The present study investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain or trigeminal neuropathic pain. Experiments were carried out on male Sprague–Dawley rats weighing between 230 and 280 g. Under anesthesia, a polyethylene tube was implanted in the atlanto-occipital membrane for intracisternal administration. IL-1β-induced inflammation was employed as an orofacial acute inflammatory pain model. IL-1β (10 ng) was injected subcutaneously into one vibrissal pad. We used the trigeminal neuropathic pain animal model produced by chronic constriction injury of the infraorbital nerve. DL-threo-β -benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block the spinal glutamate transporter and the glutamine synthetase activity in astroglia. Intracisternal administration of TBOA produced mechanical allodynia in naïve rats, but it significantly attenuated mechanical allodynia in rats with interleukin (IL)-1 β-induced inflammatory pain or trigeminal neuropathic pain. In contrast, intracisternal injection of MSO produced antiallodynic effects in rats treated with IL-1β or with infraorbital nerve injury. Intracisternal administration of MSO did not produce mechanical allodynia in naive rats. These results suggest that blockade of glutamate recycling induced pro-nociception in naïve rats, but it paradoxically resulted in anti-nociception in rats experiencing inflammatory or neuropathic pain. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic pain conditions.

In the present study, we investigated the role of peripheral ionotropic receptors in artemin-induced thermal hyperalgesia in the orofacial area. Male Sprague-Dawley rats weighting 230 to 280 g were used in the study. Under anesthesia, a polyethylene tube was implanted in the subcutaneous area of the vibrissa pad, which enabled drug-injection. After subcutaneous injection of artemin, changes in air-puff thresholds and head withdrawal latency time were evaluated. Subcutaneous injection of artemin (0.5 or 1 μg) produced significant thermal hyperalgesia in a dose-dependent manner. However, subcutaneous injection of artemin showed no effect on air-puff thresholds. IRTX (4 μg), a TRPV1 receptor antagonist, D-AP5 (40 or 80 μg), an NMDA receptor antagonist, or NBQX (20 or 40 μg), an AMPA receptor antagonist, was injected subcutaneously 10 min prior to the artemin injection. Pretreatment with IRTX and D-AP5 significantly inhibited the artemin-induced thermal hyperalgesia. In contrast, pretreatment with both doses of NBQX showed no effect on artemin-induced thermal hyperalgesia. Moreover, pretreatment with H-89, a PKA inhibitor, and chelerythrine, a PKC inhibitor, decreased the artemin-induced thermal hyperalgesia. These results suggested that artemin-induced thermal hyperalgesia is mediated by the sensitized peripheral TRPV1 and NMDA receptor via activation of protein kinases.

The present study investigated the role of central GABAA and GABAB receptors in orofacial pain in rats. Experiments were conducted on Sprague-Dawley rats weighing between 230 and 280 g. Intracisternal catheterization was performed for intracisternal injection, under ketamine anesthesia. Complete Freund's Adjuvant (CFA)-induced thermal hyperalgesia and inferior alveolar nerve injury-induced mechanical allodynia were employed as orofacial pain models. Intracisternal administration of bicuculline, a GABAA receptor antagonist, produced mechanical allodynia in naive rats, but not thermal hyperalgesia. However, CGP35348, a GABAB receptor antagonist, did not show any pain behavior in naive rats. Intracisternal administration of muscimol, a GABAA receptor agonist, attenuated the thermal hyperalgesia and mechanical allodynia in rats with CFA treatment and inferior alveolar nerve injury, respectively. On the contrary, intracisternal administration of bicuculline also attenuated the mechanical allodynia in rats with inferior alveolar nerve injury. Intracisternal administration of baclofen, a GABAB receptor agonist, attenuated the thermal hyperalgesia and mechanical allodynia in rats with CFA treatment and inferior alveolar nerve injury, respectively. In contrast to GABAA receptor antagonist, intracisternal administration of CGP35348 did not affect either the thermal hyperalgesia or mechanical allodynia. Our current findings suggest that the GABAA receptor, but not the GABAB receptor, participates in pain processing under normal conditions. Intracisternal administration of GABAA receptor antagonist, but not GABAB receptor antagonist, produces paradoxical antinociception under pain conditions. These results suggest that central GABA has differential roles in the processing of orofacial pain, and the blockade of GABAA receptor provides new therapeutic targets for the treatment of chronic pain.

The activation of glial cells in the spinal cord has been contribute to the initiation and maintenance of pain facilitation induced by peripheral inflammation and nerve injury. The present study investigated effects of botulinum toxin type A (BoNT-A), injected subcutaneously or intracisternally, on the expression of microglia and astrocytes in rats. Complete Freund’s Adjuvant (CFA)-induced inflammation was employed as an orofacial chronic inflammatory pain model. A subcutaneous injection of 40 μL CFA into the vibrissa pad was performed under 3% isoflurane anesthesia in SD rats. Immunohistochemical analysis for changes in Iba1 (a microglia marker) and GFAP (an astrocyte marker), were performed 5 days after CFA injection. Subcutaneous injection of CFA produced increases in Iba1 and GFAP expression, in the ipsilateral superficial lamia I and II in the medullary dorsal horn of rats. Subcutaneous treatment with BoNT-A attenuated the up-regulation of Iba1 and GFAP expressions induced by CFA injection. Moreover, intracisternal injection of BoNT-A also attenuated the up-regulated Iba1 and GFAP expressions. These results suggest that the anti-nociceptive action of BoNT-A is mediated by modulation activation of glial cells, including microglia and astrocyte.

The present study investigated the role of peripheral P2X receptors in inflammatory pain transmission in the orofacial area in rats. Experiments were carried out on male Sprague- Dawley rats weighing 220 to 280 g. Formalin (5%, 50 μL) and complete Freund's adjuvant (CFA, 25 μL) was applied subcutaneously to the vibrissa pad to produce inflammatory pain. TNP-ATP, a P2X2,2/3,4 receptor antagonist, or OX-ATP, a P2X7 receptor antagonist, was then injected subcutaneously at 20 minutes prior to formalin injection. One of the antagonists was administered subcutaneously at three days after CFA injection. The subcutaneous injection of formalin produced a biphasic nociceptive behavioral response. Subcutaneous pretreatment with TNP-ATP (80, 160 or 240 μg) significantly suppressed the number of scratches in the second phase produced by formalin injection. The subcutaneous injection of 50 μg of OX-ATP also produced significant antinociceptive effects in the second phase. Subcutaneous injections of CFA produced increases in mechanical and thermal hypersensitivity. Both TNP-ATP (480 μg) and OX-ATP (100 μg) produced an attenuation of mechanical hypersensitivity. However, no change was observed in thermal hypersensitivity after the injection of either chemical. These results suggest that the blockade of peripheral P2X receptors is a potential therapeutic approach to the onset of inflammatory pain in the orofacial area.