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Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor
Accepted Manuscript Title: Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor Authors: Mina Taati, Esmaeal Tamaddonfard PII: DOI: Reference:
S1734-1140(17)30610-2 https://doi.org/10.1016/j.pharep.2017.12.010 PHAREP 840
To appear in: Received date: Revised date: Accepted date:
2-9-2017 13-12-2017 20-12-2017
Please cite this article as: Mina Taati, Esmaeal Tamaddonfard, Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor (2010), https://doi.org/10.1016/j.pharep.2017.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor Mina Taati, Esmaeal Tamaddonfard*
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Division of Physiology, Department of Basic sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
Corresponding author: Esmaeal Tamaddonfard, Tel: +98 44 32770508, Fax: +98 44 32771926, Email: [email protected]; [email protected]
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Short title: oxytocin in VLOC-PAG modulation of neuropathic pain
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Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal
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gray opioid receptors
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Intra-VLOC microinjection of oxytocin attenuated mechanical allodynia. Atosiban and naloxone prevented antiallodynic effect of oxytocin. Intra-vlPAG administration of naloxone prevented intra-VLOC oxytocin-induced antiallodynia. Opioid receptors of VLOC and vlPAG may be involved in VLOC oxytocin-induced antiallodynia.
ABSTRACT
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Highlights
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Background: Oxytocin plays an important role in supraspinal modulation of pain. In the present
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study, we investigated the effects of ventrolateral orbital cortex (VLOC) microinjection of oxytocin on neuropathic pain after blockade of opioid receptors in this area and ventrolateral
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periaqueductal gray (vlPAG). Methods: Neuropathic pain was induced by complete transcection of preoneal and tibial branches of sciatic nerve. The VLOC and vlPAG were unilaterally (contralateral to the sciatic nerve-injured side) and bilaterally implanted with guide cannulas, respectively. Mechanical paw withdrawal threshold (PWT) was measured using von-Frey filaments. Area under curve (AUC) was also calculated. 1
Results: Microinjection of oxytocin (5, 10 and 20 ng/site) into the VLOC increased PWT. Antiallodynia induced by oxytocin (20 ng/site) was inhibited by prior intra-VLOC administration of atosiban (an oxytocin receptor antagonist, 100 ng/site) and naloxone (an opioid receptor antagonist, 500 ng/site). Prior microinjection of naloxone (500 ng/site) into the vlPAG also
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inhibited antiallodynia induced by intra-VLOC microinjection of oxytocin (20 ng/site). All the VLOC and vlPAG microinjected drugs did not alter locomotor activity.
Conclusions: It is concluded that oxytocin and its receptor may be involved in modulation of neuropathic pain at the VLOC level. Opioid receptors of VLOC and vlPAG might be involved in the antiallodynic effect of the VLOC-microinjected oxytocin.
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Keywords: Oxytocin; Ventrolateral orbital cortex; Periaqueductal gray; Opioid receptor;
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Neuropathic pain
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Introduction
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Oxytocin producing neurons are located in the paraventricular nucleus (PVN) and suprachiasmatic nucleus (SCN) of the hypothalamus [1]. Due to difficult blood-brain barrier crossing of oxytocin,
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the brain-related effects of this neuropeptide are originated from centrally projecting neurons [2].
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The brain areas such as amygdala, prefrontal cortex (PFC), orbital cortex (OC) subdivisions and
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periaqueductal gray (PAG) are innervated by oxytocin neurons [3]. Oxytocin is released in response to various stimuli and acts through oxytocin receptors, which are widely distributed in
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the brain [1]. This neuropeptide modulates many brain functions such as maternal and social behaviors, learning, anxiety, epilepsy and pain processing [3-7]. Some researchers have suggested
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the involvement of opioid receptors in the antinociceptive effects of supraspinal oxytocin [8, 9]. The ventrolateral orbital cortex (VLOC) has a potent role in pain processing [10, 11]. Electrical stimulation of the VLOC depressed jaw-opening reflex evoked by facial skin stimulation [12]. Using single neuron electrical activity recording, Follett and Dirks [13] found the involvement of VLOC in visceral pain modulation. In this context, prior intra-VLOC microinjection of yohimbin 2
(a selective α-2 adrenoceptor antagonist) blocked the anti-allodynic effect induced by the same site microinjection of a selective α-2 adrenoceptor agonist, clonidine [14]. The distribution of oxytocin and opioid receptors has been reported in orbitofrontal cortex (OFC)
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structures such as OC subdivisions [3]. In addition, Mansour et al. [15] reported opioid receptors distribution in the ventral PAG (vPAG) and dorsal PAG (dPAG) in rats. This study was planned to investigate the effects of microinjection of oxytocin, atosiban (an oxytocin receptor antagonist) and naloxone (an opioid receptor antagonist) into the VLOC on spared nerve injury (SNI)-induced neuropathic pain. In addition, the effect of VLOC microinjection of oxytocin was also evaluated
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after blockade of ventrolateral PAG (vlPAG) opioid receptors. The SNI model of neuropathic pain
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was introduced by Decosterd and Woolf [16]. The roles of VLOC [11, 14], and PAG [17, 18] in
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processing of SNI-induced neuropathic pain have been reported in rats. Using an electronic activity
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box, we examined the effects of the above-mentioned drugs on locomotor activity. Materials and methods
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Animals
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In the present study, a total of 96 healthy, 10-12 months old male Wistar rats weighing 250–280 g
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were used. The animals were maintained in a controlled ambient temperature laboratory (22 ± 0.5 °C) at 12 h light-dark cycles (light on at 07:00 h). Commercial food and water are available to
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them. The experiments were performed between 10:00 h and 14:00 h. Drugs
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In the present study, the following drugs were used: oxytocin, atosiban and naloxone dihydrochloride. Drugs were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA, and dissolved in sterile normal saline 30 min before intra-VLOC and intra-vlPAG microinjections. Spared nerve injury
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SNI model of neuropathic pain was performed as previously described [16]. Briefly, the rats were anaesthetized with intraperitoneal (ip) injection of a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg), and an incision was then made along the back of the left thigh. The biceps femoris
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muscle was separated to expose the tibial, peroneal and sural branches of the sciatic nerve. With no manipulation of sural nerve, the tibial and peroneal nerves were tightly ligated using 5-0 silk and transected distal to the ligation [19]. Following surgery, the rats were allowed to recover for one week before implantation of guide cannulas. Intracerebral guide cannula placement
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One week after SNI, using a stereotaxic apparatus (Stoelting, Wood Lane, IL, USA), the VLOC
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and vlPAG were implanted with guide cannulas in ketamine (80 mg/kg)-xylazine (8 mg/kg)
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anesthetized rats. A stainless-steel guide cannula (23-gauge, 12-mm) was unilaterally
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(contralateral to the sciatic nerve-injured side) inserted 1 mm above the right side of the VLOC at the following coordinates: 3.2 mm anterior to the bregma, 2.2 mm left side of the midline, and 4.6
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mm below the top of the skull [20]. The vlPAG were bilaterally implanted with two guide cannulas
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at the following coordinates: 7.8 mm posterior to the bregma, 0.8 mm left and right sides of the
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midline, and 5.5 mm below the top of the skull [20]. We used unilateral cannulation of the VLOC and bilateral cannulation of vlPAG, because spinal cord-thalamic submedius (Sm) projections are
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bilateral with a contralateral dominance [21]. In addition, the reciprocal connections between Sm and VLOC are largely ipsilateral in the rat [22]. On the other hand, the VLOC contains neurons
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projecting bilaterally to the vlPAG [23]. The cannulas were then fixed to the skull by three screws and dental acrylic. At least, seven days were allowed before intra-VLOC and intra-vlPAG microinjections. Intra-VLOC and intra-vlPAG microinjections
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Using a 30-guage, 13 mm needle attached to a 1 µl Hamilton syringe, intra-VLOC and intra-vlPAG microinjections of normal saline (control) and test drugs were performed. The VLOC and each vlPAG were microinjected with constant volumes of 0.5 and 0.25 µl of test drugs, respectively,
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over a period of 60 seconds. The injection needle was left in place for a further 60 seconds to facilitate diffusion of the drug. Mechanical paw withdrawal threshold measurement
Two weeks after SNI, paw withdrawal threshold (PWT) was measured in response to application of von Frey filaments using the up-down method [24, 25]. Animals were placed in a transparent
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plastic box (25 × 15 × 20 cm) with a metal wire mesh floor. Eleven von Frey filaments (IITC-Life
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Science Instruments, Woodland Hill, CA) were chosen (von Frey numbers: 3, 4, 5, 6, 7, 8, 9, 10,
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11, 12 and 13, equivalent to 0.4, 0.8, 1, 1.2, 1.5, 2.5, 3.6, 4, 8, 10, 20 g, respectively). Starting with
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middle filament (number 8, equivalent to 2.5 g) in the series, von Frey filaments were repeatedly applied over a 3-s time interval from below and perpendicular to the fourth and fifth toes of the
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hind paw for approximately 5-7 s. The sural nerve innervates the lateral part of the hind paw
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including parts of the 4th digit and the entire 5th digit [26]. If there was a withdrawal response to
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filament stimulation (positive), the next lower force was delivered. If response was negative, the next higher force was applied. After recording of positive and negative responses, 50% threshold
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were calculated using the following formula, which provided by Dixon, [24] and Chaplan et al. [25]: 50% threshold = 10(Xf+kδ), where Xf is the value of the final von Frey filament used (in log
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units), k is the tabular value for the pattern of positive/negative responses presented at the appendix 1 of Chaplan et al., [25] article, and δ is the mean difference between stimuli in log units (0.155). This formula, with a slight modification, has been frequently used to calculate 50% PWT in SNI model of neuropathic pain [27-29]. Values of 0.4 or 20.0 g were assigned where continuous
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positive or negative responses were observed all the way out to the end of the stimulus spectrum, respectively. Study protocol
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Fig. 1 shows drug microinjection and allodynia recording time line. To examine the role of oxytocin and opioid receptors in VLOC modulation of neuropathic pain, 66 rats were divided into 11 groups and intra-VLOC microinjections of normal saline plus normal saline (control), normal saline plus oxytocin (2.5, , 5, 10 and 20 ng/site), atosiban (10 and 100 ng/site) plus normal saline, atosiban (100 ng/site) plus oxytocin (20 ng/site), naloxone (100 and 500 ng/site) plus normal saline
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and naloxone (500 ng/site) plus oxytocin (20 ng/site) were performed (Fig. 1A). To explore the
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role of vlPAG opioid receptor in VLOC-microinjected oxytocin effect, 30 rats were divided into
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5 groups and the following intra-vlPAG and intra-VLOC were performed: normal saline (vlPAG)
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plus normal saline (VLOC), naloxone (100 ng/site, vlPAG) plus normal saline (VLOC), naloxone (500 ng/site, vlPAG) plus normal saline (VLOC), normal saline (vlPAG) plus oxytocin (20 ng/site,
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VLOC) and naloxone (500 ng/site, vlPAG) plus oxytocin (20 ng/site, VLOC). Atosiban and
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naloxone were microinjected six min before initiation of PWT recording. Oxytocin was
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microinjected three min after atosiban and naloxone microinjection (Fig. 1B). The PWT was measured immediately (time point 0), 10, 20, 30, 40, 50 and 60 min after intra-structural injection
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(Fig. 1A and 1B). The experimenters were blinded to the treatment protocol. The doses of drugs used here were according to previous studies [6, 30] and our preliminary experiments.
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Locomotor activity We used an electronic activity box (BorjSanat, Tehran, Iran) to assess locomotor activity. The animals were placed in a Plexiglas chamber (40 × 40 × 40 cm). The number of beam breaks due to animal movement was recorded from the monitor of the apparatus in a 5-min session.
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Verification of cannula tip placement Initially, 0.5 and 0.25 µl methylene blue were microinjected into the VLOC and each side of vlPAG, respectively. Animals were deeply anaesthetized with a mixture of ketamine and xylazine and perfused intracardially with physiological saline followed by 10% formalin solution. The
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brains were removed and placed in a 10% formalin solution. Twenty-four h later, we provided transverse sections (50–100 µm) and viewed under a loupe to localize the injection sites according to the atlas of Paxinos and Watson [20]. Statistical analysis
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The Graph Pad Prism version 5 (Graph Pad Software Inc, San Diego, CA USA) was used to
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analyzing the results. The data obtained from 10-min time points were analyzed using two-way
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repeated measure analysis of variance (ANOVA) and then Bonferroni post hoc test. The area under
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curve (AUC) data was analyzed by one-way analysis of variance (ANOVA), and post hoc Tukey’s
Results
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Cannulas tip verification
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test. Values are expressed as mean ± SEM. p < 0.05 was considered statistically significant.
The location of the cannulas tip placement in the VLOC (Fig. 2B) and vlPAG (Fig. 2C, right side)
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were confirmed in the brain section. The rat brain sections of VLOC (Fig. 2A) and vlPAG (Fig.
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2C, left side) were adopted from the atlas of Paxinos and Watson [20]. Effects of intra-VLOC microinjection of oxytocin, atosiban and naloxone on SNI-induced
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allodynia
The 50% PWT were approximately between 2.0 and 3.0 g (2.68± 0.23 g) at all time points after intra-VLOC microinjection of normal saline. Fig. 3A shows significant different between treatments (F(4,175) = 168.23, p < 0.05), across times (F(6, 175) = 35.38, p < 0.05), and between interactions (F(24,175) = 7.08, p < 0.05). In this context, oxytocin at a dose of 2.5 ng/site did not alter 7
50% PWT, whereas at a dose of 5 ng/site increased 50% PWT at 20, 30 and 40 min time points, and at a dose of 10 ng/site it increased 50% PWT at 10, 20, 30, 40 and 50 min time points. Oxytocin (20 ng/site) increased 50% PWT at all time points (Fig. 3A). Fig. 3B shows significant different
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between treatments (F(4,175) = 222.71, p < 0.05), across times (F(6, 175) = 10.16, p < 0.05), and between interactions (F(24,175) = 9.55, p < 0.05). Alone intra-VLO microinjection of atosiban (20 and 100 ng/site) produced no significant effects on 50% PWT. Prior microinjection of atosiban (100 ng/site) prevented oxytocin (20 ng/site)-induced antiallodynia (Fig. 3B). Fig. 3C shows significant different between treatments (F(4,175) = 249.9, p < 0.05), across times (F(6, 175) = 10.84,
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p < 0.05), and between interactions (F(24,175) = 10.47, p < 0.05). Intra-VLO microinjection of
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naloxone (100 and 500 ng/site) alone produced no significant effect on 50% PWT. Antiallodynia
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induced by intra-VLOC microinjection of oxytocin (20 ng/site) was prevented by prior
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microinjection of 500 ng/site naloxone (Fig. 3C). Analyses of AUC on PWT showed that the AUC of 5, 10 and 20 ng/site oxytocin were significantly (F(4, 29) = 20.98, p < 0.05) larger than the saline
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normal treated group (Fig. 3D). Prior microinjection of 100 ng/site atosiban (F(4,29) = 21.93, p
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<0.05) and 500 ng/site naloxone (F(4,29) = 40.38, p <0.05) significantly decreased the increased
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AUC induced by oxytocin (20 ng/site) and reached it to the saline normal treated AUC (Fig. 3D). Effects of intra-vlPAG microinjection of naloxone on antiallodynic effect induced by intra-VLOC
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microinjection of oxytocin
As shown in Fig. 4A, time-course curve were significantly different between treatments (F(4,175) =
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222.9, p < 0.05), across times (F(6, 175) = 15.36, p < 0.05), and between interactions (F(24,175) = 9.91, p < 0.05). Further analyses showed that intra-VLO microinjection of oxytocin (20 ng/site) increased 50% PWT at all time points. Allodynia was not changed after intra-vlPAG microinjection of naloxone at doses of 100 and 500 ng/site. Prior intra-vlPAG microinjection of
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naloxone (500 ng/site) prevented the antiallodynia induced by microinjection of oxytocin (20 ng/site) into the VLOC (Fig. 4A). Analyses of AUC showed that the increased AUC induced by intra-VLOC microinjection of oxytocin (20 ng/site) was significantly (F(4,29) = 35.42, p <0.05)
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decreased by prior microinjection of naloxone (500 ng/site) into the vlPAG (Fig. 4B). Effects of test drugs on locomotor activity
The numbers of photo beam break in intact, normal saline (VLOC) plus normal saline (VLOC) and normal saline (vlPAG) plus normal saline (VLOC) were 82.5 ± 5.28, 75.5 ± 6.23 and 79.2 ± 5.38, respectively. Microinjection of oxytocin, naloxone and atosiban into the VLOC and naloxone
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into the vlPAG did not alter photo beam breaks number (Data not shown).
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Discussion
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In the present study, the 50% PWT were approximately between 2.0 and 3.0 g at all time points on 14th day after induction of SNI. This is in consistent with other findings in which 1.5 – 2.0 g
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and 2.5 – 3.5 g PWT have been reported on day 14 after SNI [14, 27, 28]. The PWT less than 4.0
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g indicates a successful mechanical allodynia in SNI model of neuropathic pain [14, 27, 28]. This
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is a novel animal model of neuropathic pain developed by Decosterd and Wolf [16]. In contrast to chronic constriction injury (CCI), partial sciatic nerve ligation (PSL) and spinal nerve ligation
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(SNL) models with regenerative capacity of injured nerves leading to abnormal pain sensation and behavior, mechanical allodynia occurs within four days of injury and persists for several weeks in
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SNI model [31]. Long-term plastic changes occurring along sensory pathways, from peripheral nociceptors to spinal cord and pain-processing brain regions contribute to induction of mechanical allodynia [32]. Recent evidences have suggested plastic changes occur in frontal, retrosplenial,
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entorhinal and prefrontal cortices after SNI [33]. For this reason, SNI has been frequently used in the study of supraspinal mechanisms of neuropathic pain [14, 27, 28]. Our present results showed that microinjection of oxytocin into the VLOC suppressed mechanical
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allodynia, and prior microinjection of atosiban into the same site prevented this allodynia. The VLOC has a central role in spinal cord/medulla-Sm-VLOC-PAG-spinal cord/medulla pain pathway of acute and chronic pain modulation [11]. In addition, opioid, serotonin, dopamine and noradrenalin receptors of the VLOC participate in modulation of SNI-induced neuropathic pain [11, 14, 34, 35]. Although there are no reports showing the involvement of VLOC oxytocin in
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neuropathic pain modulation, electrical stimulation of PVN increased cerebrospinal fluid (CSF)
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concentration of oxytocin and suppressed mechanical allodynia and heat hyperalgesia in CCI
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model of neuropathic pain [36]. In this context, Kubo et al. [37] reported that direct administration
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of oxytocin into the trigeminal ganglion suppressed mechanical hypersensitivity induced by partial ligation of infraorbital nerve. Oxytocin receptor distribution and oxytocinergic fiber projection in
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pain processing areas and nuclei such as PFC, ventral tegmental area (VTA), nucleus accumbens
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(NAcc), amygdala, hippocampus, raphe nucleus (RN) and PAG, and difficult crossing of oxytocin
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through blood-brain barrier [7, 38], can explain oxytocin contribution to supraspinal mechanisms of pain. At the PAG level, pre-administration of desGly-NH2,d(CH2)5[D-Tyr2,Thr-Sup-4]OVT,
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an oxytocin antagonist, inhibited the analgesic effect of oxytocin in tail skin potassium iontophoresis model of acute pain [39]. In addition, prior microinjection of selective oxytocin
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receptor antagonist, 1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin (atosiban) into the central nucleus of amygdala prevented the suppressive effects of oxytocin on acute pain induced by thermal and mechanical stimulation of hind paw [40]. Taken together, the results of the present
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study reveal for the first time that oxytocin and its receptor at the VLOC can modulate neuropathic pain in rats. In the present study, prior microinjection of naloxone into the VLOC inhibited oxytocin-induced
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antiallodynia. Opioid receptors are distributed in the VLOC and involved in antiallodynic effects of endogenous opioid peptides in neuropathic pain [35, 41]. In addition, at the level of VLOC, opioid receptors mediate the antinociceptive effects of a variety of neurotransmitters such as gama amino butyric acid (GABA), dopamine and serotonin [11]. Opioid receptors are involved in the centrally-administered oxytocin-induced antinociception. For example, naloxone inhibited the
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suppressive effects of centrally-administration oxytocin on both phases of formalin-induced pain
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in mice [42].
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Our present results showed that prior microinjection of naloxone into the vlPAG inhibited
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antiallodynia induced by intra-VLOC microinjection of oxytocin. Neuroanatomical and functional connections exist between subdivisions of OC and PAG [10, 11]. In this context, functional
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blockade of the PAG eliminated chemically-induced activation of VLOC depresses of nocifensive
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reflexes [11]. Moreover, bilateral electrolytic lesions of the lateral and ventrolateral parts of PAG
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and bilateral microinjection of GABA into the vlPAG reduced or eliminated the VLOC-evoked inhibition of tail-flick reflex [43, 44]. The PAG receives inputs from cortical sites and has
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reciprocal connections with the amygdala as well as ascending nociceptive inputs from the spinal cord [45]. Due to these connections, the PAG play a central role in pain modulation pathways such
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as motor cortex-PAG-spinal cord and PFC-amygdala-PAG pathways [46, 47]. Mu-opioid receptors are widely distributed in dorsomedial, lateral and ventrolateral sub-regions of PAG [48]. In this context, microinjection of naloxone into the PAG decreased the inhibitory effect of motor cortex stimulation on sensory evoked potentials induced by forepaw electrical stimulation [49]. In
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addition, the suppressive effect induced by intra-arcuate nucleus microinjection of galanin was inhibited by prior microinjection of naloxone into the PAG [50]. These results showed that the VLOC oxytocin plays an important role in modulation of neuropathic pain, and opioid receptors
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of the PAG may be involved in the antiallodynia induced by VLOC microinjection of oxytocin. In conclusion, the results of the present study showed that at the VLOC level oxytocin via its receptors as well as opioid receptors can modulate SNI model of neuropathic pain in rats. One of the mechanisms by which oxytocin modulates neuropathic pain may be associated with contribution of vlPAG opioid receptors.
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The authors declare that there are no conflicts of interest.
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Conflict of interest
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Acknowledgement
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This study was financially supported by the directorate of postgraduate studies of Urmia
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Figure legends
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Fig. 1. Drug microinjection and mechanical allodynia recording time line. At -30 min adaptation
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of animals to experimental conditions was performed. Six min after intra-VLOC microinjection of
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atosiban and naloxone (Fg.1A) and intra-vlPAG microinjection of naloxone (Fig. 1B) and three
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min after intra-VLOC (Fig. 1A) and intra-vlPAG microinjection of oxytocin (Fig. 1B), mechanical
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allodynia recording were begun (time point 0), and continued at 10, 20, 30, 40, 50 and 60 min time points after microinjections.
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Fig. 2. Schematic illustrations of the rat brain showing the approximate location of the ventrolateral orbital cortex (VLOC) and ventrolateral periaqueductal grey (vlPAG) microinjection sites. Section
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of the rat brain shows the location of the permanent cannula tip (black arrow) in the VLOC (Fig.
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2B) and vlPAG (Fig. 2C, right side) of rats included in the present study. Atlas plates of VLOC (Fig. 2A) and vlPAG (Fig. 2C, left side) were adopted from Paxinos and Watson, (1986). CGD: central gray, dorsal; CGLD: central gray, lateral dorsal; CGM: central gray, medial; DR: dorsal raphe nucleus; CGLV: central gray, lateral ventral (ventrolateral PAG), LOC: lateral orbital cortex, Cl: clastrum, fmi: forceps minor corpus collasum.
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Fig. 3. The effects of intra-VLOC microinjection of oxytocin, atosiban and naloxone on 50% paw withdrawal threshold (PWT, A, B and C) and area under curve (AUC, D) in spared nerve injury (SNI) model of neuropathic pain. Atosiban and naloxone were microinjected six min before and
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oxytocin was microinjected three min before mechanical allodynia recording. Values are the mean ± SEM of six rats per group. * p < 0.05 significantly different compared with normal saline treated group.
†
p < 0.05 significantly different compared with oxytocin (20 ng/site) group. VLOC:
ventrolateral orbital cortex, PWT: paw withdrawal threshold.
Fig. 4. The effects of intra-vlPAG microinjection of naloxone alone and before intra-VLOC
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microinjection of oxytocin on 50% paw withdrawal threshold (A) and area under curve (B) in
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spared nerve injury (SNI) model of neuropathic pain. Intra-vlPAG microinjection of naloxone and
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intra-VLOC microinjection of oxytocin were preformed six and three min before mechanical
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allodynia recording, respectively. Values are the mean ± SEM of six rats per group. * p < 0.05 significantly different compared with normal saline treated group. † p < 0.05 significantly different
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compared with oxytocin (20 ng/site) group. VLOC: ventrolateral orbital cortex, PAG:
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periaqueductal gray, PWT: paw withdrawal threshold.
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S1734-1140(17)30610-2 https://doi.org/10.1016/j.pharep.2017.12.010 PHAREP 840
To appear in: Received date: Revised date: Accepted date:
2-9-2017 13-12-2017 20-12-2017
Please cite this article as: Mina Taati, Esmaeal Tamaddonfard, Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor (2010), https://doi.org/10.1016/j.pharep.2017.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor Mina Taati, Esmaeal Tamaddonfard*
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Division of Physiology, Department of Basic sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
Corresponding author: Esmaeal Tamaddonfard, Tel: +98 44 32770508, Fax: +98 44 32771926, Email: [email protected]; [email protected]
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Short title: oxytocin in VLOC-PAG modulation of neuropathic pain
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Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal
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gray opioid receptors
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Intra-VLOC microinjection of oxytocin attenuated mechanical allodynia. Atosiban and naloxone prevented antiallodynic effect of oxytocin. Intra-vlPAG administration of naloxone prevented intra-VLOC oxytocin-induced antiallodynia. Opioid receptors of VLOC and vlPAG may be involved in VLOC oxytocin-induced antiallodynia.
ABSTRACT
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Highlights
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Background: Oxytocin plays an important role in supraspinal modulation of pain. In the present
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study, we investigated the effects of ventrolateral orbital cortex (VLOC) microinjection of oxytocin on neuropathic pain after blockade of opioid receptors in this area and ventrolateral
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periaqueductal gray (vlPAG). Methods: Neuropathic pain was induced by complete transcection of preoneal and tibial branches of sciatic nerve. The VLOC and vlPAG were unilaterally (contralateral to the sciatic nerve-injured side) and bilaterally implanted with guide cannulas, respectively. Mechanical paw withdrawal threshold (PWT) was measured using von-Frey filaments. Area under curve (AUC) was also calculated. 1
Results: Microinjection of oxytocin (5, 10 and 20 ng/site) into the VLOC increased PWT. Antiallodynia induced by oxytocin (20 ng/site) was inhibited by prior intra-VLOC administration of atosiban (an oxytocin receptor antagonist, 100 ng/site) and naloxone (an opioid receptor antagonist, 500 ng/site). Prior microinjection of naloxone (500 ng/site) into the vlPAG also
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inhibited antiallodynia induced by intra-VLOC microinjection of oxytocin (20 ng/site). All the VLOC and vlPAG microinjected drugs did not alter locomotor activity.
Conclusions: It is concluded that oxytocin and its receptor may be involved in modulation of neuropathic pain at the VLOC level. Opioid receptors of VLOC and vlPAG might be involved in the antiallodynic effect of the VLOC-microinjected oxytocin.
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Keywords: Oxytocin; Ventrolateral orbital cortex; Periaqueductal gray; Opioid receptor;
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Neuropathic pain
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Introduction
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Oxytocin producing neurons are located in the paraventricular nucleus (PVN) and suprachiasmatic nucleus (SCN) of the hypothalamus [1]. Due to difficult blood-brain barrier crossing of oxytocin,
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the brain-related effects of this neuropeptide are originated from centrally projecting neurons [2].
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The brain areas such as amygdala, prefrontal cortex (PFC), orbital cortex (OC) subdivisions and
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periaqueductal gray (PAG) are innervated by oxytocin neurons [3]. Oxytocin is released in response to various stimuli and acts through oxytocin receptors, which are widely distributed in
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the brain [1]. This neuropeptide modulates many brain functions such as maternal and social behaviors, learning, anxiety, epilepsy and pain processing [3-7]. Some researchers have suggested
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the involvement of opioid receptors in the antinociceptive effects of supraspinal oxytocin [8, 9]. The ventrolateral orbital cortex (VLOC) has a potent role in pain processing [10, 11]. Electrical stimulation of the VLOC depressed jaw-opening reflex evoked by facial skin stimulation [12]. Using single neuron electrical activity recording, Follett and Dirks [13] found the involvement of VLOC in visceral pain modulation. In this context, prior intra-VLOC microinjection of yohimbin 2
(a selective α-2 adrenoceptor antagonist) blocked the anti-allodynic effect induced by the same site microinjection of a selective α-2 adrenoceptor agonist, clonidine [14]. The distribution of oxytocin and opioid receptors has been reported in orbitofrontal cortex (OFC)
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structures such as OC subdivisions [3]. In addition, Mansour et al. [15] reported opioid receptors distribution in the ventral PAG (vPAG) and dorsal PAG (dPAG) in rats. This study was planned to investigate the effects of microinjection of oxytocin, atosiban (an oxytocin receptor antagonist) and naloxone (an opioid receptor antagonist) into the VLOC on spared nerve injury (SNI)-induced neuropathic pain. In addition, the effect of VLOC microinjection of oxytocin was also evaluated
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after blockade of ventrolateral PAG (vlPAG) opioid receptors. The SNI model of neuropathic pain
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was introduced by Decosterd and Woolf [16]. The roles of VLOC [11, 14], and PAG [17, 18] in
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processing of SNI-induced neuropathic pain have been reported in rats. Using an electronic activity
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box, we examined the effects of the above-mentioned drugs on locomotor activity. Materials and methods
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Animals
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In the present study, a total of 96 healthy, 10-12 months old male Wistar rats weighing 250–280 g
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were used. The animals were maintained in a controlled ambient temperature laboratory (22 ± 0.5 °C) at 12 h light-dark cycles (light on at 07:00 h). Commercial food and water are available to
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them. The experiments were performed between 10:00 h and 14:00 h. Drugs
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In the present study, the following drugs were used: oxytocin, atosiban and naloxone dihydrochloride. Drugs were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA, and dissolved in sterile normal saline 30 min before intra-VLOC and intra-vlPAG microinjections. Spared nerve injury
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SNI model of neuropathic pain was performed as previously described [16]. Briefly, the rats were anaesthetized with intraperitoneal (ip) injection of a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg), and an incision was then made along the back of the left thigh. The biceps femoris
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muscle was separated to expose the tibial, peroneal and sural branches of the sciatic nerve. With no manipulation of sural nerve, the tibial and peroneal nerves were tightly ligated using 5-0 silk and transected distal to the ligation [19]. Following surgery, the rats were allowed to recover for one week before implantation of guide cannulas. Intracerebral guide cannula placement
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One week after SNI, using a stereotaxic apparatus (Stoelting, Wood Lane, IL, USA), the VLOC
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and vlPAG were implanted with guide cannulas in ketamine (80 mg/kg)-xylazine (8 mg/kg)
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anesthetized rats. A stainless-steel guide cannula (23-gauge, 12-mm) was unilaterally
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(contralateral to the sciatic nerve-injured side) inserted 1 mm above the right side of the VLOC at the following coordinates: 3.2 mm anterior to the bregma, 2.2 mm left side of the midline, and 4.6
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mm below the top of the skull [20]. The vlPAG were bilaterally implanted with two guide cannulas
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at the following coordinates: 7.8 mm posterior to the bregma, 0.8 mm left and right sides of the
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midline, and 5.5 mm below the top of the skull [20]. We used unilateral cannulation of the VLOC and bilateral cannulation of vlPAG, because spinal cord-thalamic submedius (Sm) projections are
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bilateral with a contralateral dominance [21]. In addition, the reciprocal connections between Sm and VLOC are largely ipsilateral in the rat [22]. On the other hand, the VLOC contains neurons
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projecting bilaterally to the vlPAG [23]. The cannulas were then fixed to the skull by three screws and dental acrylic. At least, seven days were allowed before intra-VLOC and intra-vlPAG microinjections. Intra-VLOC and intra-vlPAG microinjections
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Using a 30-guage, 13 mm needle attached to a 1 µl Hamilton syringe, intra-VLOC and intra-vlPAG microinjections of normal saline (control) and test drugs were performed. The VLOC and each vlPAG were microinjected with constant volumes of 0.5 and 0.25 µl of test drugs, respectively,
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over a period of 60 seconds. The injection needle was left in place for a further 60 seconds to facilitate diffusion of the drug. Mechanical paw withdrawal threshold measurement
Two weeks after SNI, paw withdrawal threshold (PWT) was measured in response to application of von Frey filaments using the up-down method [24, 25]. Animals were placed in a transparent
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plastic box (25 × 15 × 20 cm) with a metal wire mesh floor. Eleven von Frey filaments (IITC-Life
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Science Instruments, Woodland Hill, CA) were chosen (von Frey numbers: 3, 4, 5, 6, 7, 8, 9, 10,
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11, 12 and 13, equivalent to 0.4, 0.8, 1, 1.2, 1.5, 2.5, 3.6, 4, 8, 10, 20 g, respectively). Starting with
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middle filament (number 8, equivalent to 2.5 g) in the series, von Frey filaments were repeatedly applied over a 3-s time interval from below and perpendicular to the fourth and fifth toes of the
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hind paw for approximately 5-7 s. The sural nerve innervates the lateral part of the hind paw
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including parts of the 4th digit and the entire 5th digit [26]. If there was a withdrawal response to
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filament stimulation (positive), the next lower force was delivered. If response was negative, the next higher force was applied. After recording of positive and negative responses, 50% threshold
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were calculated using the following formula, which provided by Dixon, [24] and Chaplan et al. [25]: 50% threshold = 10(Xf+kδ), where Xf is the value of the final von Frey filament used (in log
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units), k is the tabular value for the pattern of positive/negative responses presented at the appendix 1 of Chaplan et al., [25] article, and δ is the mean difference between stimuli in log units (0.155). This formula, with a slight modification, has been frequently used to calculate 50% PWT in SNI model of neuropathic pain [27-29]. Values of 0.4 or 20.0 g were assigned where continuous
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positive or negative responses were observed all the way out to the end of the stimulus spectrum, respectively. Study protocol
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Fig. 1 shows drug microinjection and allodynia recording time line. To examine the role of oxytocin and opioid receptors in VLOC modulation of neuropathic pain, 66 rats were divided into 11 groups and intra-VLOC microinjections of normal saline plus normal saline (control), normal saline plus oxytocin (2.5, , 5, 10 and 20 ng/site), atosiban (10 and 100 ng/site) plus normal saline, atosiban (100 ng/site) plus oxytocin (20 ng/site), naloxone (100 and 500 ng/site) plus normal saline
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and naloxone (500 ng/site) plus oxytocin (20 ng/site) were performed (Fig. 1A). To explore the
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role of vlPAG opioid receptor in VLOC-microinjected oxytocin effect, 30 rats were divided into
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5 groups and the following intra-vlPAG and intra-VLOC were performed: normal saline (vlPAG)
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plus normal saline (VLOC), naloxone (100 ng/site, vlPAG) plus normal saline (VLOC), naloxone (500 ng/site, vlPAG) plus normal saline (VLOC), normal saline (vlPAG) plus oxytocin (20 ng/site,
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VLOC) and naloxone (500 ng/site, vlPAG) plus oxytocin (20 ng/site, VLOC). Atosiban and
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naloxone were microinjected six min before initiation of PWT recording. Oxytocin was
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microinjected three min after atosiban and naloxone microinjection (Fig. 1B). The PWT was measured immediately (time point 0), 10, 20, 30, 40, 50 and 60 min after intra-structural injection
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(Fig. 1A and 1B). The experimenters were blinded to the treatment protocol. The doses of drugs used here were according to previous studies [6, 30] and our preliminary experiments.
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Locomotor activity We used an electronic activity box (BorjSanat, Tehran, Iran) to assess locomotor activity. The animals were placed in a Plexiglas chamber (40 × 40 × 40 cm). The number of beam breaks due to animal movement was recorded from the monitor of the apparatus in a 5-min session.
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Verification of cannula tip placement Initially, 0.5 and 0.25 µl methylene blue were microinjected into the VLOC and each side of vlPAG, respectively. Animals were deeply anaesthetized with a mixture of ketamine and xylazine and perfused intracardially with physiological saline followed by 10% formalin solution. The
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brains were removed and placed in a 10% formalin solution. Twenty-four h later, we provided transverse sections (50–100 µm) and viewed under a loupe to localize the injection sites according to the atlas of Paxinos and Watson [20]. Statistical analysis
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The Graph Pad Prism version 5 (Graph Pad Software Inc, San Diego, CA USA) was used to
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analyzing the results. The data obtained from 10-min time points were analyzed using two-way
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repeated measure analysis of variance (ANOVA) and then Bonferroni post hoc test. The area under
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curve (AUC) data was analyzed by one-way analysis of variance (ANOVA), and post hoc Tukey’s
Results
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Cannulas tip verification
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test. Values are expressed as mean ± SEM. p < 0.05 was considered statistically significant.
The location of the cannulas tip placement in the VLOC (Fig. 2B) and vlPAG (Fig. 2C, right side)
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were confirmed in the brain section. The rat brain sections of VLOC (Fig. 2A) and vlPAG (Fig.
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2C, left side) were adopted from the atlas of Paxinos and Watson [20]. Effects of intra-VLOC microinjection of oxytocin, atosiban and naloxone on SNI-induced
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allodynia
The 50% PWT were approximately between 2.0 and 3.0 g (2.68± 0.23 g) at all time points after intra-VLOC microinjection of normal saline. Fig. 3A shows significant different between treatments (F(4,175) = 168.23, p < 0.05), across times (F(6, 175) = 35.38, p < 0.05), and between interactions (F(24,175) = 7.08, p < 0.05). In this context, oxytocin at a dose of 2.5 ng/site did not alter 7
50% PWT, whereas at a dose of 5 ng/site increased 50% PWT at 20, 30 and 40 min time points, and at a dose of 10 ng/site it increased 50% PWT at 10, 20, 30, 40 and 50 min time points. Oxytocin (20 ng/site) increased 50% PWT at all time points (Fig. 3A). Fig. 3B shows significant different
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between treatments (F(4,175) = 222.71, p < 0.05), across times (F(6, 175) = 10.16, p < 0.05), and between interactions (F(24,175) = 9.55, p < 0.05). Alone intra-VLO microinjection of atosiban (20 and 100 ng/site) produced no significant effects on 50% PWT. Prior microinjection of atosiban (100 ng/site) prevented oxytocin (20 ng/site)-induced antiallodynia (Fig. 3B). Fig. 3C shows significant different between treatments (F(4,175) = 249.9, p < 0.05), across times (F(6, 175) = 10.84,
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p < 0.05), and between interactions (F(24,175) = 10.47, p < 0.05). Intra-VLO microinjection of
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naloxone (100 and 500 ng/site) alone produced no significant effect on 50% PWT. Antiallodynia
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induced by intra-VLOC microinjection of oxytocin (20 ng/site) was prevented by prior
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microinjection of 500 ng/site naloxone (Fig. 3C). Analyses of AUC on PWT showed that the AUC of 5, 10 and 20 ng/site oxytocin were significantly (F(4, 29) = 20.98, p < 0.05) larger than the saline
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normal treated group (Fig. 3D). Prior microinjection of 100 ng/site atosiban (F(4,29) = 21.93, p
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<0.05) and 500 ng/site naloxone (F(4,29) = 40.38, p <0.05) significantly decreased the increased
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AUC induced by oxytocin (20 ng/site) and reached it to the saline normal treated AUC (Fig. 3D). Effects of intra-vlPAG microinjection of naloxone on antiallodynic effect induced by intra-VLOC
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microinjection of oxytocin
As shown in Fig. 4A, time-course curve were significantly different between treatments (F(4,175) =
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222.9, p < 0.05), across times (F(6, 175) = 15.36, p < 0.05), and between interactions (F(24,175) = 9.91, p < 0.05). Further analyses showed that intra-VLO microinjection of oxytocin (20 ng/site) increased 50% PWT at all time points. Allodynia was not changed after intra-vlPAG microinjection of naloxone at doses of 100 and 500 ng/site. Prior intra-vlPAG microinjection of
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naloxone (500 ng/site) prevented the antiallodynia induced by microinjection of oxytocin (20 ng/site) into the VLOC (Fig. 4A). Analyses of AUC showed that the increased AUC induced by intra-VLOC microinjection of oxytocin (20 ng/site) was significantly (F(4,29) = 35.42, p <0.05)
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decreased by prior microinjection of naloxone (500 ng/site) into the vlPAG (Fig. 4B). Effects of test drugs on locomotor activity
The numbers of photo beam break in intact, normal saline (VLOC) plus normal saline (VLOC) and normal saline (vlPAG) plus normal saline (VLOC) were 82.5 ± 5.28, 75.5 ± 6.23 and 79.2 ± 5.38, respectively. Microinjection of oxytocin, naloxone and atosiban into the VLOC and naloxone
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into the vlPAG did not alter photo beam breaks number (Data not shown).
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Discussion
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In the present study, the 50% PWT were approximately between 2.0 and 3.0 g at all time points on 14th day after induction of SNI. This is in consistent with other findings in which 1.5 – 2.0 g
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and 2.5 – 3.5 g PWT have been reported on day 14 after SNI [14, 27, 28]. The PWT less than 4.0
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g indicates a successful mechanical allodynia in SNI model of neuropathic pain [14, 27, 28]. This
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is a novel animal model of neuropathic pain developed by Decosterd and Wolf [16]. In contrast to chronic constriction injury (CCI), partial sciatic nerve ligation (PSL) and spinal nerve ligation
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(SNL) models with regenerative capacity of injured nerves leading to abnormal pain sensation and behavior, mechanical allodynia occurs within four days of injury and persists for several weeks in
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SNI model [31]. Long-term plastic changes occurring along sensory pathways, from peripheral nociceptors to spinal cord and pain-processing brain regions contribute to induction of mechanical allodynia [32]. Recent evidences have suggested plastic changes occur in frontal, retrosplenial,
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entorhinal and prefrontal cortices after SNI [33]. For this reason, SNI has been frequently used in the study of supraspinal mechanisms of neuropathic pain [14, 27, 28]. Our present results showed that microinjection of oxytocin into the VLOC suppressed mechanical
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allodynia, and prior microinjection of atosiban into the same site prevented this allodynia. The VLOC has a central role in spinal cord/medulla-Sm-VLOC-PAG-spinal cord/medulla pain pathway of acute and chronic pain modulation [11]. In addition, opioid, serotonin, dopamine and noradrenalin receptors of the VLOC participate in modulation of SNI-induced neuropathic pain [11, 14, 34, 35]. Although there are no reports showing the involvement of VLOC oxytocin in
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neuropathic pain modulation, electrical stimulation of PVN increased cerebrospinal fluid (CSF)
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concentration of oxytocin and suppressed mechanical allodynia and heat hyperalgesia in CCI
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model of neuropathic pain [36]. In this context, Kubo et al. [37] reported that direct administration
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of oxytocin into the trigeminal ganglion suppressed mechanical hypersensitivity induced by partial ligation of infraorbital nerve. Oxytocin receptor distribution and oxytocinergic fiber projection in
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pain processing areas and nuclei such as PFC, ventral tegmental area (VTA), nucleus accumbens
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(NAcc), amygdala, hippocampus, raphe nucleus (RN) and PAG, and difficult crossing of oxytocin
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through blood-brain barrier [7, 38], can explain oxytocin contribution to supraspinal mechanisms of pain. At the PAG level, pre-administration of desGly-NH2,d(CH2)5[D-Tyr2,Thr-Sup-4]OVT,
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an oxytocin antagonist, inhibited the analgesic effect of oxytocin in tail skin potassium iontophoresis model of acute pain [39]. In addition, prior microinjection of selective oxytocin
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receptor antagonist, 1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin (atosiban) into the central nucleus of amygdala prevented the suppressive effects of oxytocin on acute pain induced by thermal and mechanical stimulation of hind paw [40]. Taken together, the results of the present
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study reveal for the first time that oxytocin and its receptor at the VLOC can modulate neuropathic pain in rats. In the present study, prior microinjection of naloxone into the VLOC inhibited oxytocin-induced
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antiallodynia. Opioid receptors are distributed in the VLOC and involved in antiallodynic effects of endogenous opioid peptides in neuropathic pain [35, 41]. In addition, at the level of VLOC, opioid receptors mediate the antinociceptive effects of a variety of neurotransmitters such as gama amino butyric acid (GABA), dopamine and serotonin [11]. Opioid receptors are involved in the centrally-administered oxytocin-induced antinociception. For example, naloxone inhibited the
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suppressive effects of centrally-administration oxytocin on both phases of formalin-induced pain
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in mice [42].
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Our present results showed that prior microinjection of naloxone into the vlPAG inhibited
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antiallodynia induced by intra-VLOC microinjection of oxytocin. Neuroanatomical and functional connections exist between subdivisions of OC and PAG [10, 11]. In this context, functional
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blockade of the PAG eliminated chemically-induced activation of VLOC depresses of nocifensive
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reflexes [11]. Moreover, bilateral electrolytic lesions of the lateral and ventrolateral parts of PAG
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and bilateral microinjection of GABA into the vlPAG reduced or eliminated the VLOC-evoked inhibition of tail-flick reflex [43, 44]. The PAG receives inputs from cortical sites and has
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reciprocal connections with the amygdala as well as ascending nociceptive inputs from the spinal cord [45]. Due to these connections, the PAG play a central role in pain modulation pathways such
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as motor cortex-PAG-spinal cord and PFC-amygdala-PAG pathways [46, 47]. Mu-opioid receptors are widely distributed in dorsomedial, lateral and ventrolateral sub-regions of PAG [48]. In this context, microinjection of naloxone into the PAG decreased the inhibitory effect of motor cortex stimulation on sensory evoked potentials induced by forepaw electrical stimulation [49]. In
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addition, the suppressive effect induced by intra-arcuate nucleus microinjection of galanin was inhibited by prior microinjection of naloxone into the PAG [50]. These results showed that the VLOC oxytocin plays an important role in modulation of neuropathic pain, and opioid receptors
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of the PAG may be involved in the antiallodynia induced by VLOC microinjection of oxytocin. In conclusion, the results of the present study showed that at the VLOC level oxytocin via its receptors as well as opioid receptors can modulate SNI model of neuropathic pain in rats. One of the mechanisms by which oxytocin modulates neuropathic pain may be associated with contribution of vlPAG opioid receptors.
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The authors declare that there are no conflicts of interest.
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Conflict of interest
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Acknowledgement
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This study was financially supported by the directorate of postgraduate studies of Urmia
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Figure legends
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Fig. 1. Drug microinjection and mechanical allodynia recording time line. At -30 min adaptation
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of animals to experimental conditions was performed. Six min after intra-VLOC microinjection of
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atosiban and naloxone (Fg.1A) and intra-vlPAG microinjection of naloxone (Fig. 1B) and three
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min after intra-VLOC (Fig. 1A) and intra-vlPAG microinjection of oxytocin (Fig. 1B), mechanical
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allodynia recording were begun (time point 0), and continued at 10, 20, 30, 40, 50 and 60 min time points after microinjections.
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Fig. 2. Schematic illustrations of the rat brain showing the approximate location of the ventrolateral orbital cortex (VLOC) and ventrolateral periaqueductal grey (vlPAG) microinjection sites. Section
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of the rat brain shows the location of the permanent cannula tip (black arrow) in the VLOC (Fig.
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2B) and vlPAG (Fig. 2C, right side) of rats included in the present study. Atlas plates of VLOC (Fig. 2A) and vlPAG (Fig. 2C, left side) were adopted from Paxinos and Watson, (1986). CGD: central gray, dorsal; CGLD: central gray, lateral dorsal; CGM: central gray, medial; DR: dorsal raphe nucleus; CGLV: central gray, lateral ventral (ventrolateral PAG), LOC: lateral orbital cortex, Cl: clastrum, fmi: forceps minor corpus collasum.
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Fig. 3. The effects of intra-VLOC microinjection of oxytocin, atosiban and naloxone on 50% paw withdrawal threshold (PWT, A, B and C) and area under curve (AUC, D) in spared nerve injury (SNI) model of neuropathic pain. Atosiban and naloxone were microinjected six min before and
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oxytocin was microinjected three min before mechanical allodynia recording. Values are the mean ± SEM of six rats per group. * p < 0.05 significantly different compared with normal saline treated group.
†
p < 0.05 significantly different compared with oxytocin (20 ng/site) group. VLOC:
ventrolateral orbital cortex, PWT: paw withdrawal threshold.
Fig. 4. The effects of intra-vlPAG microinjection of naloxone alone and before intra-VLOC
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microinjection of oxytocin on 50% paw withdrawal threshold (A) and area under curve (B) in
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spared nerve injury (SNI) model of neuropathic pain. Intra-vlPAG microinjection of naloxone and
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intra-VLOC microinjection of oxytocin were preformed six and three min before mechanical
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allodynia recording, respectively. Values are the mean ± SEM of six rats per group. * p < 0.05 significantly different compared with normal saline treated group. † p < 0.05 significantly different
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compared with oxytocin (20 ng/site) group. VLOC: ventrolateral orbital cortex, PAG:
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periaqueductal gray, PWT: paw withdrawal threshold.
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