Peripheral glutamate receptors are required for hyperalgesia induced by capsaicin.

Transient receptor potential vanilloid1 (TRPV1) and glutamate receptors (GluRs) are located in small diameter primary afferent neurons (nociceptors), and it was speculated that glutamate released in the peripheral tissue in response to activation of TRPV1 might activate nociceptors retrogradely. But, it was not clear which types of GluRs are functioning in the nociceptive sensory transmission. In the present study, we examined the c-Fos expression in spinal cord dorsal horn following injection of drugs associated with glutamate receptors with/without capsaicin into the hindpaw. The subcutaneous injection of capsaicin or glutamate remarkably evoked c-Fos expression in ipsilateral sides of spinal cord dorsal horn. This capsaicin evoked increase of c-Fos expression was significantly prevented by concomitant administration of MK801, CNQX, and CPCCOEt. On the other hand, there were not any significant changes in coinjection of capsaicin and MCCG or MSOP. These results reveal that the activation of iGluRs and group I mGluR in peripheral afferent nerves play an important role in mechanisms whereby capsaicin evokes/maintains nociceptive responses.

Capsaicin-induced glutamate release is implicated in nociceptive processing through activation of ionotropic glutamate receptors and group I metabotropic glutamate receptor in primary afferent fibers.

Glutamate (Glu) is the major excitatory neurotransmitter in the central nervous system. The role of peripheral Glu and Glu receptors (GluRs) in nociceptive transmission is, however, still unclear. In the present study, we examined Glu levels released in the subcutaneous perfusate of the rat hind instep using a microdialysis catheter and the thermal withdrawal latency using the Plantar Test following injection of drugs associated with GluRs with/without capsaicin into the hindpaw. The injection of capsaicin into the rat hind instep caused an increase of Glu level in the s.c. perfusate. Capsaicin also significantly decreased withdrawal latency to irradiation. These effects of capsaicin were inhibited by pretreatment with capsazepine, a transient receptor potential vanilloid receptor 1 (TRPV1) competitive antagonist. Capsaicin-induced Glu release was also suppressed by combination with each antagonist of ionotropic GluRs (iGluRs: NMDA/AMPA receptors) and group I metabotropic GluR (mGluR), but not group II and group III mGluRs. Furthermore, these GluRs antagonists showed remarkable inhibition against capsaicin-induced thermal hyperalgesia. These results suggest that Glu is released from the peripheral endings of small-diameter afferent fibers by noxious stimulation and then activates peripheral iGluRs and group I mGluR in development and/or maintenance of nociception. Furthermore, the activation of peripheral NMDA/AMPA receptors and group I mGluR may be important in mechanisms whereby capsaicin evokes nociceptive responses.

Alteration in sensitivity of ionotropic glutamate receptors and tachykinin receptors in spinal cord contribute to development and maintenance of nerve injury-evoked neuropathic pain.

Allodynia or hyperalgesia induced by peripheral nerve injury may be involved in changes in the sensitivity of neurotransmitters at the spinal cord level. In order to clarify the functional role of neurotransmitters in peripheral nerve injury, we used rats with nerve injury induced by chronic constriction of the sciatic nerve (CCI rat model) and estimated the effects of the intrathecal injection of drugs known to affect glutamate and tachykinin receptors. In sham-operated rats, the NMDA receptor agonist NMDA and AMPA-kinate receptor agonist RS-(5)-bromowillardin reduced withdrawal latency. The non-competitive NMDA receptor antagonist MK-801, competitive NMDA receptor antagonist AP-5 and AMPA-kinate receptor antagonist NBQX increased withdrawal latency. Substance P (SP) increased the withdrawal latency but only transitorily. The NK1 receptor antagonist RP67580 increased withdrawal latency, but the NK2 receptor antagonist SR48968 did not show an effect. In CCI rats, RS-(5)-bromowillardin reduced withdrawal latency, but NMDA did not show an effect. NBQX increased withdrawal latency, while MK-801 and AP-5 showed little or no effect. SP reduced withdrawal latency, and both RP67580 and SR48968 increased it. These results indicate that the alteration in sensitivity of ionotropic glutamate receptors and tachykinin receptors in the spinal cord contribute to development and maintenance of nerve injury-evoked neuropathic pain.

Mechanisms of orofacial pain control in the central nervous system.

Recent advances in the study of pain have revealed somatotopic- and modality-dependent processing and the integration of nociceptive signals in the brain and spinal cord. This review summarizes the uniqueness of the trigeminal sensory nucleus (TSN) in structure and function as it relates to orofacial pain control. The oral nociceptive signal is primarily processed in the rostral TSN above the obex, the nucleus principalis (Vp), and the subnuclei oralis (SpVo) and interpolaris (SpVi), while secondarily processed in the subnucleus caudalis (SpVc). In contrast, the facial nociceptive signal is primarily processed in the SpVc. The neurons projecting to the thalamus are localized mostly in the Vp, moderately in the SpVi, and modestly in the ventrolateral SpVo and the SpVc. Orofacial sensory inputs are modulated in many different ways: by interneurons in the TSN proper, through reciprocal connection between the TSN and rostral ventromedial medulla, and by the cerebral cortex. A wide variety of neuroactive substances, including substance P, gamma-aminobutyric acid, serotonin and nitric oxide (NO) could be involved in the modulatory functions of these curcuits. The earliest expression of NO synthase (NOS) in the developing rat brain is observed in a discrete neuronal population in the SpVo at embryonic day 15. NOS expression in the SpVc is late at postnatal day 10. The neurons receiving intraoral signals are intimately related with the sensorimotor reflexive function through the SpVo. In summary, a better understanding of the trigeminal sensory system--which differs from the spinal system--will help to find potential therapeutic targets and lend to developing new analgesics for orofacial-specific pain with high efficacy and fewer side effects.

Nitric oxide-induced increase of excitatory amino acid levels in the trigeminal nucleus caudalis of the rat with tactile hypersensitivity evoked by the loose-ligation of the inferior alveolar nerves.

To investigate whether or not N-methyl-D-aspartate (NMDA)/nitric oxide (NO) pathway in the trigeminal system is involved in the development and/or maintenance of such pathological pain states as the hyperalgesia and allodynia observed after dental surgery, we examined the alteration patterns of excitatory amino acid (EAA) level in the superficial layer of subnucleus caudalis of the brain-stem trigeminal sensory nuclear complex (SpVc-I,II) by in vivo microdialysis. A very high EAA release response was observed immediately after the start of the perfusion in ligated animals compared with sham-operated rats. The EAA level evoked by application of the 40-V tooth pulp-stimulation or 1% capsaicin cream was significantly higher in the ligated animals than those in the sham-operated animals. This increase of EAA level induced by capsaicin cream was inhibited by adding carboxy-PTIO (100 microM) to the perfusate. The applications of SNAP (2 mM) into the perfusate enhanced the level of EAAs in ligated animals and sham-operated animals. However, SNAP-evoked EAA levels in ligated animals were not significantly different compared with those of sham-operated animals. These results suggest that alterations in the stimulus-evoked raised EAA levels that occur in the site of the first synaptic relay of the dental pain pathway and which are expressed via endogenous NO, and which play an important role in development and/or maintenance of pathological pain states following dental peripheral nerve injury.

Effects of deep hypothermia on nitric oxide-induced cytotoxicity in primary cultures of cortical neurons.

Nitric oxide (NO) is thought to play a major role during cerebral ischemia. However, the protective efficacy of hypothermia against NO-induced neurotoxicity remains to be examined. In the present study, the degree of neurotoxicity induced by NO was analyzed in two temperature groups (normothermia, 37 degrees C; deep hypothermia, 22 degrees C) of cultured E16 Wistar rat cortical neurons. Two different NO donors, 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC-12) and 1-hydroxy-2-oxo-3-(3-amynopropyl)-3-isopropyl-1-triazene (NOC-5), that have equal half-lives at 37 degrees C and 22 degrees C, respectively, were used. Cultured neurons in each temperature group were exposed to 30 and 100 micro M NOC for three different time courses, 6 hr, 12 hr, and 24 hr. The survival rates of neurons were evaluated by assessing viable neurons on photomicrographs before and after the experiments. The highest survival rate (approximately 93%) was seen in both temperature groups when neurons were exposed to 30 micro M NOC for 6 hr and 12 hr, and there was no significant difference observed between these two groups (P > 0.05). Almost equal survival rates were observed in both temperature groups following exposure to 30 micro M NOC for 24 hr (at 37 degrees C, 80.4% +/- 2.6%; at 22 degrees C, 83.2% +/- 1.6%; P > 0.05). During exposure to 100 micro M NOC, although the survival rate linearly decreased (approximately from 70% to 5%) in both temperature groups when exposed for 6-24 hr, there were no significant intergroup differences observed (P > 0.05). In conclusion, hypothermia does not provide adequate protection to the neurons by acting on the mechanisms evoked by NO, so we speculate that hypothermia may not confer neuroprotetcion once NO is released during ischemia.

Involvement of NMDA-nitric oxide pathways in the development of tactile hypersensitivity evoked by the loose-ligation of inferior alveolar nerves in rats.

To investigate whether or not NMDA/nitric oxide (NO) pathways in the trigeminal system are involved in the development and/or maintenance of such pathological pain states as the hyperalgesia and allodynia observed after dental surgery, loose-ligation on the left inferior alveolar nerves of rats were performed. The responses to mechanical stimulation were then measured using von Frey filaments. Hypersensitivity to tactile stimulation developed on the ipsilateral side in ligated animals 5 days after surgery and lasted for at least 30 days. In addition, the effects of drugs on these pain states during the period 2-3 weeks following surgery were investigated. As a result, it was observed that tactile hypersensitivity was inhibited by the intraperitoneal (i.p.) administration of both MK-801 hydrogen maleate (0.05-0.1 mg/kg) and N(G)-monomethyl-L-arginine acetate (L-NMMA: 10-100 mg/kg). Still further, NO production and the number of neuronal NO synthase (nNOS)-positive neurons in the trigeminal nucleus caudalis (SpVc) was evaluated. As a result of these experiments, it was found that the changes in NO levels evoked by the intravenous infusions of N-methyl-D-aspartate (NMDA; 10 mg/kg) and MK-801 (0.5 mg/kg) were significantly larger in the loose-ligated rats compared to the sham-operated rats. Moreover, the number of nNOS-positive neurons was found to have increased on the ipsilateral side in layers I/II of the SpVc. These results would suggest that tactile hypersensitivity develops after inferior alveolar nerve injury and that NMDA receptor/NOS/NO production pathways in the SpVc may be involved in the development of such pathophysiological states.

Involvement of the NMDA-nitric oxide pathway in the development of hypersensitivity to tactile stimulation in dental injured rats.

To investigate mechanisms in pathological pain conditions as the hyperalgesia and allodynia observed after dental surgery, we employed a rat dental-injury model involving the simultaneous pulpectomy to a lower incisor and extraction of an ipsilateral upper incisor. We found that hypersensitivity to tactile stimulation developed on both ipsilateral and contralateral sides in the dental-injured rats 5 days after the surgery and that this lasted for at least 30 days. Recovery from hypersensitivity to tactile stimulation was achieved by the intraperitoneal (i.p.) administration of MK-801 (0.05 mg/kg) or N(G)-monomethyl-L-arginine monoacetate (L-NMMA: 10 - 100 mg/kg), but not attained by N(G)-monomethyl-D-arginine monoacetate (D-NMMA: 100 mg/kg). This recovery effect of L-NMMA (50 mg/kg) was inhibited by pretreatment with L-arginine (600 mg/kg). In the trigeminal nucleus caudalis (SpVc), the changes in nitric oxide (NO) levels invoked by the intravenous (i.v.) administration of N-methyl-D-aspartate (NMDA; 10 mg/kg) were found to be significantly larger in the dental-injured rats than in sham-operated rats. The number of neuronal NO synthase (nNOS)-positive neurons increased in layers I-II and III-IV in the SpVc on both sides of the dental-injured rats. These results suggest that hypersensitivity to tactile stimulation developed following dental injury, and that NMDA receptor/NOS/NO production pathways in the SpVc may be involved in pathological conditions.

A possible involvement of the central endorphin system in the autoanalgesia induced by chronic administration of Freund's adjuvant solution in rats.

Chronic pain was induced in rats by daily injections of complete Freund's adjuvant into hind paws. Daily changes of pain threshold and endorphin (ED) content and their receptors in 4 divided parts: cortex, diencephalon-mesencephalon containing striatum (D-M), pons-medulla (P-M) and spinal cord, were measured. Decrease in pain responsiveness was observed in the adjuvant-injected group with concomitant increase of ED content in P-M and spinal cord. This decrease in pain responsiveness in the adjuvant-injected group was significantly different from that in the non-treated control group being partially reversed by naloxone. Furthermore, [3H]met-enkephalin binding sites increase in number in P-M of the adjuvant-injected group when maximal decrease of pain responsiveness was observed, returning to control level thereafter. Scatchard analysis revealed the increase of the low affinity binding site in P-M of the adjuvant-injected group. In cortex and D-M, on the other hand, ED content tended to decrease and no change was observed in number of [3H]met-enkephalin binding sites. These results indicate that the ED system in P-M and spinal cord may be more substantially involved in autoanalgesia than in cortex and D-M.