Differential Regulation of Intracisternally Injected Angiotensin II-Induced Mechanical Allodynia and Thermal Hyperalgesia in Rats.

The present study examined the underlying mechanisms of mechanical allodynia and thermal hyperalgesia induced by the intracisternal injection of angiotensin (Ang) II. Intracisternal Ang II injection decreased the air puff threshold and head withdrawal latency. To determine the operative receptors for each distinct type of pain behavior, we intracisternally injected Ang II receptor antagonists 2 h after Ang II injection. Losartan, an Ang II type 1 receptor (AT1R) antagonist, alleviated mechanical allodynia. Conversely, PD123319, an Ang II type 1 receptor (AT2R) antagonist, blocked only thermal hyperalgesia. Immunofluorescence analyses revealed the co-localization of AT1R with the astrocyte marker GFAP in the trigeminal subnucleus caudalis and co-localization of AT2R with CGRP-positive neurons in the trigeminal ganglion. Intracisternal pretreatment with minocycline, a microglial inhibitor, did not affect Ang II-induced mechanical allodynia, whereas L-α-aminoadipate, an astrocyte inhibitor, significantly inhibited Ang II-induced mechanical allodynia. Furthermore, subcutaneous pretreatment with botulinum toxin type A significantly alleviated Ang II-induced thermal hyperalgesia, but not Ang II-induced mechanical allodynia. These results indicate that central Ang II-induced nociception is differentially regulated by AT1R and AT2R. Thus, distinct therapeutic targets must be regulated to overcome pain symptoms caused by multiple underlying mechanisms.

TNF-α-Mediated RIPK1 Pathway Participates in the Development of Trigeminal Neuropathic Pain in Rats.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) participates in the regulation of cellular stress and inflammatory responses, but its function in neuropathic pain remains poorly understood. This study evaluated the role of RIPK1 in neuropathic pain following inferior alveolar nerve injury. We developed a model using malpositioned dental implants in male Sprague Dawley rats. This model resulted in significant mechanical allodynia and upregulated RIPK1 expression in the trigeminal subnucleus caudalis (TSC). The intracisternal administration of Necrosatin-1 (Nec-1), an RIPK1 inhibitor, blocked the mechanical allodynia produced by inferior alveolar nerve injury The intracisternal administration of recombinant rat tumor necrosis factor-α (rrTNF-α) protein in naive rats produced mechanical allodynia and upregulated RIPK1 expression in the TSC. Moreover, an intracisternal pretreatment with Nec-1 inhibited the mechanical allodynia produced by rrTNF-α protein. Nerve injury caused elevated TNF-α concentration in the TSC and a TNF-α block had anti-allodynic effects, thereby attenuating RIPK1 expression in the TSC. Finally, double immunofluorescence analyses revealed the colocalization of TNF receptor and RIPK1 with astrocytes. Hence, we have identified that astroglial RIPK1, activated by the TNF-α pathway, is a central driver of neuropathic pain and that the TNF-α-mediated RIPK1 pathway is a potential therapeutic target for reducing neuropathic pain following nerve injury.

Mannitol Enhances the Antinociceptive Effects of Diphenhydramine as an Alternative Local Anesthetic.

Mannitol has recently been reported to be effective in enhancing the antinociceptive efficacy of lidocaine. No single study to date, however, has compared diphenhydramine with and without mannitol for nociceptive processing as an alternative local anesthetic. In this study, we examined the antinociceptive efficacy enhancements of diphenhydramine when combined with mannitol. Male Sprague-Dawley rats weighing 230-260 g were used in a hot plate test to evaluate the antinociceptive effects of diphenhydramine. All chemicals were dissolved in isotonic normal saline and administered subcutaneously into the plantar surface of the right hind paw at 10 min before the hot plate test. A subcutaneous injection of 0.5% or 1% diphenhydramine produced significant inhibition of the withdrawal latency time compared with the vehicle treatment. Antinociceptive effects appeared 10 min after the diphenhydramine injections and persisted for over 30 min. The antinociceptive effects of 1% diphenhydramine were not statistically different from those of 1% lidocaine. Although a subcutaneous injection of a 0.5 M mannitol solution alone did not affect the withdrawal latency time, 1% diphenhydramine with 0.5 M mannitol significantly enhanced antinociception. A subcutaneous injection of 1% diphenhydramine with epinephrine (1 : 100,000) solution did not increase the antinociceptive effect of the diphenhydramine. These results suggest that diphenhydramine with mannitol can be used as an alternative local anesthetic.

Early Blockade of EphA4 Pathway Reduces Trigeminal Neuropathic Pain.

BACKGROUND: Although the Eph receptor plays an important role in the development of neuropathic pain following nerve injury, there has been no evidence of the participation of the ephrin A4 receptor (EphA4) in the development of trigeminal neuropathic pain. The present study investigated the role of EphA4 in central nociceptive processing in rats with inferior alveolar nerve injury. MATERIALS AND METHODS: Male Sprague-Dawley rats were used in all our experiments. A rat model for trigeminal neuropathic pain was produced using malpositioned dental implants. The left mandibular second molar was extracted under anesthesia, followed by the placement of a miniature dental implant to injure the inferior alveolar nerve. RESULTS: Our current findings show that nerve injury induced by malpositioned dental implants evokes significant mechanical allodynia and up-regulation of EphA4 expression in the ipsilateral trigeminal subnucleus caudalis. Although daily treatment with EphA4-Fc, an EphA4 antagonist, did not produce prolonged anti-allodynic effects after the chronic neuropathic pain had been already established, an early treatment protocol with repeated EphA4-Fc administration significantly attenuated mechanical allodynia before initiation of chronic neuropathic pain. Finally, we confirmed the participation of the central EphA4 pathway in the development of trigeminal neuropathic pain by reducing EphA4 expression using EphA4 siRNA. This suppression of EphA4 produced significantly prolonged anti-allodynic effects. CONCLUSION: These results suggest that early blockade of central EphA4 signaling provides a new therapeutic target for the treatment of trigeminal neuropathic pain.

Preemptive application of QX-314 attenuates trigeminal neuropathic mechanical allodynia in rats.

The aim of the present study was to examine the effects of preemptive analgesia on the development of trigeminal neuropathic pain. For this purpose, mechanical allodynia was evaluated in male Sprague-Dawley rats using chronic constriction injury of the infraorbital nerve (CCI-ION) and perineural application of 2% QX-314 to the infraorbital nerve. CCI-ION produced severe mechanical allodynia, which was maintained until postoperative day (POD) 30. An immediate single application of 2% QX-314 to the infraorbital nerve following CCI-ION significantly reduced neuropathic mechanical allodynia. Immediate double application of QX-314 produced a greater attenuation of mechanical allodynia than a single application of QX-314. Immediate double application of 2% QX-314 reduced the CCI-ION-induced upregulation of GFAP and p-p38 expression in the trigeminal ganglion. The upregulated p-p38 expression was co-localized with NeuN, a neuronal cell marker. We also investigated the role of voltage-gated sodium channels (Navs) in the antinociception produced by preemptive application of QX-314 through analysis of the changes in Nav expression in the trigeminal ganglion following CCI-ION. Preemptive application of QX-314 significantly reduced the upregulation of Nav1.3, 1.7, and 1.9 produced by CCI-ION. These results suggest that long-lasting blockade of the transmission of pain signaling inhibits the development of neuropathic pain through the regulation of Nav isoform expression in the trigeminal ganglion. Importantly, these results provide a potential preemptive therapeutic strategy for the treatment of neuropathic pain after nerve injury.

A role for the purinergic receptor P2X3 in astrocytes in the mechanism of craniofacial neuropathic pain.

The purinergic receptor P2X3, expressed in the central terminals of primary nociceptive neurons in the brainstem, plays an important role in pathological pain. However, little is known about expression of P2X3 in the brainstem astrocytes and its involvement in craniofacial pathologic pain. To address this issue, we investigated the expression of P2X3 in astrocytes in the trigeminal caudal nucleus (Vc) in a rat model of craniofacial neuropathic pain, chronic constriction injury of infraorbital nerve (CCI-ION). We found that 1) P2X3-immunoreactivity is observed in the brainstem astrocytes, preferentially in their fine processes, 2) the number of P2X3-positive fine astrocytic processes and the density of P2X3 in these processes were increased significantly in CCI-ION rats, compared to control rats, and 3) administration of MPEP, a specific mGluR5 antagonist, alleviated the mechanical allodynia and abolished the increase in density of P2X3 in fine astrocytic processes caused by CCI-ION. These findings reveal preferential expression of P2X3 in the fine astrocytic processes in the brainstem, propose a novel role of P2X3 in the fine astrocytic process in the mechanism of craniofacial neuropathic pain, and suggest that the expression of astrocytic P2X3 may be regulated by astrocytic mGluR5.

Participation of central GABAA receptors in the trigeminal processing of mechanical allodynia in rats.

Here we investigated the central processing mechanisms of mechanical allodynia and found a direct excitatory link with low-threshold input to nociceptive neurons. Experiments were performed on male Sprague-Dawley rats weighing 230-280 g. Subcutaneous injection of interleukin 1 beta (IL-1ß) (1 ng/10 µL) was used to produce mechanical allodynia and thermal hyperalgesia. Intracisternal administration of bicuculline, a gamma aminobutyric acid A (GABAA) receptor antagonist, produced mechanical allodynia in the orofacial area under normal conditions. However, intracisternal administration of bicuculline (50 ng) produced a paradoxical anti-allodynic effect under inflammatory pain conditions. Pretreatment with resiniferatoxin (RTX), which depletes capsaicin receptor protein in primary afferent fibers, did not alter the paradoxical anti-allodynic effects produced by the intracisternal injection of bicuculline. Intracisternal injection of bumetanide, an Na-K-Cl cotransporter (NKCC 1) inhibitor, reversed the IL-1ß-induced mechanical allodynia. In the control group, application of GABA (100 µM) or muscimol (3 µM) led to membrane hyperpolarization in gramicidin perforated current clamp mode. However, in some neurons, application of GABA or muscimol led to membrane depolarization in the IL-1ß-treated rats. These results suggest that some large myelinated Aß fibers gain access to the nociceptive system and elicit pain sensation via GABAA receptors under inflammatory pain conditions.

Progesterone produces antinociceptive and neuroprotective effects in rats with microinjected lysophosphatidic acid in the trigeminal nerve root.

BACKGROUND: In our present study, we studied the role of demyelination of the trigeminal nerve root in the development of prolonged nociceptive behavior in the trigeminal territory. RESULTS: Under anesthesia, the Sprague-Dawley rats were mounted onto a stereotaxic frame and 3 µL of lysophosphatidic acid (LPA, 1 nmol) was injected into the trigeminal nerve root to produce demyelination. This treatment decreased the air-puff thresholds, persisted until postoperative day 130, and then returned to the preoperative levels 160 days after LPA injection. The LPA-treated rats also showed a significant hyper-responsiveness to pin-prick stimulation. We further investigated the antinociceptive and neuroprotective effects of progesterone in rats undergoing demyelination of the trigeminal nerve root. Progesterone (8, 16 mg/kg/day) was administered subcutaneously, beginning on the operative day, for five consecutive days in the LPA-treated rats. Treatment with progesterone produced significant early anti-allodynic effects and delayed prolonged anti-allodynic effects. The expression of protein zero (P0) and peripheral myelin protein 22 (PMP22) were significantly down-regulated in the trigeminal nerve root on postoperative day 5 following LPA injection. This down-regulation of the P0 and PMP22 levels was blocked by progesterone treatment. CONCLUSIONS: These results suggest that progesterone produces antinociceptive effects through neuroprotective action in animals with LPA-induced trigeminal neuropathic pain. Moreover, progesterone has potential utility as a novel therapy for trigeminal neuropathic pain relief at an appropriate managed dose and is therefore a possible future treatment strategy for improving the recovery from injury.

WITHDRAWN: Peripheral mGluR5 antagonist attenuated craniofacial muscle pain and inflammation but not mGluR1 antagonist in lightly anesthetized rats.

The present study investigated the role of peripheral group I metabotropic glutamate receptors (mGluRs) in MO-induced nociceptive behaviour and inflammation in the masseter muscles of lightly anesthetized rats. Experiments were carried out on male Sprague-Dawley rats weighing 300-400 g. After initial anesthesia with sodium pentobarbital (40 mg/kg, i.p.), one femoral vein was cannulated and connected to an infusion pump for 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 over 10s. After 30 microl injection of 5, 10, 15, or 20% MO into the masseter muscle, the total number of hindpaw shaking behaviour and extravasated Evans' blue dye concentration in the masseter muscle were significantly higher in the MO-treated group in a dose-dependent manner compared with the vehicle (mineral oil)-treated group. Intramuscular pretreatment with 3 or 5% lidocaine reduced MO-induced hindpaw shaking behaviour and increases in extravasated Evans' blue dye concentration. Intramuscular pretreatment with 5mM MCPG, non-selective group I/II mGluR antagonist, or MPEP, a selective group I mGluR5 antagonist, produced a significant attenuation of MO-induced hindpaw shaking behaviour and increases in extravasated Evans' blue dye concentration in the masseter muscle while LY367385, a selective group I mGluR1 antagonist, did not affect MO-induced nociceptive behaviour and inflammation in the masseter muscle. These results indicate that peripheral mGluR5 plays important role in mediating MO-induced nociceptive behaviour and inflammation in the craniofacial muscle.

Peripheral mGluR5 antagonist attenuated craniofacial muscle pain and inflammation but not mGluR1 antagonist in lightly anesthetized rats.

The present study investigated the role of peripheral group I metabotropic glutamate receptors (mGluRs) in MO-induced nociceptive behaviour and inflammation in the masseter muscles of lightly anesthetized rats. Experiments were carried out on male Sprague-Dawley rats weighing 300-400 g. After initial anesthesia with sodium pentobarbital (40 mg/kg, i.p.), one femoral vein was cannulated and connected to an infusion pump for 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 over 10s. After 30 microl injection of 5, 10, 15, or 20% MO into the masseter muscle, the total number of hindpaw shaking behaviour and extravasated Evans' blue dye concentration in the masseter muscle were significantly higher in the MO-treated group in a dose-dependent manner compared with the vehicle (mineral oil)-treated group. Intramuscular pretreatment with 3 or 5% lidocaine reduced MO-induced hindpaw shaking behaviour and increases in extravasated Evans' blue dye concentration. Intramuscular pretreatment with 5 mM MCPG, non-selective group I/II mGluR antagonist, or MPEP, a selective group I mGluR5 antagonist, produced a significant attenuation of MO-induced hindpaw shaking behaviour and increases in extravasated Evans' blue dye concentration in the masseter muscle while LY367385, a selective group I mGluR1 antagonist, did not affect MO-induced nociceptive behaviour and inflammation in the masseter muscle. These results indicate that peripheral mGluR5 plays important role in mediating MO-induced nociceptive behaviour and inflammation in the craniofacial muscle.

Intracisternal administration of mitogen-activated protein kinase inhibitors reduced IL-1beta-induced mirror-image mechanical allodynia in the orofacial area of rats.

The present study investigated the role of central mitogen-activated protein kinases (MAPKs) in interleukin-1beta (IL-1beta)-induced mirror-image mechanical allodynia in the orofacial area. Experiments were carried out on Sprague-Dawley rats. Under pentobarbital sodium anesthesia, a polyethylene tube was implanted in the subcutaneous area of one vibrissa pad, which enabled us to inject IL-1beta. For an intracisternal injection, each anesthetized rat was mounted on a stereotaxic frame and a polyethylene tube was implanted. Animals were given a recovery time of at least 72 h from surgery. After a subcutaneous administration of 0.01, 0.1, 1, or 10 pg of IL-1beta, we examined the face withdrawal behavioral responses produced by 10 successive trials of air puffs ipsilateral or contralateral to the IL-1beta injection site. Normal animals did not respond to pressure less than 40 psi. The thresholds of air puffs ipsilateral and contralateral to the IL-1beta injection site were significantly lower in the IL-1beta-treated group, compared with the vehicle-treated group. The decrease in the threshold of air puffs appeared 10 min after an IL-1beta injection and persisted for over 3h. Intracisternal pretreatment with PD98059, a p44/42 MAPK inhibitor, or SB203580, a p38 MAPK inhibitor, significantly reduced the decrease in the threshold of air puffs ipsilateral to the IL-1beta injection site produced by 10 pg of IL-1beta. IL-1beta-induced mirror-image mechanical allodynia was also reduced significantly by intracisternal pretreatment with both PD98059 and SB203580. These results indicate that central MAPK pathways mediate IL-1beta-induced mirror-image mechanical allodynia in the orofacial area.

Interleukin-1 beta injected intracisternally inhibited NMDA-evoked behavioral response in the orofacial area of freely moving rats.

The brain-derived interleukin-1beta (IL-1beta) has been involved in the modulation of nociceptive processing. The direction of the effects, however, analgesia or hyperalgesia, is controversial. Here, we report the role of IL-1beta injected intracisternally in orofacial pain transmission. Experiments were carried out on 90 male SD rats and surgical procedures were performed under pentobarbital sodium. Intracisternal injection of 0.3 or 0.6 microg of N-methyl-d-aspartic acid (NMDA) produced intense scratching behavioral responses including vocalization, agitation and a desire to escape in a dose-related manner. The intracisternal injection of 1 or 10 ng IL-1beta significantly decreased the NMDA-evoked scratching behavioral responses. Pretreatment with an IL-1 receptor antagonist or naloxone, an opioid receptor antagonist, blocked the IL-1beta-induced antinociceptive response. These results suggest that cytokine injected intracisternally seems to produce antinociceptive effects in the NMDA-evoked pain model of the orofacial area and the antinociceptive effect seems to be mediated by an opioid pathway.

Intracisternal NMDA produces analgesia in the orofacial formalin test of freely moving rats.

The present study was performed to investigate the antinociceptive response to the intracisternal administration of NMDA in the orofacial area. To achieve this purpose, the effects of NMDA injected intracisternally on the orofacial formalin test were monitored in freely moving rats. We also investigated underlying the mechanisms of NMDA-induced antinociceptive response. Experiments were carried out on 80 male SD rats and surgical procedures were performed under pentobarbital sodium (40 mg/kg, i.p.). Fifty microliters of 5% formalin was applied subcutaneously to the vibrissa pad without any restraining of the animals. For each animal, the number of noxious behavioral responses and the time spent grooming, rubbing, and/or scratching the facial region proximal to the injection site were recorded for nine successive 5-min intervals. The orofacial formalin responses showed two distinct phases separated by a time of relative inactivity. Intracisternal administration of NMDA produced intense scratching behavioral responses with dose related manner. NMDA injected intracisternally 30 min prior to formalin injection, however, inhibited noxious behavioral responses produced by a formalin injection significantly. Pretreatment with naloxone 20 min prior to NMDA injection abolished the inhibition of number of scratches and the duration of scratching produced by the intracisternal injection of NMDA in the late phase. Pretreatment with L-NAME, NO synthesis inhibitor, however, did not affect the antinociceptive response produced by NMDA injected intracisternally. These results suggest that NMDA injected intracisternally produces brief pain behavioral responses and also produces delayed antinociceptive effects in the orofacial formalin test. The opioid pathway seems to be involved in the NMDA-induced antinociception in the orofacial area.

Effects of TNF-alpha injected intracisternally on the nociceptive jaw-opening reflex and orofacial formalin test in freely moving rats.

The present study was performed to investigate the effects of central cytokines on the modulation of nociception in the orofacial area. A nociceptive jaw-opening reflex (JOR) and an orofacial formalin test were monitored after intracisternal administration of tumor necrosis factor (TNF)-alpha in freely moving rats. Experiments were carried out on 83 male rats weighing 300-350 g and surgical procedures were performed under pentobarbital sodium. After intracisternal injection of Tnf-alpha, digastric electromyogram (dEMG) and noxious behavioral responses were monitored. In the nociceptive JOR, dEMG was not significantly changed after intracisternal injection of 200 pg and 2 ng Tnf-alpha. However, 20 ng Tnf-alpha suppressed dEMG to 72+/-6% of the control values. The orofacial formalin responses showed two distinct phases separated by a time of relative inactivity with an early short-lasting response (0-9 min, first phase) and a continuous prolonged response (10-45 min, second phase). In the inflammatory orofacial formalin test, intracisternal injection of 20 pg Tnf-alpha did not change the number of noxious behavioral responses produced by formalin injection. However, 200 pg Tnf-alpha injected intracisternally significantly increased the number of noxious behavioral responses produced by formalin injection in both the early and late phases, and 2 ng Tnf-alpha increased formalin induced noxious behavioral responses in only the late phase. A higher dose of 20 ng Tnf-alpha did not change the number of noxious behavioral responses produced by formalin injection. The hyperalgesic action of Tnf-alpha injected intracisternally was blocked by pretreatment with the interleukin-1 (IL-1) receptor antagonist. These results suggest that central Tnf-alpha modulates the transmission of nociceptive information in the orofacial area. However, the hypo/hyperalgesic response of central Tnf-alpha seems to depend on the orofacial pain model or in a dose-related manner. The hyperalgesic response of central Tnf-alpha seems to be mediated by the IL-1 receptor.

Effects of intracisternal injection of interleukin-6 on nociceptive jaw opening reflex and orofacial formalin test in freely moving rats.

The present study was performed to investigate the effects of central cytokines on the modulation of nociception in the orofacial area. To achieve this purpose, a nociceptive jaw opening reflex and an orofacial formalin test were monitored before and after intracisternal administration of interleukin-6 (IL-6) in freely moving rats. In the nociceptive jaw opening reflex, the digastric electromyogram (dEMG) was not significantly changed after intracisternal injection of 200 pg and 2 ng IL-6. However, 20 ng IL-6 suppressed dEMG to 74+/-7% of the control values. In the inflammatory orofacial formalin test, intracisternal injection of 200 pg and 2 ng IL-6 did not change the number of noxious behavioral responses produced by formalin injection. However, 20 ng IL-6 injected intracisternally significantly increased the number of noxious behavioral responses produced by formalin. The hyperalgesic action of intracisternal IL-6 in the orofacial formalin test was blocked by pretreatment with interleukin-1 (IL-1) receptor antagonist. These results suggest that IL-6 injected intracisternally modulates the transmission of nociceptive information in the orofacial area. However, the hypo/hyper-algesic response of central cytokines seems to depend on the orofacial pain model. The hyperalgesic response of central IL-6 seems to be mediated by the IL-1 receptor.