared with intra-PAG administration of morphine (.5 mL of 20 mM, 5 minutes before the formalin injection). Morphine decreased the formalin-induced nociceptive behaviors in all phases. To investigate whether the orexin has a special action on the early part of the second phase, or its delayed effects are related to its pharmacokinetics, the orexin-A was injected into the PAG, 10 minutes before the formalin injection. No difference was observed between 5 and 10 minutes injection of orexin-A prior to formalin injection. The antinociceptive effect of orexin was blocked by intra-PAG injection of SB-334867, a putative type 1 orexin receptor antagonist, suggesting the involvement of orexin receptor type 1 in antinociception produced with intra-PAG injection of orexin-A. The results showed that the orexin-A plays an antinociceptive role in PAG in the interphase and the late phase of formalin test through type 1 orexin receptor dependent mechanism. Perspective: Orexin is produced exclusively in the lateral hypothalamus, where it is known to modulate the pain processing through PAG. The antinociceptive effect of orexin in PAG may provide a role for this neurotransmitter in the up-down modulating pain system and further support the development of orexin-1 agonists for pain treatment. ª 2011 by the American Pain Society Key words: Orexin-A, periaqueductal gray matter, orexin receptor type 1, SB-334867, formalin test.
Received March 9, 2010; Revised July 21, 2010; Accepted September 17, 2010. Supported by grants from the Neuroscience Research Center, Shahid Beheshti University, and Iran National Science Foundation (INSF), Tehran, Iran. Address reprint requests to Saeed Semnanian, Department of Physiology, School of Medical Sciences, Tarbiat Modares University, P.O. Box 14115116, Tehran, Iran. E-mail: [email protected] 1526-5900/$36.00 ª 2011 by the American Pain Society doi:10.1016/j.jpain.2010.09.006
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he neuropeptides orexin-A and -B (also called hypocretin-1 and -2) are expressed in neurons of the lateral hypothalamus (LH), with a few orexin cells extending into the dorsomedial hypothalamic nucleus (DMH).21 Orexin-A is a 33 amino acid peptide and orexinB a 28 amino acid peptide.21 Sakurai et al21 described 2 orexin receptors coupled to G proteins. Orexin-A binds equally to both orexin receptor type 1 and 2, while orexin-B has a preferential affinity for orexin receptor type 2. The broad projections of the orexinergic system have led to its implication in a variety of functions,
Azhdari Zarmehri et al including feeding, sleep-wake cycle, cardiovascular function, hormone secretion,8,20,22,23 and, more recently, the modulation of nociceptive processing.4,5,18,24,26,27 Initial support for this idea came from a study in which intracerebroventricular (ICV) Orexin-A (3–30 mg) produced a dose-related analgesia in the hotplate test in rats.4 Systemic (iv) administration of orexin-A also proved to be effective in this rat model and also in the equivalent mouse paradigm at doses between 3 and 30 mg/kg.4 In other mouse models, orexin-A inhibited the visceral nociception (abdominal constriction; 10 and 30 mg/kg) and thermal hyperalgesia (in intraplantar carrageenan model; 3–30 mg/kg), with an efficacy equivalent to the opioid analgesic morphine.4 Morphological studies have established that the orexin-containing neurons and fibers as well as orexin receptors (orexin receptor type 1 and 2) are distributed along all parts of pain circuitry, including the periaqueductal gray matter (PAG) region which is considered to be important for pain modulation.13,19,25 Stimulation of the lateral hypothalamus elicits antinociception via relays to the PAG and the rostral ventromedial medulla (RVM), which ultimately triggers the activation of descending noradrenergic pathways.3 Electrical stimulation applied in the PAG produced antinociception analogous to 10 mg/kg morphine.28 Like electrical stimulation, opioid administration into the ventrolateral parts of PAG also produces robust behavioral signs of antinociception.16 Although orexin-A exhibits analgesic effects when administered via ICV or intrathecal microinjection,4,17,26 the effect of orexin-A in the PAG is unclear. In the present study, the effect of orexin microinjected into the PAG region on the tail-flick test and formalin test was investigated. The tail-flick test was used to evaluate the acute nociceptive transmission, and the formalin test to reflect an inflammatory pain condition to measure its therapeutic potential. Portions of these data were presented as abstracts at Neuro2010.1
Methods Subjects All experiments involving the animals were conducted according to the policy of Iranian Convention for the Protection of Vertebrate Animals used for experiments and the protocol was approved by the Ethics Committee of the School of Medical Sciences, Tarbiat Modares University (TMU), Tehran, Iran. Efforts were made throughout the experiments to minimize the animal discomfort and to reduce the number of animals used. Adult male, Sprague-Dawley rats (220–300 g) purchased from Razi Institute (Hesarak Karj, Iran), were housed in groups of 3 in a temperature controlled room, under a 12 hour light/ dark cycle with lights on at 0700 to 1900. Food and water were provided ad libitum. During the experiments, attention was strictly paid to the regulations of local authorities for handling laboratory animals.
Surgical Preparation for Intra-PAG and ICV Microinjections To perform direct intra-PAG and ICV administrations of drugs or the respective vehicle (saline), rats were anaes-
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thetized with Ketamine (100 mg/kg)/Xylazine (10 mg/ kg). The rats were placed in a stereotaxic apparatus (Narishige, Japan), holes were drilled in the skull over the PAG, and the dura was removed to allow the placement of a guide cannula. 23-gauge, 5 mm-long stainless steel guide cannula was stereotaxically lowered until its tip was 2 mm above the PAG by applying coordinates from the atlas of Paxinos and Watson10 (for PAG: A, 7.8 mm from bregma; L, .5 mm; V, 5.7 mm below the bregma and for ICV A, .9 mm from bregma; L, .1.8 mm; V, 3.8 mm below the bregma). The cannula was anchored with dental cement to a stainless steel screw in the skull. The guide cannula for intra-PAG microinjection was implanted 7 days before the experiment for pain formalin or tail-flick tests in awake rats. Immediately after waking from surgery, rats were returned to their home cages to await the formalin test procedure. Direct intra-PAG administration of drugs, or respective vehicle, was conducted with a stainless steel cannula (30 gauge) connected via a polyethylene tube to a Hamilton syringe, inserted through the guide cannula, and extended 2 mm beyond the tip of the guide cannula to reach the PAG. At day 7, while the animals were habituated, volumes of .5 mL of drug solutions or vehicles were injected into the PAG over a period of 60 seconds or 5 mL orexin-A 10 nM or vehicles injected into the ICV over a period of 100 seconds and the injection cannula was gently removed later. After 5 minutes, formalin was injected into the plantar surface of the right hind paw using a disposable insulin syringe with a fixed 30-gauge needle. A separate group of animals were used for conducting tail-flick test.
Tail-Flick Test Latency to tail-flick against heat was used as a measure of nociceptive responsiveness. At day 7, rats were kept in a glass restrainer and the dorsal surface of the tail between 4 and 6 cm from the tip of the tail was exposed to a beam of light generated from an automated analgesia meter (Harvard Tail-flick Analgesia Meter, Holliston, MA). The timer stopped when the animal flicked its tail away from the beam of light. Tail-flick latency was measured at 5-minute intervals until a stable baseline was obtained over 3 consecutive trials. The latency was measured 10 minutes after intra-PAG orexin-A injection up to 60 minutes. Orexin-A (.01–10 nM) and saline were given intra-PAG, in experimental and vehicle groups, respectively. Latencies were determined twice at 10-minutes interval after the administration of orexin-A or vehicle, and the average of 2 frequent readings was taken as the predrug latency. To avoid tissue damage, a 10-second cut-off was used.
Formalin-Induced Nociceptive Behavior Formalin-induced nociceptive behavior is a widely used animal model of persistent pain.2,7 Formalininduced nociceptive behavior was used because it employs an adequate painful stimulus; the animals show a spontaneous response and it is sensitive to the commonly used analgesics. Moreover, the pain stimulus
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is a continuous rather than a transient one and thus may have resemblance to some kinds of clinical pain, and the observations were obtained from the animals which were under mild or no stress. Also, the test employs a chemical nociceptive stimulus that has 2 distinct phases which possibly indicate different types of pain modulations.14,15 Rats were moved to the test room at least 1 hour before the commencement of the experiment. In the present study, the rats were first acclimatized for 30
Orexin-A Decreased Formalin-Induced Nociceptive Behaviors minutes in an acrylic observation chamber (30 cm in diameter and in height) and then 50 mL of 2% formalin was injected subcutaneously into the plantar surface of the right hind paw with a 30-gauge needle. Each rat was then immediately returned to the observation box, and the behavioral recording commenced. A mirror, placed at a 45 angle beneath the box, permitted the observation of behaviors without moving the box. Pain behavior was scored as follows: 0, the injected paw was not favored; 1, the injected paw had little or no weight
Figure 1. Time scores of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 to 8 rats per group) following intraperiaqueductal gray matter (PAG) infusion of the saline (.5 mL, A1) or orexin-A and control site (A2-A6) measured every 3 minutes for 60 minutes (A) and bar chart for different doses of orexin-A 1, 5, 10, 20 nM/.5 mL) in the formalin test and control site (B1-B6). The columns represent the mean of nociceptive score in each phase: phase 1 (minutes 1–7), interphase (minutes 8–14), and phase 2 (minutes 15–60) (B1-B6). Rats received orexin-A into the PAG 5 minutes before formalin injection in the right hind paw. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. For clarity, each dose of orexin-A was plotted in separate graphs, with the same vehicle group in each. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the orexin-A groups, open circles correspond to the vehicle group. All microinjections were made on the right side of the PAG, and are separated here for clarity only. *P < .05; ***P < .001 in comparison with saline.
Azhdari Zarmehri et al placed on it; 2, the injected paw was elevated and not in contact with any surface; and 3, the injected paw was licked or bit. Scores were continuously observed for the duration of the experiment (60 minutes). The nociceptive behaviors score for each 3-minute interval was calculated as the weighted average of the number of seconds spent in each nociceptive behavioral, from the start of the experiment. The scores were recorded in normal rats as well as in those who received orexin-A. In each group, the behavioral response of each rat during the first phase (1–7 minutes), interphase (8–14), and the second phase (15–60) was separately evaluated.
Drugs Two percent formalin was prepared by diluting 37% formaldehyde (ie, the commercially available saturated aqueous solution of formaldehyde, (Temad, Iran) 2:100 with sterile physiological saline solution. Morphine sulfate (Temad, Iran) was dissolved in saline. Orexin-A (molecular weight = 3,561, Sigma) was dissolved in saline, an orexin-1 receptor agonist; SB 334867 (N-(2-methyl-6benzorexin-Azolyl)-N00 -1,5-naphthyridin-4-yl urea; (molecular weight = 356, Tocris), was dissolved in dimethyl sulfoxide (DMSO) and diluted in saline to reach the final concentration. All drugs were diluted in saline immediately before use on the morning of an experiment.
Histology At the end of the experiments, the animals were deeply anaesthetized with Ketamine overdose, a volume of .5 mL of pontamin sky blue (.2%) was also injected in the cannula 10 to 20 minutes before sacrificing the rat. Rats were then perfused intracardially with 100 mL of 4% formalin solution and the brain was removed and sectioned. Only those rats whose microinjection site and diffusion were located within the PAG were included in the results.10 In all experiments, the orexin-A was microinjected into the right PAG and the formalin injected into the plantar surface of the right hind paw.
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Data Analysis Data are presented as mean 6 S.E.M. and analyzed by 1-way analysis of variance and t-test between groups. The mean nociceptive score in each phase (I, interphase, II) of the formalin test for the effect of each dose of orexin-A was analyzed using 1-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Phase 1 (1–7 minutes), the interphase (8–14 minutes), and the phase 2 (15–60 minutes) of the formalin test were analyzed separately. The defined level for statistical significance was P < .05.
Results Effects of Different Doses of Orexin-A Injected into the PAG on FormalinInduced Nociceptive Behaviors Formalin produced typical biphasic pain responses. The first and second phases were separated by a brief interphase where little to no nociceptive behavior was observed in control group. Microinjections of saline (.5 mL), into the PAG, 5 minutes before the formalin, failed to change the pattern of the early and late hyperalgesic behaviors induced by formalin (Fig 1). While the intra-PAG microinjection of 5, 10, and 20 nM orexin-A decreased the formalin-induced nociceptive behaviors in phase 2 compared with saline-treated rats (Fig 1), the injection of 10 nM of orexin-A caused a decrease in formalin-induced nociceptive behaviors in the interphase (Fig 1). Control injections of orexin-A into the sites near the PAG resulted in less or no reduction of pain, indicating that the analgesia observed is probably due to a site of action within the PAG rather than the surrounding neural structures (Fig 1; A6, B6, and C6, n = 12). In this study, the selective effect of orexin-A on the early second phase response was intriguing and the effect of orexin on the second phase showed to be clearly time dependent. To investigate whether the orexin has a special action on the early part of the second phase, or its delayed
Figure 2. Time scores of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 to 8 rats per group) following intraperiaqueductal gray matter (PAG) infusion of the orexin-A (10 nM) measured every 3 minutes for 60 minutes (A) and bar chart (B). The columns represent the mean of nociceptive score in each phase: phase 1 (minutes 1–7), interphase (minutes 8–14), and phase 2 (minutes 15–60). Rats received orexin-A into the PAG 5 and 10 minutes before formalin injection in the right hind paw. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the orexin-A (5 minutes before formalin test) group, open circles correspond to the orexin-A (10 minutes before formalin test) group. All microinjections were made on the right side of the PAG, and are separated here for clarity only.
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effects are related to its pharmacokinetics, orexin-A was injected into the PAG, 10 minutes before the formalin injection. No difference between 5- and 10-minute injections of orexin-A prior to formalin injection was demonstrated (Fig 2, P > .05).
Dose-Response Relationships of Orexin-A Injected into the PAG on Formalin-Induced Nociceptive Behaviors The dose-response relationships of intra-PAG administered orexin-A on the first phase, interphase and the second phase of the formalin-induced nociceptive behaviors is shown in Fig 3.
Comparing the Effects of Morphine and Orexin-A Injected into the PAG on Formalin-Induced Nociceptive Behaviors Intra-PAG microinjection of morphine (.5 mL of 20 mM) vigorously decreased the formalin-induced nociceptive behaviors in phase 1, interphase as well as the phase 2 of the formalin test (Fig 4). As mentioned earlier, 10-nM orexin-A decreased the formalin-induced nociceptive behaviors in the interphase and phase 2, but not in phase 1.
Effects of SB-334867 (a Type 1 Orexin Receptor Antagonist) Injected into the PAG on Orexin-A-Induced Attenuation of Nociceptive Behaviors in Formalin Test Pretreatment with SB-334867 (100 nM/.5 mL) antagonized the effect of 10-nM orexin-A on the interphase and phase 2 nociceptive behaviors (Fig 5). Intra-PAG microinjection of SB-334867 produced no effect on phase 1, interphase, and the phase 2 in formalin test compared
Figure 3. Dose-response curves of formalin-induced nociceptive behaviors during phase 1 (minutes 1–7), interphase (minutes 8–14), and phase 2 (minutes 15–60) after microinjection of different dose of orexin-A. The ordinate scale shows percent 6 S.E.M. relative to the control group in formalin-induced nociceptive behaviors. Drugs were administered intraperiaqueductal gray matter (PAG) 5 minutes before formalin injection in the right hind paw. Orexin-A in 10 nM reduced formalin-induced nociceptive behaviors in the inter-phase (P < .001) and phase 2 (P < .05), and in 5 and 20 nM reduced formalin-induced nociceptive behaviors only phase 2 (P < .05). *P < .05; ***P < .001 in comparison with saline.
Orexin-A Decreased Formalin-Induced Nociceptive Behaviors with the saline-treated rats (Fig 5). The SB 334867 was microinjected 5 minutes before the orexin-A injection.
Effects Of Orexin-A Injected into the ICV on Formalin-Induced Nociceptive Behaviors and Orexin-A Injected into the PAG on Tail-Flick Withdrawal Test To determine the effect of ICV injection of orexin-A on formalin test, the orexin-A (10 nM, 5 mL) was injected into the right ventricle and the formalin-induced nociceptive behaviors were monitored. In this experiment, orexin-A failed to elicit significant changes in formalin-induced nociceptive behaviors (for phase 1: 1.89 6 .16, F > .05; for interphase: .98 6 .35, F > .05; for phase 2: 1.78 6 .11, F > .05; n = 10). Unilateral injections of saline (.5 mL) into the right PAG also caused no significant changes in latency of the tail-flick test. Orexin-A, injected unilaterally into the PAG at the concentrations up to 10 nM, demonstrated no significant effect on thermal tail-flick test (Table 1).
Discussion In this study we investigated the role of the midbrain PAG as a common substrate in orexin-A anti-nociception in formalin and tail-flick tests. The injection of orexin-A into the nuclei of the descending antinociceptive pathway may provide an opportunity to investigate the role of this neurotransmitter in the up-down modulating pain system. Orexin is produced exclusively in the dorsal and lateral hypothalamus,6,21 the regions known to modulate pain processing through the PAG.3 In addition, Orexin projections are widespread within the brain regions that play an important role in descending pain modulation such as PAG.13,19,25 We clearly demonstrated that the microinjection of orexin-A (5 nM) into the PAG reduced the formalin-evoked nociceptive behaviors in the interphase and the late phase but not the initial phase. Orexin-A had no effect on tail flick up to a dose of 10 nM that was effective in formalin test. Moreover, the SB-334867, a selective type 1 orexin receptor antagonist, antagonized the antinociceptive effect of orexin-A in the formalin test. Microinjection of morphine produced persistent and profound analgesia in all phases of the formalin test and the effect of orexin was less than the effect observed with morphine. In previous studies, the systemic administration of orexin-A produced a dose-related analgesia in hot plate analgesic test in rats4 and inhibited the visceral nociception (10 and 30 mg/kg) and thermal hyperalgesia induced with carrageenan (3–30 mg/kg), with an efficacy equivalent to the opioid analgesic morphine.4,26 Our findings on the antinociceptive effects of orexin-A are consistent with previous studies in which the intrathecal delivery of orexin-A was reported to have inhibited the pain perception.26 The site(s) of analgesic action of orexin-A following iv and ICV administration remains to be established. It has been suggested that the antinociceptive effects of
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Figure 4. Time scores of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 rats per group) following intra-periaqueductal
gray matter (PAG) infusion of the orexin-A (10 nM/.5 mL) and morphine (20 mM/.5 mL) measured every 3 minutes for 60 minutes (A) and bar chart (B). The columns represent the mean of nociceptive scores in each phase: phase 1 (minutes 1–7), inter-phase (minutes 8–14), and phase 2 (minutes 15–60). Rats received orexin-A 5 minutes before formalin injection. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the orexin-A group, open circles correspond to the vehicle group and tingle correspond morphine group. All microinjections were made on the right side of the PAG, and are separated here for clarity only. *P < .05; ** P< .01; ***P < .001 in comparison with saline.
orexin-A and B are mediated via spinal and supraspinal mechanisms.4,26 According to dense interaction between the LH and PAG,3 the orexinergic innervations and its receptors expression in PAG,19 it can be suggested that the orexinergic pathway may mediate the interaction between these 2 sites. The difference between their effects on the first and second phase of the test may well be due to the mechanisms by which the formalin induces nociceptive behaviors in 2 phases. Subcutaneous injection of formalin generates typical biphasic nociceptive response that is characterized by an early phase (minutes 0–7), a quiescent interphase, and a second prolonged phase (minutes 15–55).2,7 The early phase, the quiescent interval, and the second delayed phase are all active phases. The capacity of orexin-A to attenuate the second phase of the nociceptive behaviors suggests that it recruits the putative descending inhibitory control pathways and also influences the formalin-induced spinal nociceptive processing during the interphase and the late phase.
It seems that the effect of orexin-A in the PAG may not be related to control of acute nociception, as there was no effect on the tail-flick test and the first phase of the formalin test. On the other hand, it may play a critical role in the modulation of the active inhibition on the interphase as well as the neuronal process occurring during the late phase of the formalin test. The period between the 2 phases of nociceptive responses is largely overlooked, probably as it is generally considered to be a phase of inactivity. There is some evidence that this interphase in the formalin test may be an active process due to the action of endogenous pain-suppressing mechanisms.9,11,12 Franklin et al9 reported that pentobarbital, diazepam, and alcohol inhibit the ‘‘inter-phase diminution’’ and argued that these GABAA receptor agonists unmask the pain that is suppressed by some inhibitory mechanism. Henry et al12 showed that after 2 injections of formalin (with 20-minute interval), there was a second diminution of nociceptive scores after the second formalin injection, rather than an additive sum of the
Figure 5. Time course of formalin-induced nociceptive behaviors (mean 6 S.E.M. of 6 to 8 rats per group) following intra-
periaqueductal gray matter (PAG) infusions of the vehicle 1 orexin-A (10 nM/.5 mL), SB-334867 (100 nM/.5 mL) 1 vehicle and orexin-A 1 SB-334867 measured every 3 minutes for 60 minutes (A) and bar chart for each phase (B). The columns represent the mean of nociceptive scores in each phase: phase 1 (minutes 1–7), interphase (minutes 8–14), and phase2 (minutes 15–60) (B). Rats received orexin-A 5 minutes before formalin injection. Recording of the nociceptive behaviors began immediately after formalin injection (time 0) and was continued for 60 minutes. Histological location10 of PAG microinjection sites (C). Fill circles correspond to the vehicle 1 orexin-A group, open circles correspond to the vehicle 1 vehicle group, square correspond SB334867 1 vehicle group and tingle correspond SB 334867 1 orexin-A group. All microinjections were made on the right side of the PAG. *P < .05; ***P < .001 in comparison with vehicle group.
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Mean Tail-Flick Latencies in Seconds (Mean 6 S.E.M. of 6 to 7 Rats per Group) of Test Given 2 Every 10 Minutes for 1 Hour Following Infusions of Orexin-A or Saline into the Periaqueductal Gray Matter (PAG)
Table 1.
REACTION TIME IN SECOND GROUPS
DOSE
PRE INJ.
10 MIN
20 MIN
30 MIN
40 MIN
50 MIN
60 MIN
Vehicle Orexin Orexin Orexin
0 .01 nM 1 nM 10 nM
6.3 6 .55 4.6 6 .20 5.7 6 .51 5.0 6 .43
5.8 6 .56 4.5 6 .32 5.1 6 .91 5.1 6 .46
6.2 6 .49 4.8 6 .38 5.2 6 .49 4.6 6 .31
5.9 6 .39 4.7 6 .07 5.1 6 .44 5.3 6 .84
5.5 6 .35 4.7 6 .4 5.3 6 .65 4.6 6 .47
5.9 6 .4 4.8 6 .26 5.6 6 1.06 5.4 6 .25
5.5 6 .32 4.9 6 .06 5.1 6 .69 5.5 6 .96
nociceptive responses to the 2 injections. Consistent with their suggestions that the diminution that normally occurs after the first phase of the second injection of formalin test is due to an active inhibitory process, the intra-PAG orexin-A injection increases the inhibitory period during this inter-phase.12 The active inhibitory phase may be considered as a form of nonopioid autoanalgesia brought about by the descending inhibitory controls arising above the PAG.9,12 The reduction in the nociceptive behaviors recorded during the first part of phase 2 may reflect a prolongation of the interphase in response to orexin microinjection or a short action on the phase 2 nociceptive mechanisms; even there are significant differences when analyzing the mean response for phase 2. In agreement with another report regarding the formalin and hot plate tests,4,26 the SB-334867, a selective type 1 orexin receptor antagonist, has no effect on formalin test when used alone, which suggests that a tonic orexin receptor type 1 mediated inhibitory system does not exist in the PAG during this test. It has been reported that the SB-334867 is acting as a pro-hyperalgesic in the mouse carrageenan assay (3 and 10 mg/kg IP), and this suggests a tonically activated type 1 orexin receptor mediated inhibitory system is present during a carrageenan-induced thermal hyperalgesia test.26 This may reflect the strain differences, different route of administration, differences between the models used (carrageenan hyperalgesia test and the formalin test), methodological differences (eg, lighting, noise, odors, handling stress, or anaesthesia prior to formalin injection, all of which are known to influence the test). Our
data corroborate the previous studies, all demonstrating that SB-334867 restores the analgesic effect of both ICV and intrathecal administration of orexin-A in mice and rats, respectively.4,26 It has been suggested that the antinociceptive effects of orexin-A are mediated via spinal and supraspinal mechanisms. Subsequently, we tested the antinociceptive activity of orexin-A and morphine in the formalin test. Microinjection of morphine produced persistent and profound analgesia in first, interphase, and late phase of formalin test. The present study has shown that the orexin causes a decrease in formalin induced nociceptive behaviors in interphase and late phase, although its antinociceptive effect is lower than morphine. In conclusion, the intra-PAG injection of orexin-A depresses the inter-phase and phase 2 in formalininduced nociceptive behaviors in rats without having any effect on tail-flick latency. These effects of orexin-A are mediated by the activation of type 1 orexin receptors expressed in the PAG. These data might suggest a potential therapeutic approach in treatment of pain.
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