Mouse maternal odontogenic infection with Porphyromonas gingivalis induces cognitive decline in offspring.

Introduction: Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, causes intrauterine infection/inflammation. Offspring exposed to intrauterine infection/inflammation have an increased risk of neurological disorders, regardless of gestational age. However, the relationship between maternal periodontitis and offspring functional/histological changes in the brain has not yet been elucidated. Methods: In this study, we used a gestational mouse model to investigate the effects of maternal odontogenic infection of P. gingivalis on offspring behavior and brain tissue. Results: The step-through passive avoidance test showed that the latency of the acquisition trial was significantly shorter in the P. gingivalis group (p < 0.05), but no difference in spontaneous motor/exploratory parameters by open-field test. P. gingivalis was diffusely distributed throughout the brain, especially in the hippocampus. In the hippocampus and amygdala, the numbers of neuron cells and cyclic adenosine monophosphate response element binding protein-positive cells were significantly reduced (p < 0.05), whereas the number of ionized calcium binding adapter protein 1-positive microglia was significantly increased (p < 0.05). In the hippocampus, the number of glial fibrillary acidic protein-positive astrocytes was also significantly increased (p < 0.05). Discussion: The offspring of P. gingivalis-infected mothers have reduced cognitive function. Neurodegeneration/neuroinflammation in the hippocampus and amygdala may be caused by P. gingivalis infection, which is maternally transmitted. The importance of eliminating maternal P. gingivalis-odontogenic infection before or during gestation in maintenance healthy brain function in offspring should be addressed in near future.

High-frequency near-infrared semiconductor laser irradiation suppressed experimental tooth movement-induced inflammatory pain markers in the periodontal ligament tissues of rats.

High-frequency near-infrared (NIR) semiconductor laser-irradiation has an unclear effect on nociception in the compressed lateral periodontal ligament region, a peripheral nerve region. This study aimed to investigate the effects of NIR semiconductor laser irradiation, with a power of 120 J, on inflammatory pain markers and neuropeptides induced in the compressed lateral periodontal ligament area during ETM. A NIR semiconductor laser [910 nm wavelength, 45 W maximum output power, 300 mW average output power, 30 kHz frequency, and 200 ns pulse width (Lumix 2; Fisioline, Verduno, Italy)] was used. A nickel-titanium closed coil that generated a 50-g force was applied to the maxillary left-side first molars and incisors in 7-week-old Sprague-Dawley (280-300 g) rats to induce experimental tooth movement (ETM) for 24 h. Ten rats were divided into two groups (ETM + laser, n = 5; ETM, n = 5). The right side of the ETM group (i.e., the side without induced ETM) was evaluated as the untreated group. We performed immunofluorescent histochemistry analysis to quantify the interleukin (IL)-1ß, cyclooxygenase-2 (COX2), prostaglandin E2 (PGE2), and neuropeptide [calcitonin gene-related peptide (CGRP)] expression in the compressed region of the periodontal tissue. Post-hoc Tukey-Kramer tests were used to compare the groups. Compared with the ETM group, the ETM + laser group showed significant suppression in IL-1ß (176.2 ± 12.3 vs. 310.8 ± 29.5; P < 0.01), PGE2 (104.4 ± 14.34 vs. 329.6 ± 36.52; P < 0.01), and CGRP (36.8 ± 4.88 vs. 78.0 ± 7.13; P < 0.01) expression. High-frequency NIR semiconductor laser irradiation exerts significant effects on ETM-induced inflammation. High-frequency NIR semiconductor laser irradiation can reduce periodontal inflammation during orthodontic tooth movement.

Effects of high-frequency near infrared laser irradiation on experimental tooth movement-induced pain in rats.

Discomfort and dull pain are known side effects of orthodontic treatment. Pain is expected to be reduced by near-infrared (NIR) lasers; however, the mechanism underlying effects of short-pulse NIR lasers in the oral and maxillofacial area remains unclear. This study aimed to examine the effects of high-frequency NIR diode laser irradiation on pain during experimental tooth movement (ETM) on 120 J. NIR laser with 910 nm wavelength, 45 W maximum output power, 300 mW average output power, and 200 ns pulse width (Lumix 2; (Lumix 2; Fisioline, Verduno CN, Italy) was used for the experiment. A nickel-titanium-closed coil was used to apply a 50-gf force between the maxillary left-side first molar and incisor in 7-week-old Sprague-Dawley rats (280-300 g) to induce ETM. We measured facial-grooming frequency and vacuous chewing movement (VCM) period between laser-irradiation and ETM groups. We performed immunofluorescent histochemistry analysis to quantify levels of Iba-1, astrocytes, and c-fos protein-like immunoreactivity (Fos-IR) in the trigeminal spinal nucleus caudalis (Vc). Compared with the ETM group, the laser irradiation group had significantly decreased facial-grooming frequency (P = 0.0036), VCM period (P = 0.043), Fos-IR (P = 0.0028), Iba-1 levels (P = 0.0069), and glial fibrillary acidic protein (GFAP) levels (P = 0.0071). High-frequency NIR diode laser irradiation appears to have significant analgesic effects on ETM-induced pain, which involve inhibiting neuronal activity, microglia, and astrocytes, and it inhibits c-fos, Iba-1, and GFAP expression, reducing ETM-induced pain in rats. High-frequency NIR diode laser application could be applied to reduce pain during orthodontic tooth movement.

Osteoclastogenesis from bone marrow cells during estrogen-induced medullary bone formation in Japanese quails.

Osteoclasts are differentiated from hematopoietic mononuclear cells by regulation of the receptor activator of nuclear factor kappa-B (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) system. Medullary bone (MB) that forms in the bone marrow of female birds is remodeled under the control of circulating estrogen (E2) during the laying period. Although the osteoclasts of MB are differentiated from mononuclear cells, the mechanism of osteoclastogenesis is not known. We investigated whether MB osteoclastogenesis is regulated by the RANK/RANKL/OPG system using MB from male quails induced with E2. Bone marrow cells (BMCs) differentiate into osteoclasts that have the ability of bone resorption via stimulation of RANKL/M-CSF, but this ability is suppressed by OPG and differentiation is inhibited by calcinurin inhibitors. We found that BMCs at 3 days after E2 administration had high bone osteoclastogenesis ability and colony forming unit-granulocyte/macrophage (CFU-GM)/colony forming unit-macrophage (CFU-M) formation abilities. We conclude that MB osteoclasts are differentiated from BMCs by the RANK/RANKL/OPG system, and that precursor cells of osteoclasts are increased during MB formation.

A3 adenosine receptor agonist attenuates neuropathic pain by suppressing activation of microglia and convergence of nociceptive inputs in the spinal dorsal horn.

Peripheral nerve injuries cause glial activation and neuronal hyperactivity in the spinal dorsal horn. These changes have been considered to be involved in the underlying mechanisms for the development and maintenance of neuropathic pain. Using double immunofluorescence labeling, we previously demonstrated that spinal microglial activation induced by nerve injury enhanced convergence of nociceptive inputs in the spinal dorsal horn from uninjured afferents. The adenosine A3 receptor (A3AR) agonists have been shown to have antinociceptive activities in several experimental neuropathic pain models. However, the mechanisms underlying these antinociceptive actions of the A3AR agonist are still not fully explored. In this study, the effects of the A3AR agonist (i.e., IB-MECA) on microglial activation, enhancement of convergent nociceptive inputs, and nocifensive behaviors were examined after tibial nerve injury. Injury to the tibial nerve initially caused hyposensitivity to touch stimulus at 3 days, and then resulted in tactile allodynia at 14-day post-injury. The daily systemic administration of IB-MECA (0.1 mg/kg/day) for 8 days in a row starting on the day of nerve injury or 7 days after nerve injury prevented the development of behaviorally assessed hypersensitivities, and spinal microglial activation induced by nerve injury. These treatments also suppressed anomalous convergence of nociceptive primary inputs in the spinal dorsal horn. The present findings indicate that the A3AR agonist attenuates neuropathic pain states by suppressing enhanced microglial activation, and anomalous convergence of nociceptive inputs in the spinal dorsal horn from uninjured afferents after injury to the peripheral nerve.

Differential induction of c-Fos and phosphorylated ERK by a noxious stimulus after peripheral nerve injury.

PURPOSE: In this study, we compared induction of c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) in the spinal dorsal horn after peripheral nerve injury. MATERIALS AND METHODS: We examined the spinal dorsal horn for noxious heat-induced c-Fos and p-ERK protein-like immunoreactive (c-Fos- and p-ERK-IR) neuron profiles after tibial nerve injury. The effect of administration of a MEK 1/2 inhibitor (PD98059) on noxious heat-induced c-Fos expression was also examined after tibial nerve injury. RESULTS: A large number of c-Fos- and p-ERK-IR neuron profiles were induced by noxious heat stimulation to the hindpaw in sham-operated animals. A marked reduction in the number of c-Fos- and p-ERK-IR neuron profiles was observed in the medial 1/3 (tibial territory) of the dorsal horn at 3 and 7 days after nerve injury. Although c-Fos-IR neuron profiles had reappeared by 14 days after injury, the number of p-ERK-IR neuron profiles remained decreased in the tibial territory of the superficial dorsal horn. Double immunofluorescence labeling for c-Fos and p-ERK induced by noxious heat stimulation to the hindpaw at different time points revealed that a large number of c-Fos-IR, but not p-ERK-IR, neuron profiles were distributed in the tibial territory after injury. Although administration of a MEK 1/2 inhibitor to the spinal cord suppressed noxious heat-induced c-Fos expression in the peroneal territory, this treatment did not alter c-Fos induction in the tibial territory after nerve injury. CONCLUSIONS: ERK phosphorylation may be involved in c-Fos induction in normal nociceptive responses, but not in exaggerated c-Fos induction after nerve injury.

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats.

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4-L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.

Activated microglia contribute to convergent nociceptive inputs to spinal dorsal horn neurons and the development of neuropathic pain.

The activation of microglia in the spinal dorsal horn following peripheral nerve injury has been reported previously, and this change has been proposed to contribute to the development of a neuropathic pain state. We recently demonstrated that peripheral nerve injury activated convergent nociceptive inputs to spinal dorsal horn neurons. The present study was designed to further examine the role of microglia in the activation of convergent nociceptive inputs as well as development of a neuropathic pain state after peripheral nerve injury. Tibial nerve injury initially induced hyposensitivity at 3 days post-injury, and this was followed by hypersensitivity to tactile and thermal stimuli at 14 days. The intraperitoneal administration of minocycline (30 mg/kg), an inhibitor of microglial activation, for 8 days starting on the day of surgery prevented increases in OX-42 immunofluorescence labeling in the spinal dorsal horn and the development of tactile and thermal hypersensitivity at 14 days post-injury. The same minocycline treatment (day 0-7) also reduced the nerve injury-induced convergence of nociceptive inputs to spinal dorsal horn neurons, as revealed by double immunofluorescence labeling for c-Fos induced by noxious heat stimulation of the hindpaw and phosphorylated extracellular signal-regulated kinase induced by electrical stimulation of the injured tibial nerve. However, the administration of minocycline for 8 days starting 7 days after surgery did not prevent nerve injury-induced microglial activation, convergent nociceptive inputs, or tactile and thermal hypersensitivity. These results suggest that microglial activation in the early stage following peripheral nerve injury plays an important role in the anomalous convergence of nociceptive signals to spinal dorsal horn neurons and the development of neuropathic pain.

Peripheral nerve injury activates convergent nociceptive input to dorsal horn neurons from neighboring intact nerve.

Previous studies demonstrated that peripheral nerve injury induced excessive nociceptive response of spinal cord dorsal horn neurons and such change has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the spinal dorsal horn for convergence of nociceptive input to second-order neurons deafferented by peripheral nerve injury. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input to spinal dorsal horn neurons after the saphenous nerve injury. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and by electrical stimulation of the injured or uninjured saphenous nerve, respectively. Within the central terminal field of the saphenous nerve, the number of c-Fos protein-like immunoreactive (c-Fos-IR) cell profiles was significantly decreased at 3 days and returned to the control level by 14 days after the injury. p-ERK immunoreactive (p-ERK-IR) cell profiles were distributed in the central terminal field of the saphenous nerve, and the topographic distribution pattern and number of such p-ERK-IR cell profiles remained unchanged after the nerve injury. The time course of changes in the number of double-labeled cell profiles was similar to that of c-Fos-IR cell profiles after the injury. These results indicate that convergent primary nociceptive input through neighboring intact nerves contributes to increased responsiveness of spinal dorsal horn nociceptive neurons.

Assessment of intraoral mucosal pain induced by the application of capsaicin.

OBJECTIVE: To develop an objective method for assessing nociceptive behaviour in an animal model of capsaicin-induced intraoral pain. Changes in nociceptive responses were also examined after injury to the inferior alveolar nerve (IAN). DESIGN: Nociceptive responses evoked by the intraoral application of various doses of capsaicin were analyzed in lightly anaesthetized rats. The number of c-Fos protein-like immunoreactive (Fos-LI) neurons in the medullary dorsal horn (MDH) induced by the intraoral application of capsaicin was measured. Behavioural and c-Fos responses were also examined 14 days after injury to the IAN. RESULTS: Larger doses of intraoral capsaicin (1, 10 and 100µg) induced vigorous licking behaviour and c-Fos response in the MDH in a reproducible manner. The magnitudes of both behavioural activity and the c-Fos response from the 10 and 100µg doses of capsaicin were significantly greater than that by the 1µg dose. Injury to the IAN exaggerated the behavioural and c-Fos responses evoked by intraoral capsaicin. CONCLUSIONS: The intraoral application of capsaicin is a valid and reliable method for studying intraoral pain and hyperalgesia following nerve injury.

Effect of adenosine A1 receptor agonist on the enhanced excitability of spinal dorsal horn neurons after peripheral nerve injury.

Neuronal hyperactivity has been implicated in abnormal pain sensation following peripheral nerve injuries. Previous studies have indicated that the activation of adenosine A1 receptors (A1R) in the central and peripheral nervous systems produces an antinociceptive effect. However, the mechanisms involved in the peripheral effect are still not fully understood. The effects of the local application of the selective A1R agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) on neuronal hyperactivity were examined in this study using a neuropathic pain model induced by a tibial nerve injury. We utilized Fos protein-like immunoreactivity induced by noxious heat stimulation to examine changes in the number of Fos protein like immunoreactive (Fos-LI) neuron profiles in the spinal dorsal horn, and behavioral analysis for mechanical and thermal sensitivities. The nerve injury induced an exaggerated Fos response to noxious heat stimulation. The number of Fos-LI neuron profiles was significantly decreased and their distribution was restricted to the central terminal field of the spared peroneal nerve 3 days after the injury. The number of Fos-LI neuron profiles returned to control levels and a large number of these profiles were observed in the central terminal field of the injured tibial nerve 14 days after the injury. These enhanced Fos responses were attenuated by the local application of CCPA. The nerve injury also resulted in mechanical allodynia and thermal hyperalgesia. The local application of CCPA inhibited thermal hyperalgesia, but was less effective against mechanical allodynia. These results indicated that activation of peripheral A1R plays a role in the regulation of nerve injury-induced hyperalgesia.

Fos protein-like immunoreactive neurons induced by electrical stimulation in the trigeminal sensory nuclear complex of rats with chronically injured peripheral nerve.

The rat trigeminal sensory nuclear complex (TSNC) was examined for Fos protein-like immunoreactive (Fos-LI) neurons induced by electrical stimulation (ES) of the lingual nerve (LN) at 2 weeks after injury to the LN or the inferior alveolar nerve (IAN). Intensity-dependent increase in the number of Fos-LI neurons was observed in the subnucleus oralis (Vo) and caudalis (Vc) of the spinal trigeminal tract nucleus irrespective of nerve injury. The number of Fos-LI neurons induced by ES of the chronically injured LN at A-fiber intensity (0.1 mA) was significantly increased in the Vo but not the Vc. On the other hand, in rats with chronically injured IAN, the number of Fos-LI neurons induced by ES of the LN at C-fiber intensity (10 mA) was significantly increased in the Vc but not the Vo. These results indicated that injury of a nerve innervating intraoral structures increased the c-Fos response of Vo neurons to A-fiber intensity ES of the injured nerve. A similar nerve injury enhanced the c-Fos response of Vc neurons to C-fiber intensity ES of a spared uninjured nerve innervating an intraoral territory neighboring that of the injured nerve. The present result show that nerve injury causes differential effects on c-Fos expression in the Vo and Vc, which may explain complexity of neuropathic pain symptoms in clinical cases.

Kallikrein 8 is involved in skin desquamation in cooperation with other kallikreins.

Kallikrein type serine proteases, KLK8/neuropsin, KLK6, and KLK7, have been implicated in the proliferation and differentiation of epidermal keratinocytes and in the pathogenesis of psoriasis. However, their mechanistic roles in these processes remain largely unknown. We applied 12-O-tetradecanoylphorbol-13-acetate on the wild type (WT) and the Klk8 gene-disrupted (Klk8(-/-)) mouse skin, inducing keratinocyte proliferation similar to the human psoriatic lesion. Klk8 mRNA as well as Klk6 and Klk7 mRNA were up-regulated after 12-O-tetradecanoylphorbol-13-acetate application in the WT mice. In contrast, Klk8(-/-) mice showed minimum increases of Klk6 and Klk7 transcripts, the proteins, and enzymatic activities. Relative to the WT, the Klk8(-/-) skin showed less proliferation and an increase in the number of cell layers in the stratum corneum. However, overexpression of Klk8 by adenovirus vector in knock-out keratinocytes did not result in an increase in Klk6 or Klk7 mRNA. The inefficient cleavage of adhesion molecules DSG1 and CDSN in Klk8(-/-) skin contributes to a delay in corneocyte shedding, resulting in the hyperkeratosis phenotype. We propose that in psoriatic lesion, KLK8 modulates hyperproliferation and prevents excessive hyperkeratosis by shedding the corneocytes.

Implications of protease M/neurosin in myelination during experimental demyelination and remyelination.

Protease M/neurosin is a serine protease expressed by oligodendrocytes (OLGs) in the central nervous system (CNS). To investigate the role of protease M/neurosin during experimental demyelination and remyelination, mice were fed cuprizone (bis-cyclohexanon oxaldihydrazone). Semi-quantitative RT-PCR analysis and immunohistochemistry revealed that the expressions of protease M/neurosin mRNA and protein were rapidly reduced in demyelination, whereas the expression of protease M/neurosin was increased in pi form of glutathione-S-transferases (GST-pi)-positive OLGs during remyelination. Cultured primary OLGs displayed a strong correlation between protease M/neurosin and myelin basic protein (MBP). After tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma stimulation, these proteins showed colocalization in the oligodendroglial process. The suppression of protease M/neurosin using RNAi reduced the level of MBP mRNA in cultured OLGs. In contrast, the reduced level of protease M/neurosin was not associated with oligodendroglial cell death or differentiation in cultured OLGs. This study identifies that protease M/neurosin in OLGs is closely associated with the expression of the MBP and the PLP gene. Our data emphasize that the maintenance of myelination is an important function of protease M/neurosin in OLGs, suggesting its relation to the oligodendroglial response to myelin disorders.

Involvement of neuropsin in the pathogenesis of experimental autoimmune encephalomyelitis.

Inflammation, demyelination, and axonal damage of the central nervous system (CNS) are major pathological features of multiple sclerosis (MS). Proteolytic digestion of the blood-brain barrier and myelin protein by serine proteases is known to contribute to the development and progression of MS. Neuropsin, a serine protease, has a role in neuronal plasticity, and its expression has been shown to be upregulated in response to injury to the CNS. To determine the possible involvement of neuropsin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model for MS. Neuropsin mRNA expression was induced in the spinal cord white matter of mice with EAE. Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing neuropsin mRNA showed immunoreactivity for CNPase, a cell-specific marker for oligodendrocytes. Mice lacking neuropsin (neuropsin-/-) exhibited an altered EAE progression characterized by delayed onset and progression of clinical symptoms as compared to wild-type mice. Neuropsin-/- mice also showed attenuated demyelination and delayed oligodendroglial death early during the course of EAE. These observations suggest that neuropsin is involved in the pathogenesis of EAE mediated by demyelination and oligodendroglial death.

Differential expression of protease M/neurosin in oligodendrocytes and their progenitors in an animal model of multiple sclerosis.

To determine the possible involvement of protease M/neurosin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model of multiple sclerosis (MS). In situ hybridization, immunohistochemistry and quantitative real-time polymerase chain reaction (PCR) demonstrated that EAE caused an increase in the expression of protease M/neurosin mRNA and its protein product throughout the white and gray matter surrounding demyelinating lesions. Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing protease M/neurosin mRNA within control spinal cord showed immunoreactivity for CNPase or NG2, cell-specific markers for oligodendrocytes and their progenitors, respectively. In the spinal cord from mice with EAE, the expression of protease M/neurosin mRNA in CNPase-positive cells appeared to be increased while double-labeled cells positive for protease M/neurosin mRNA and NG2 were rarely found in areas associated with demyelinating lesions. Although a prominent accumulation of inflammatory cells including T-cells was observed in the vicinity of demyelinated lesions, these cells were not associated with protease M/neurosin mRNA expression. The levels of protease M/neurosin mRNA expression were unchanged in the spleen and even decreased in the thymus during the course of EAE. These observations suggest that the differential expression of protease M/neurosin in mature oligodendrocytes and their progenitors is involved in the pathogenesis of MS and EAE.

Differential rostral projections of caudal brainstem neurons receiving trigeminal input after masseter inflammation.

To understand the functional significance of orofacial injury-induced neuronal activation, this study examined the rostral projection of caudal brainstem neurons that were activated by masseteric inflammation. Rats were injected with a retrograde tracer, Fluorogold, into the nucleus submedius of the thalamus (Sm), parabrachial nucleus (PB), lateral hypothalamus (LH), or medial ventroposterior thalamic nucleus (VPM) 7 days before injection of an inflammatory agent, complete Freund's adjuvant (CFA), into the masseter muscle. Rats were perfused at 2 hours after inflammation, and brainstem tissues were processed for Fos-Fluorogold double immunocytochemistry. Although there was no difference in Fos expression among the four groups (n=4 per site), the rostral projection of Fos-positive neurons showed dramatic differences. In the ventral portion of the trigeminal subnuclei interpolaris/caudalis (Vi/Vc) transition zone, the percentage of Fos-positive neurons projecting to the Sm (39.7%) was significantly higher than that projecting to the LH (5.4%) or VPM (5.6%; P<.001). The anesthesia alone also induced Fos expression in ventral Vi/Vc neurons, but these neurons did not project to Sm. In the caudal laminated Vc and dorsal Vi/Vc, the PB was the major site of rostral projection of Fos-positive neurons. In the caudal ventrolateral medulla and nucleus tractus solitarius, Fos-positive neurons projected to the Sm, PB, and LH. Most VPM-projecting neurons examined did not show Fos-like immunoreactivity after masseter inflammation. These findings emphasize the importance of the trigeminal Vi/Vc transition zone in response to orofacial deep tissue injury. Furthermore, the results differentiate the ventral and dorsal portions of the Vi/Vc transition zone, in that the Sm received projection mainly from activated neurons in the ventral Vi/Vc. The activation of Vi/Vc neurons and associated ascending pathways may facilitate somatoautonomic and somatovisceral integration and descending pain modulation after orofacial deep tissue injury.

Differential effects of NMDA and AMPA/KA receptor antagonists on c-Fos or Zif/268 expression in the rat spinal dorsal horn induced by noxious thermal or mechanical stimulation, or formalin injection.

The involvement of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate (KA) receptors in the induction of c-Fos and Zif/268 expression in spinal dorsal horn neurons following noxious thermal or mechanical stimulation, or formalin injection into the rat hind paw was examined by intrathecal administration of a competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV) or an AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or both, 30 min prior to noxious stimulation. APV caused a significant reduction in the level of c-Fos expression in the superficial layer induced by each of these three noxious stimuli. The effects of APV on Zif/268 expression or of CNQX on c-Fos or Zif/268 expression in the superficial layer induced by these three noxious stimuli were dependent on the type of stimulus applied to the rat hind paw. The noxious thermal stimulus-evoked c-Fos expression level was reduced by APV and/or CNQX, while Zif/268 expression was hardly changed. Both c-Fos and Zif/268 expressions following formalin injection were reduced by APV alone and APV+CNQX, but not by CNQX alone. Zif/268 expression following noxious mechanical stimulation was significantly reduced only by APV+CNQX although APV or CNQX alone did not affect the expression, while c-Fos expression was reduced by APV and APV+CNQX but not by CNQX alone. These findings suggest that NMDA and AMPA/KA receptors are differentially involved in c-Fos and Zif/268 expression in the spinal dorsal horn following noxious thermal, formalin and mechanical stimulation.

Effect of ionotropic glutamate receptor antagonists on Fos-like immunoreactivity in the dorsal horn following transection of the rat sciatic nerve.

Fos-like immunoreactivity (FLI) was investigated in the lumbar dorsal horn 2 h after transection of the rat sciatic nerve and sham operation. FLI following nerve transection was distributed through the medio-lateral extension of the superficial layer of the dorsal horn, while FLI after sham operation, tissue injury, was restricted to the lateral one-third of this layer. The number of FLI neurons in the lateral one-third was similar in the two operations, indicating that neurons expressing FLI in the medial two-thirds and in the lateral one-third of the superficial layer after nerve transection are derived from nerve injury and tissue injury, respectively. FLI in the lateral one-third, but not the medial two-thirds, after nerve transection was significantly reduced by pretreatment with NMDA and AMPA/KA receptor antagonists, indicating that there is a considerable difference in the contributions of ionotropic glutamate receptors to FLI in this layer induced by nerve injury and tissue injury.