Blockade of microglial activation reduces mechanical allodynia in rats with compression of the trigeminal ganglion.

The present study investigated the role of microglia and p38 MAPK in the development of mechanical allodynia in rats with compression of the trigeminal ganglion. Male Sprague-Dawley rats weighing 250-260 g were used. Under pentobarbital sodium anesthesia, the animals were mounted onto a stereotaxic frame and given injections of 4% agar solution (10 µL) to compress the trigeminal ganglion. The air-puff thresholds significantly decreased after compression of the trigeminal ganglion. On postoperative day 14, immunoreactivity to both OX-42 and p-p38 MAPK was up-regulated in the medullary dorsal horn as compared to the sham group. P-p38 MAPK was found to be co-localized with OX-42, but not with NeuN, a neuronal cell marker, or with GFAP, an astroglial cell marker. Intracisternal administration of 100 µg of minocycline significantly inhibited both mechanical allodynia and activation of microglia produced by compression of the trigeminal ganglion. Intracisternal administration of 0.1, 1, or 10 µg of SB203580, a p38 MAPK inhibitor, also significantly decreased mechanical allodynia and p38 MAPK activation in the trigeminal ganglion-compressed group. These results suggest that activation of p38 MAPK in the microglia is an important step in the development of mechanical allodynia in rats with compression of the trigeminal ganglion and that the targeted blockade of microglial p38 MAPK pathway is a potentially important new treatment strategy for trigeminal neuralgia-like nociception.

Peripheral administration of NR2 antagonists attenuates orofacial formalin-induced nociceptive behavior in rats.

The present study investigated the role of the peripheral NR2 subunits of N-methyl-d-aspartatic acid (NMDA) receptors in inflammatory orofacial pain. Experiments were carried out using adult male Sprague-Dawley rats weighing 220 to 280 g. Formalin (5%, 50 µl) was applied subcutaneously to the vibrissa pad. For each animal, the number of noxious behavioral responses, including rubbing or scratching of the facial region proximal to the injection site, was recorded for 9 sequential 5 min intervals. NR2 subunit antagonists were injected subcutaneously at 20 min prior to formalin injection. The subcutaneous injection of 100 or 200 µg of memantine significantly suppressed the number of scratches in the second phase of the behavioral responses to formalin. The subcutaneous injection of 0.25, 2.5, or 25 µg of 5,7-dichlorokynurenic acid also produced significant antinociceptive effects in the second phase. The subcutaneous injection of AP-5 at high dose produced significant antinociceptive effects in the second phase. The subcutaneous injection of PPPA and Ro 25-6981 both significantly suppressed the number of scratches in the second phase. The antinociceptive doses of memantine (200 µg), 5,7-dichlorokynurenic acid (25 µg), AP-5 (20 µg), PPPA (2.5 µg), or Ro 25-6981 (50 µg) injected into the contralateral hind paw did not affect the number of scratches in both the first and second phases. Moreover, the peripheral administration of NR2 subunit antagonists, including other NMDA receptor blockers, did not produce any motor dysfunction. These results indicate that a targeted blockade of peripheral NR2 receptors is a potentially important new method of treating inflammatory pain in the orofacial area.

Intracisternal administration of NR2 antagonists attenuates facial formalin-induced nociceptive behavior in rats.

AIMS: To examine the antinociceptive effects of N-Methyl-D-aspartate (NMDA) receptor NR2 subunit antagonists in a rat model of the facial formalin test. METHODS: Experiments were carried out on adult male Sprague-Dawley rats weighing 220 to 280 g. Anesthetized rats were individually mounted on a stereotaxic frame and a polyethylene tube was implanted for intracisternal injection and, 72 hours later, formalin tests were performed. NMDA receptor antagonists were administered intracisternally 10 minutes prior to subcutaneous injection of 5% formalin (50 MicroL) into the vibrissal pad. RESULTS: The intracisternal administration of 25, 50, or 100 Microg of memantine, an antagonist that acts at the NMDA ion channel site, significantly suppressed the number of scratches in the second phase of the behavioral responses to formalin. Intracisternal administration of a range of doses of 5,7-dichlorokynurenic acid, a glycine site antagonist, or DL-2-amino-5-phosphonopentanoate (AP-5), a nonselective NMDA site antagonist, produced significant antinociceptive effects in the second phase. Intracisternal administration of 1, 2.5, or 5 Microg of (2R,4S)-4-(3 Phosphonopropyl)-2-piperidine_carboxylic acid (PPPA), a competitive NR2A antagonist, significantly suppressed the number of scratches in the second phase, while only the highest dose of PPPA (5 Microg) significantly suppressed the number of scratches in the first phase. The antinociceptive effects of intracisternal injection of (alphaR, betaS)-alpha-(4Hydroxyphenyl)-_ methyl-4-(phenylmethyl)-1-Piperidinepropanol maleate(Ro 25-6981), a selective NR2B antagonist, were similar to those of PPPA. Injection of memantine, AP-5, Ro 25-6981, or vehicle did not result in any motor dysfunction. A low dose of PPPA (1 microg) or 5,7-dichlorokynurenic acid (2.5 microg) did not affect motor function. However, higher doses of PPPA and 5,7-dichlorokynurenic acid produced motor dysfunction. CONCLUSION: The present results suggest that central NR2 subunits play an important role in orofacial nociceptive transmission. Moreover, this data also indicate that targeted inhibition of the NMDA receptor NR2 subunit is a potentially important new treatment approach for inflammatory pain originating in the orofacial area.

Intracisternal and intraperitoneal administration of morphine attenuates mechanical allodynia following compression of the trigeminal ganglion in rats.

AIMS: To investigate the effects of morphine on mechanical allodynia following compression of the trigeminal ganglion in the rat. METHODS: Experiments were carried out on male Sprague-Dawley rats weighing between 250 and 260 g. For compression, a 4% agar solution (8 microL) was injected into the trigeminal ganglion. In the control group, rats were sham operated without agar injections. The authors evaluated the effects of intraperitoneal or intracisternal administration of morphine on mechanical allodynia evoked by air-puff stimulation of the vibrissa pad area 14 days following compression of the trigeminal ganglion. RESULTS: Mechanical allodynia was established within 3 days and lasted beyond postoperative day 24. Intraperitoneal administration of morphine (2 or 5 mg/kg) significantly blocked mechanical allodynia ipsilateral to the compression of the trigeminal ganglion. Intraperitoneal administration of morphine also inhibited mechanical allodynia on the contralateral side. Moreover, intracisternal administration of morphine (5 microg) strongly suppressed both ipsilateral and contralateral mechanical allodynia. The antiallodynic effects of morphine were blocked by pretreatment with naloxone, an opioid receptor antagonist. CONCLUSION: These results suggest that the application of a high dose of morphine may be of great benefit in treating trigeminal neuralgia-like nociception.

Intratrigeminal ganglionic injection of LPA causes neuropathic pain-like behavior and demyelination in rats.

We have previously reported a novel method for producing chronic nociceptive behavior in rats following compression of the trigeminal ganglion. In the present study, we have further studied the role of demyelination in the development of prolonged nociceptive behavior in the trigeminal territory. For this purpose, lysophosphatidic acid (LPA) was injected into the trigeminal ganglia of male Sprague-Dawley rats weighing between 250 and 260 g. Under pentobarbital sodium anesthesia, the rats were mounted onto a stereotaxic frame and 3 microL of LPA (1 nmol) solution was injected into the trigeminal ganglion to produce demyelination. This treatment decreased the air-puff thresholds both ipsilateral and contralateral to the injection site, which persisted until postoperative day 100 and returned to the preoperative levels 130 days after the LPA injection. The LPA injection also produced a significant ipsilateral hyper-responsiveness to pin-prick stimulation. The effects of DGPP, an LPA1/3 receptor antagonist, and Y-27632, a Rho kinase inhibitor, upon LPA-induced mechanical allodynia and hyperalgesia were also investigated. Pretreatment with DGPP blocked both mechanical allodynia and ipsilateral hyperalgesia. However, pretreatment with Y-27632 blocked only ipsilateral and contralateral mechanical allodynia. These results thus indicate that a targeted blockade of LPA receptor and Rho kinase pathways are potentially important new treatments for demyelination-induced trigeminal neuralgia-like nociception.

Intracisternal administration of COX inhibitors attenuates mechanical allodynia following compression of the trigeminal ganglion in rats.

The purpose of the present study was to investigate the role of central cyclooxygenase (COX) pathways in the modulation of mechanical allodynia following compression of the left trigeminal ganglion. Experiments were carried out on male Sprague-Dawley rats mounted onto a stereotaxic frame under anesthesia. For compression, a 4% agar solution (10 microl) was injected into the trigeminal ganglion. In the control group, rats were sham operated without agar injections. Ipsilateral and contralateral air-puff thresholds significantly decreased following trigeminal ganglion compression. Mechanical allodynia was established within 3 days and lasted beyond postoperative day 30, returning to preoperative levels at approximately 55 days following compression. Intracisternal administration of indomethacin, a non-selective COX inhibitor, SC-560, a selective COX-1 inhibitor, or NS-398, a selective COX-2 inhibitor, significantly inhibited mechanical allodynia. The individual anti-allodynic effects of the three COX inhibitors persisted for 6 h and returned to pretreatment values within 24 h. Based on these results, the blockade of central COX pathways may comprise a potential new therapeutic tool for the treatment of trigeminal ganglion compression-induced nociception.

Compression of the trigeminal ganglion produces prolonged nociceptive behavior in rats.

The present study is the first demonstration of prolonged nociceptive behavior in the trigeminal region following compression of the trigeminal ganglion in rats. Experiments were carried out on male Sprague-Dawley rats mounted onto a stereotaxic frame under pentobarbital sodium anesthesia. For compression of the trigeminal ganglion, a 4% agar solution (8microl) was injected into the trigeminal ganglion through a stainless steel injector (24 gauge), which extended 2mm beyond the end of a guide cannula (21 gauge). Following agar injection, the injector and guide cannula were removed. In the control group, rats were sham operated without agar injection. Air-puff thresholds (mechanical allodynia), pin prick responses (mechanical hyperalgesia), and spontaneous scratching behavior were examined 3 days before surgery and at 3, 7, 10, 14, 17, 21, 24, 30, and 40 days after surgery. Data were analyzed using a repeated measures ANOVA followed by multiple group comparisons using the LSD post-hoc test. Air-puff thresholds significantly decreased after compression of the trigeminal ganglion. Mechanical allodynia was established within 3 days and lasted beyond postoperative day 24. Mechanical hyperalgesia was also evident 3 days after compression and persisted until the 40th postoperative day. Although mechanical allodynia and hyperalgesia appeared bilaterally, the ipsilateral side was significantly more sensitive. Intraperitoneal treatment with carbamazepine significantly blocked mechanical allodynia produced by compression of the trigeminal ganglion. These findings suggest that prolonged nociceptive behavior following compression of the trigeminal ganglion may mimic trigeminal neuralgia in this animal model.

Low doses of cannabinoids enhance the antinociceptive effects of intracisternally administered mGluRs groups II and III agonists in formalin-induced TMJ nociception in rats.

This study provides the first demonstration that central cannabinoids modulate the antinociceptive actions of metabotropic glutamate receptors (mGluRs) on formalin-induced temporomandibular joint (TMJ) nociception. Noxious scratching behavior induced by formalin injection in the TMJ was used as a model of pain. Intracisternal injection of 30mug of WIN 55,212-2, a non-subtype selective cannabinoid receptor agonist, attenuated the number of scratches by 75% as compared with the vehicle-treated group, whereas vehicle alone or 3 or 10 microg of WIN 55,212-2 had no effect. To explore the postulated interaction between central cannabinoid receptors and mGluRs, effects of combined administration of sub-analgesic doses of WIN 55,212-2 and group II or III mGluR agonists were tested. Group II or III mGluRs agonists were administered intracisternally 10 min after intracisternal administration of WIN 55,212-2. Neither 100 nmol APDC, a group II mGluRs agonist, nor L-AP4, a group III mGluR agonist, altered nociceptive behavior when given alone but significantly inhibited the formalin-induced nociceptive behavior in the presence of a sub-threshold dose ( 3microg) of WIN 55,212-2. The ED50 value of APDC or L-AP4 was significantly reduced upon co-treatment with WIN 55,212-2 than in the vehicle-treated group, highlighting the important therapeutic potential of the combined administration of group II or III mGluR agonists with cannabinoids to effectively treat inflammatory pain associated with the TMJ. Potentiating effects of group II or III mGluRs agonists will likely permit the administration of cannabinoids at doses that do not achieve significant accumulation to produce undesirable motor dysfunction.

Intramuscular administration of morphine reduces mustard-oil-induced craniofacial-muscle pain behavior in lightly anesthetized rats.

The present study investigated the role of peripheral opioid receptors in mustard oil-induced nociceptive behavior and inflammation in the masseter muscles of lightly anesthetized rats. Experiments were carried out on male Sprague-Dawley rats weighing between 300 and 400 g. After initial anesthesia with sodium pentobarbital (40 mg/kg, i.p.), one femoral vein was cannulated and connected to an infusion pump for the intravenous infusion of sodium pentobarbital. The rate of infusion was adjusted to provide a constant level of anesthesia. Mustard oil (MO, 30 microl) was injected into the mid-region of the left masseter muscle via a 30-gauge needle. Intramuscularly-administered morphine significantly reduced shaking behavior but not MO-induced inflammation. Intramuscular pretreatment with naloxone, an opioid receptor antagonist, reversed antinociception produced by intramuscularly-administered morphine, while intracisternal administration of naloxone did not affect the antinociception of peripheral morphine. Pretreatment with d-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a mu opioid receptor antagonist, but not naltrindole, a delta opioid receptor antagonist, nor norbinaltorphimine (nor-BNI), a kappa opioid receptor antagonist, reversed intramuscularly-administered morphine-induced antinociception. These results indicate that intramuscularly-administered morphine produces antinociception in craniofacial muscle nociception and that this intramuscularly-administered morphine-induced antinociception is mediated by a peripheral mu opioid receptor. Our observations further support the clinical approach of administering opioids in the periphery for the treatment of craniofacial muscle nociception.

Intracisternal administration of mitogen-activated protein kinase inhibitors reduced mechanical allodynia following chronic constriction injury of infraorbital nerve in rats.

The present study investigated the role of mitogen-activated protein kinase (MAPK) in orofacial neuropathic pain following chronic constriction injury of the infraorbital nerve (ION-CCI). Experiments were carried out on male Sprague-Dawley rats weighing between 200 and 230 g. The ION was separated from adhering tissue, and two ligatures (5-0 chromic gut) were tied loosely around it. We examined the air-puff thresholds (mechanical allodynia), scores of pinprick (mechanical hyperalgesia), and face grooming frequency for acetone application (hypersensitivity for cold stimulation) - 3, 3, 6, 9, 12, 15, 20, 25, 30, and 40 days after surgery. ION-CCI produced mechanical allodynia, hyperalgesia, and cold hypersensitivity. We investigated whether administration of MAPKs inhibitors blocks ION-CCI-induced mechanical allodynia. Intracisternal administration with PD98059 or SB203580, a MEK inhibitor or a p38 MAPK inhibitor, respectively, significantly inhibited ION-CCI-induced mechanical allodynia in the orofacial area. These results indicate that the ION-CCI produced behavioral alterations in the orofacial area and those central MAPKs pathways contribute to orofacial neuropathic pain. Our findings suggest that MAPKs inhibitors have a potential role in treatment for orofacial neuropathic pain.

Blockade of central cyclooxygenase (COX) pathways enhances the cannabinoid-induced antinociceptive effects on inflammatory temporomandibular joint (TMJ) nociception.

The present study is the first to investigate the participation of central cyclooxygenase (COX) pathways in modulating the antinociceptive effects of intracisternally administered cannabinoid on nociception induced by inflammation of the temporomandibular joint (TMJ) in freely moving rats. Following intra-articular injection of 5% formalin in the TMJ, nociceptive scratching behavior was recorded for nine successive 5-min intervals in Sprague-Dawley rats. Intracisternal injection of 30 microg of WIN 55,212-2, a synthetic non-subtype-selective CB1/2 agonist, administered 20 min prior to formalin injection significantly reduced the number of scratches and duration of scratching induced by formalin compared with the vehicle-treated group. Antinociceptive effect of WIN 55,212-2 was blocked by intracisternal injection of 10 microg of AM251, a CB1 receptor-selective antagonist, but not by AM630, a CB2 receptor-selective antagonist. A 10 microg dose of WIN 55,212-2 that was ineffective in producing antinociception became effective following intracisternal administration of NS-398, a selective COX-2 inhibitor; indomethacin, a non-selective COX 1/2 inhibitor; acetaminophen, a putative COX-3 inhibitor, but not following pretreatment with the selective COX-1 inhibitor, SC-560. The ED(50) value of WIN 55,212-2 in the NS-398-treated group was significantly lower than that in the vehicle-treated group. Importantly, administration of low doses of COX inhibitors alone did not attenuate nociception. These results indicate that inhibition of central COX pathways, presumably via COX-2 inhibition, reduces inflammatory pain by enhancing the cannabinoid-induced antinociceptive effect. Based on our observations, combined administration of cannabinoids with COX inhibitors may hold a therapeutic promise in the treatment of inflammatory TMJ pain.

Participation of peripheral group I and II metabotropic glutamate receptors in the development or maintenance of IL-1beta-induced mechanical allodynia in the orofacial area of conscious rats.

The present study investigated the role of peripheral groups I and II metabotropic glutamate receptors (mGluRs) in interleukin (IL)-1beta-induced mechanical allodynia in the orofacial area of rats. Subcutaneous injection of 10 pg of IL-1beta decreased air-puff thresholds ipsilateral or contralateral to the injection site. The decrease in air-puff thresholds appeared 10 min after the injection of IL-1beta and IL-1beta-induced mechanical allodynia persisted for over 3 h. Pre-treatment with 7-(hydroxyimino) cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt) or 2-methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP), a mGluR1 or mGluR5 antagonist, blocked IL-1beta-induced mechanical allodynia and mirror-image mechanical allodynia produced by a subcutaneous injection of 10 pg of IL-1beta. However, post-treatment with CPCCOEt or MPEP did not affect changes in behavioral responses, which were produced by the IL-1beta injection. Pre-treatment, as well as post-treatment with (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC), a group II mGluR agonist, blocked either IL-1beta-induced mechanical allodynia or mirror-image mechanical allodynia. The anti-allodynic effects of APDC were abolished by pre-treatment with (2S)-2-amino-2[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), a group II mGluR antagonist. These results indicate that peripheral group II mGluRs are involved in the development and maintenance of IL-1beta-induced mechanical allodynia, while peripheral group I mGluRs are involved in the development of IL-1beta-induced mechanical allodynia. Based on our observations, the peripheral application of group II mGluR agonists may be of therapeutic value in treating inflammatory pain.