Blockade of spinal glutamate recycling produces paradoxical antinociception in rats with orofacial inflammatory pain.

In our current study, we investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain. DL-threo-ß-benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block spinal glutamate transporter and glutamine synthetase activity in astroglia. Intracisternal administration of high dose TBOA (10 µg) produced thermal hyperalgesia in naïve rats but significantly attenuated the thermal hyperalgesia in rats that had been pretreated with interleukin (IL)-1ß or Complete Freund's Adjuvant (CFA). In contrast, intracisternal injection of MSO produced anti-hyperalgesic effects against thermal stimuli in CFA-treated rats only. To confirm the paradoxical antinociceptive effects of TBOA and MSO, we examined changes in c-Fos expression in the medullary dorsal horn produced by thermal stimulation in naïve, IL-1ß-, or CFA-treated rats, after intracisternal injections of TBOA and MSO. Intracisternal administration of TBOA significantly increased c-Fos immunoreactivity in naïve rats. In contrast, intracisternal administration of TBOA significantly decreased the up-regulation of c-Fos immunoreactivity in the medullary dorsal horn of IL-1ß- and CFA-treated rats. However, intracisternal injection of MSO blocked the up-regulation of c-Fos immunoreactivity in CFA-treated rats only. We also investigated the effects of botulinum toxin type A (BoNT-A) on TBOA-induced paradoxical antinociception in CFA-treated rats, as BoNT-A inhibits the release of neurotransmitters, including glutamate. BoNT-A treatment reversed behavioral responses produced by intracisternal administration of TBOA in CFA-treated rats. These results suggest that the paradoxical responses produced by blocking glutamate transporters under inflammatory pain conditions are mediated by the modulation of glutamate release from presynaptic terminals. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic inflammatory pain conditions.

Differential regulation of peripheral IL-1ß-induced mechanical allodynia and thermal hyperalgesia in rats.

This study examined the differential mechanisms of mechanical allodynia and thermal hyperalgesia after injection of interleukin (IL) 1ß into the orofacial area of male Sprague-Dawley rats. The subcutaneous administration of IL-1ß produced both mechanical allodynia and thermal hyperalgesia. Although a pretreatment with iodoresiniferatoxin (IRTX), a transient receptor potential vanilloid 1 (TRPV1) antagonist, did not affect IL-1ß-induced mechanical allodynia, it significantly abolished IL-1ß-induced thermal hyperalgesia. On the other hand, a pretreatment with D-AP5, an N-methyl-d-aspartate (NMDA) receptor antagonist, and NBQX, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, blocked IL-1ß-induced mechanical allodynia. Pretreatment with H89, a protein kinase A (PKA) inhibitor, blocked IL-1ß-induced mechanical allodynia but not thermal hyperalgesia. In contrast, pretreatment with chelerythrine, a protein kinase C (PKC) inhibitor, inhibited IL-1ß-induced thermal hyperalgesia. Subcutaneous injections of 2% lidocaine, a local anesthetic agent, blocked IL-1ß-induced thermal hyperalgesia but not IL-1ß-induced mechanical allodynia. In the resiniferatoxin (RTX)-pretreated rats, a subcutaneous injection of IL-1ß did not produce thermal hyperalgesia due to the depletion of TRPV1 in the primary afferent fibers. Double immunofluorescence revealed the colocalization of PKA with neurofilament 200 (NF200) and of PKC with the calcitonin gene-related peptide (CGRP) in the trigeminal ganglion. Furthermore, NMDA receptor 1 (NR1) and TRPV1 predominantly colocalize with PKA and PKC, respectively, in the trigeminal ganglion. These results suggest that IL-1ß-induced mechanical allodynia is mediated by sensitized peripheral NMDA/AMPA receptors through PKA-mediated signaling in the large-diameter primary afferent nerve fibers, whereas IL-1ß-induced thermal hyperalgesia is mediated by sensitized peripheral TRPV1 receptors through PKC-mediated signaling in the small-diameter primary afferent nerve fibers.

The glial-neuronal GRK2 pathway participates in the development of trigeminal neuropathic pain in rats.

UNLABELLED: This study examined the role of the glial-neuronal G protein-coupled receptor kinase 2 (GRK2) pathway in the development of trigeminal neuropathic pain. Male Sprague Dawley rats, weighing 220 to 240 g, were anesthetized with ketamine (0.2 g/kg) and xylazine (0.02 g/kg). Under anesthesia, the left lower second molar was extracted, followed by the placement of a mini-dental implant to intentionally injure the inferior alveolar nerve. This injury produced mechanical allodynia along with the downregulation of neuronal GRK2 expression in the medullary dorsal horn. On the other hand, early intracisternal treatment with MDL28170, a calpain inhibitor, produced prolonged antiallodynic effects and blocked this downregulation of neuronal GRK2 expression. The intracisternal infusion of minocycline, a microglia inhibitor, and l-α-aminoadipic acid, an astrocytic specific inhibitor, also blocked the induced mechanical allodynia and downregulated neuronal GRK2 expression, respectively. Double immunofluorescence showed that the interleukin (IL)-1ß and IL-1R signals colocalize with the astrocytes and neurons, respectively, in the medullary dorsal horn following an inferior alveolar nerve injury. In addition, the intracisternal infusion of an IL-1 receptor antagonist also produced antiallodynic effects and blocked the downregulation of neuronal GRK2 expression. These results suggest that the glial-neuronal GRK2 pathway is a potentially important new target for treating neuropathic pain. Moreover, the IL-1ß expressed in astrocytes plays a significant role in modulating this pathway. PERSPECTIVE: This study showed that the glial-neuronal GRK2 pathway participates in the development of trigeminal neuropathic pain in rats. These results suggest that the glial-neuronal GRK2 pathway is a potentially important new target for the treatment of neuropathic pain.

Participation of the central p38 and ERK1/2 pathways in IL-1ß-induced sensitization of nociception in rats.

This study examined the participation of central mitogen-activated protein kinases (MAPKs) in the central sensitization produced by a subcutaneous injection of interleukin-1ß (IL-1ß) in male Sprague-Dawley rats. Formalin-induced responses were evaluated 24h after an IL-1ß injection. A subcutaneous injection of 10ng of IL-1ß elevated the formalin-induced scratching response significantly in the second phase compared to the vehicle-treated group. Pretreatment with an IL-1 receptor antagonist reduced the IL-1ß-induced sensitization. Pretreatment with IL-1ß increased the p-ERK and p-p38 expression induced by the formalin injection. Double immunofluorescence data revealed increases in phospho-extracellular signal-regulated kinase (p-ERK) immunoreactive cells that co-localize with neuronal nuclei (NeuN), a neuronal marker, and in phospho-p38 (p-p38) immunoreactive cells that co-localize with NeuN and OX42, a microglia marker. The intracisternal administration of minocycline (50µg), a microglia inhibitor, attenuated the increased formalin-induced scratching responses in the IL-1ß-treated rats. The intracisternal administration of PD98059 (1, 10µg), a MEK inhibitor, and SB203580 (1, 5µg), a p38 inhibitor, also attenuated the number of formalin-induced scratches in the second phase in the IL-1ß-treated rats. These results suggest that the IL-1ß-induced central sensitization of nociception is mediated by the central MAPK pathways, which are activated differentially in the neurons and microglia under inflammatory pain conditions. Therefore, blockade of the MAPK pathways can be as a potential therapeutic target for the central sensitization of inflammatory pain.

A novel trigeminal neuropathic pain model: compression of the trigeminal nerve root produces prolonged nociception in rats.

We demonstrate the establishment of a novel animal model for trigeminal neuropathic pain following compression of the trigeminal nerve root, which produces prolonged nociceptive behavior and demyelination of the trigeminal nerve root. Under anesthesia, male Sprague-Dawley rats (200-230 g) were mounted onto a stereotaxic frame and injections of a 4% agar solution (10 µl) were given to achieve compression of the trigeminal nerve root. A sham operation was performed using identical procedures but without agar injections. Nociceptive behavior was examined 3 days before and then at 3, 7, 10, 14, 17, 21, 24, 30, 40, 55, and 70 days after the surgery. Compression of the trigeminal nerve root caused mechanical allodynia, hyperalgesia, and cold hypersensitivity. Mechanical allodynia was established within 3 days and recovered to preoperative levels on postoperative day (POD) 40. Mechanical hyperalgesia and cold hypersensitivity persisted until 55 days following compression. The compression produced focal demyelination in the trigeminal nerve root. In the medullary dorsal horn, phospho-p38 (p-p38) mitogen-activated protein kinase (MAPK) was found to be exclusively expressed in the microglia on POD 14. Furthermore, intraperitoneal administration of carbamazepine (50mg/kg) significantly blocked mechanical allodynia and reduced p38 MAPK activation induced by the compression of the trigeminal nerve root. Our findings suggest that prolonged nociceptive behavior following compression of the trigeminal nerve root may mimic trigeminal neuralgia in this animal model and that the activation of p38 MAPK in the microglia contributes to pain hypersensitivity in rats that have undergone compression of the trigeminal nerve root.