An electromyographic (EMG) study was carried out in 51 anesthetized rats to assess if neurokinin,... more An electromyographic (EMG) study was carried out in 51 anesthetized rats to assess if neurokinin, NK-1 and NK-2, receptor mechanisms and tachykinins were involved in the increased jaw muscle activity which can be reflexly evoked by injection of the small-fiber excitant and inflammatory irritant mustard oil (MO) into the temporomandibular joint (TMJ) region. A baseline level of EMG activity was recorded bilaterally for 20 min from digastric (DIG) and masseter (MASS) muscles and then each animal was treated with NK-1 or NK-2 antagonist or vehicle. In one series of experiments either the NK-1 antagonist CP-99,994 (20 microg approximately 54 nmol), the NK-2 antagonist MEN-10,376 (10 microg approximately 9 nmol or 20 microg approximately 18 nmol) or vehicle (control) was administrated into the lateral ventricle (i.c.v.); in another series the NK-1 antagonist (4 mg/kg approximately 3-4 micromol/rat) or vehicle (control) was given intravenously (i.v.). After 10 min, MO (20 microl, 20%) was...
Currently available methods for studying the morphology of physiologically characterized primary ... more Currently available methods for studying the morphology of physiologically characterized primary afferents are limited by difficulties inherent in impaling thin fibers and by the limited distances over which conventional tracers move during the course of a recording session. We have encountered an alternative method that overcomes these limitations. Neurobiotin (NB; Vector) injections into rat trigeminal (V) primary afferents in the brain stem or V ganglion provided rapid, long-range staining with recording and electrophoretic parameters that are commonly used to eject horseradish peroxidase (HRP) or Phaseolus vulgaris leucoagglutinin (PHA-L). When NB was injected into brain stem fibers responsive to vibrissal deflection with A-beta conduction velocities, collaterals were darkly stained in each of the 4 V subnuclei, as well as the cervical dorsal horn. Labeled fibers were also seen in the V root and peripherally in the infra-orbital nerve for a distance up to 15 mm from the injectio...
The effect of food hardness during mastication on nociceptive transmission in the spinal cord was... more The effect of food hardness during mastication on nociceptive transmission in the spinal cord was studied by analyzing complete Freund's adjuvant (CFA) induced nocifensive behavior and Fos expression. The behavioral study showed that the shortening of the withdrawal latency following CFA injection into the hind paw was depressed after a change in the given food hardness from soft to hard. The depression of nocifensive behavior in the rats with hard food was reversed after i.v. injection of naloxone. Fos protein-like immunoreactive cells (Fos protein-LI cells) were expressed in the superficial and deep laminae of the L4 -6 spinal dorsal horn after s.c. injection of CFA into the hind paw during soft food mastication. The number of Fos protein-LI cells was decreased in the rats with hard food mastication followed by soft food. This reduction of Fos protein-LI cells following change in food hardness was reversed after i.v. application of naloxone. Furthermore, the depression of Fos protein-LI cells following hard food intake was significantly inhibited after bilateral inferior alveolar nerve transection or bilateral ablation of the somatosensory cortex. These findings suggest that the change in food hardness during mastication might drive an opioid descending system through the trigeminal sensory pathway and somatosensory cortex resulting in an antinociceptive effect on chronic pain. However, IAN transection and cortical ablation did not induce 100% reversal of Fos expression, suggesting other than trigeminal sensory system may be involved in this phenomena, such as the pathway through the brainstem reticular formation.
We have previously shown that injection of the excitatory amino glutamate into the rat temporoman... more We have previously shown that injection of the excitatory amino glutamate into the rat temporomandibular joint (TMJ) evokes reflex activity in both anterior digastric (DIG) and masseter (MASS) muscles that can be attenuated by prior TMJ injection of a N-methyl-Daspartate (NMDA) receptor antagonist. The aim of the present study was to test if jaw muscle activity could also be evoked by P2X receptor agonist injection into the rat TMJ region and if the reflex activity could be modulated by TMJ injection of P2X receptor antagonist or NMDA receptor antagonist. The selective P2X subtype agonist α,β-methylene adenosine 5′-triphosphate (α,β-me ATP) and vehicle (phosphate-buffered saline) or the selective P2X antagonist, 2′-(or-3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) or selective NMDA antagonist (±)-D-2-amino-5-phosphonovalerate(APV) were injected into the rat TMJ region. Electromyographic (EMG) reflex activity was recorded in both DIG and MASS muscles. Compared with the baseline EMG activity, α,β-me-ATP injection into the TMJ (but not its systemic administration) following pre-injection of the vehicle significantly increased the magnitude and the duration of ipsilateral DIG and MASS EMG activity in a dose-dependent manner. The α,β-me-ATP-evoked responses could be antagonized by pre-injection of TNP-ATP into the same TMJ site but contralateral TMJ injection of TNP-ATP proved ineffective. Furthermore, the α,β-me-ATP-evoked responses could also be antagonized by APV injected into the same TMJ site but not by its systemic injection. These results indicate the interaction of peripheral purinergic as well as glutamatergic receptor mechanisms in the processing of TMJ nociceptive afferent inputs that evoke reflex activity in jaw muscles.
We have examined the effect of the peripheral application of glutamate and capsaicin to deep cran... more We have examined the effect of the peripheral application of glutamate and capsaicin to deep craniofacial tissues in influencing the activation and peripheral sensitization of deep craniofacial nociceptive afferents. The activity of single trigeminal nociceptive afferents with receptive fields in deep craniofacial tissues were recorded extracellularly in 55 halothane-anesthetized rats. The mechanical activation threshold (MAT) of each afferent was assessed before and after injection of 0.5M glutamate (or vehicle) and 1% capsaicin (or vehicle) into the receptive field. A total of 68 afferents that could be activated by blunt noxious mechanical stimulation of the deep craniofacial tissues (23 masseter, 5 temporalis, 40 temporomandibular joint) were studied. When injected alone, glutamate and capsaicin activated and induced peripheral sensitization reflected as MAT reduction in many afferents. Following glutamate injection, capsaicin-evoked activity was greater than that evoked by capsaicin alone, whereas following capsaicin injection, glutamate-evoked responses were similar to glutamate alone. These findings indicate that peripheral application of glutamate or capsaicin may activate or induce peripheral sensitization in a subpopulation of trigeminal nociceptive afferents innervating deep craniofacial tissues, as reflected in changes in MAT and other afferent response properties. The data further suggest that peripheral glutamate and capsaicin receptor mechanisms may interact to modulate the activation and peripheral sensitization in some deep craniofacial nociceptive afferents.
receptors have been suggested to be expressed on the central terminals of A␦-afferent fibers inne... more receptors have been suggested to be expressed on the central terminals of A␦-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X 2ϩ3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X 1 , P2X 3 homomers, and P2X 2ϩ3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined ␣,-methylene-ATP (␣meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. ␣meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 M TNP-ATP (n ϭ 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 M TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 M ␣meATP could be largely inhibited by 10 M iso-pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A␦-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.
Growing evidence suggests that astroglia are involved in pain states, but no studies have tested ... more Growing evidence suggests that astroglia are involved in pain states, but no studies have tested their possible involvement in modulating the activity of nociceptive neurons per se. This study has demonstrated that the central sensitization induced in functionally identified nociceptive neurons in trigeminal subnucleus caudalis (the medullary dorsal horn) by application of an inflammatory irritant to the rat's tooth pulp can be significantly attenuated by continuous intrathecal superfusion of methionine sulfoximine (MSO; 0.1 mm), an inhibitor of the astroglial enzyme glutamine synthetase that is involved in the glutamate–glutamine shuttle. Simultaneous superfusion of MSO and glutamine (0.25 mm) restored the irritant-induced central sensitization. In control experiments, superfusion of either MSO or glutamine alone, or vehicle, did not produce any significant changes in neuronal properties. These findings suggest that the astroglial glutamate–glutamine shuttle is essential for th...
The aim of this study was to determine if lingual nerve trauma affects the features of face prima... more The aim of this study was to determine if lingual nerve trauma affects the features of face primary motor cortex (MI) defined by intracortical microstimulation (ICMS). The left lingual nerve was transected in adult male rats by an oral surgical procedure; sham rats (oral surgery but no nerve transection) as well as naive intact rats served as control groups. ICMS was applied at post-operative days 0, 7, 14, 21, and 28 to map the jaw and tongue motor representations in face MI by analyzing ICMS-evoked movements and electromyographic activity recorded in the genioglossus (GG) and anterior digastric (AD) muscles. There were no statistically significant effects of acute (day 0) nerve transection or sham procedure (p40.05). The surgery in the sham animals was associated with limited post-operative change; this was reflected in a significant (p50.05) increase in the number of GG sites in left MI at post-operative day 14 compared to day 0. However, nerve transection was associated with significant increases in the total number of AD and GG sites in left or right MI or specifically the number of GG sites in rats at post-operative days 21 or 28 compared to earlier time periods. There were also significant differences between nerve-transected and sham groups at post-operative days 7, 14, or 21. These findings suggest that lingual nerve transection is associated with significant time-dependent neuroplastic changes in the tongue motor representations in face MI.
The aim of this study was to examine the possible role of N-methyl-D-aspartate (NMDA)receptor mec... more The aim of this study was to examine the possible role of N-methyl-D-aspartate (NMDA)receptor mechanismsin responsesinducedby the small-fibreexcitantand inflammatoryirritantmustardoil injectedinto the temporomandibular joint (TMJ)region of rats. The effectsof the non-competitiveNMDAantagonistMK-801weretested on the mustardoil-evoked increases in electromyog- rapbic (EMG) activity of the masseter and digastric muscles and Evans Blue plasma extravasation. Five minutes before the mustard oil injection, MK-801 or its vehicle was administered systemically (iv.), into the third ventricle (i.c.v.), or locally into the TMJ region. Compared with control animals receiving vehicle, the rats receiving MK-801 at an iv. dose of 0.5 mgikg (n= 5) showed a significant reduction in the incidence and magnitude of EMG responsesas wellas in the plasmaextravasationevoked by mustard oil; MK-801 at an iv. dose of 0.1 mg/kg (n= 5) had no significant effect on plasma extravasation or on the incidence and magnitude of EMG responsesbut did significantly increase the latency of EMG responses. An i.c.v. dose of 0.1 mglkg (n = 5) or 0.01 mgllcg(n = 5) had no significant effect on plasma extravasation or incidence of EMG responses but did significantly reduce the magnitudes of the masseter EMG response; the 0.01 mgkg dose also significantly increased the latency of the digastric EMG response. The magnitudes of both the masseter and digastric EMG responses were also significantly reduced by MK-801 administered into the TMJ region at a dose of 0.1 mgkg (n= 5) but not by 0.01 mg/kg (n= 5); neither dose significantly affected the incidence of EMG responses or the plasma extravasation. These data suggest that both central and peripheral NMDA receptor mechanisms may play an important role in EMG responses evoked by the small-fibre excitant and inflammatory irritant mustard oil, but that different neurochemical mechanisms may be involved in the plasma extravasation induced by mustard oil.
The aim of this study was to determine if the application of mustard oil (MO), a small-fiber exci... more The aim of this study was to determine if the application of mustard oil (MO), a small-fiber excitant and inflammatory irritant, or other algesic chemicals (capsaicin, CAP, and bradykinin, BK) to the rat maxillary molar tooth pulp induces electromyographic (EMG) activity of the masseter and digastric muscles, and also to determine if endogenous opioid mechanisms may be involved in any documented EMG changes. Application of MO to the tooth pulp induced a significant increase in EMG activity of the ipsilateral masseter up to 30 min. The application of mineral oil to the pulp or MO application to the pulp-extirpated tooth did not induce any significant EMG increases. The application of CAP or BK to the pulp in contrast had much weaker effects on EMG activity of the jaw muscles. CAP produced a small but prolonged increase in masseter EMG activity, and BK induced a short-lasting increase in digastric EMG activity. The systemic administration of the opiate antagonist naloxone significantly reactivated (i.e. rekindled) the EMG response evoked by MO application to the pulp. Naloxone did not produce any such significant rekindling effect on EMG activity following CAP, BK or mineral oil application to the pulp or following MO application to the pulp-extirpated tooth. The MO, BK and especially CAP groups showed histological evidence of vasodilatation and polymorphonuclear leukocyte infiltration in the pulp tissue and a significant increase in plasma extravasation of Evans Blue dye, whereas mineral oil did not induce these changes. These findings suggest that pulp afferent inputs to the central nervous system evoked by BK, CAP and especially MO may induce enhanced jaw muscle activity. In addition, the naloxone data suggest that an opioid suppressive mechanism may be induced by the pulpal afferent inputs evoked by MO, and may serve to limit the jaw muscle activity elicited by these inputs.
Modulatory effects of the new antimigraine drug naratriptan, a 5-HT 1 -receptor-agonist, on neuro... more Modulatory effects of the new antimigraine drug naratriptan, a 5-HT 1 -receptor-agonist, on neurons of the nucleus raphe  magnus were examined in rat by extracellular recordings. In the nucleus raphe  magnus neuronal activity decreased in on-cells and increased in off-cells after intravenous administration of naratriptan. The modulatory effects of naratriptan were similar to the well-known effects of morphine on neurons in the nucleus raphe  magnus. The results of this study suggest central actions of naratriptan and may point to an involvement of the endogenous pain control system in the antinociceptive effects of the 5-HT 1 -receptor-agonist.
The aim of this study was to determine whether trigeminal subnucleus caudalis (Vc) neurons show d... more The aim of this study was to determine whether trigeminal subnucleus caudalis (Vc) neurons show differential inhibitory effects produced by parabrachial area (PBA) or nucleus raphe magnus (NRM) conditioning stimulation (CS) on their A-fiber versus C-fiber inputs electrically evoked from cutaneous and deep tissues. A total of 55 Vc neurons in urethane/chloralose-anesthetized rats were functionally classified as low-threshold mechanoreceptive (LTM; n = 12), nociceptive (wide-dynamic-range, WDR, and nociceptive-specific, NS; n = 111, deep (D; n = 6), or nociceptive convergent neurons receiving skin and deep inputs (S + D; n = 26). Most neuronal responses were tested for both PBA and NRM CS (20-40 PA) delivered 50 msec prior to test stimulation (1.1-1.5 x threshold) of their mechanoreceptive field (RF) or the hypoglossal nerve (X11). Inhibition of responses to both cutaneous and deep A-fiber and C-fiber inputs occurred in over two-thirds of the Vc neurons tested in each neuronal class, and facilitation of responses was only occasionally found. In general, both PBA (ipsi-and contralateral) and NRM CS produced profound inhibition of electrically evoked responses in al1 neuron types, e.g., responses reduced to mean values of 50-53% of control in LTM neurons, 35-46% in WDR, NS, and S + D neurons, and 61-63% in D neurons. NO significant differente in the magnitude of their inhibitory effects was found between PBA and NRM, and between Vc neuronal responses to A-and C-fiber inputs irrespective of their origin from skin or deep tissues. However, in a group of 6 S + D Vc neurons, responses elicited by deep inputs were more powerfully inhibited by PBA stimulation than those elicited by cutaneous inputs, although the magnitude of NRM-induced inhibition of the responses to deep and cutaneous inputs were comparable. These data confirm earlier findings that Vc neuronal responses to cutaneous A-and C-fiber inputs are subject to PBA-and NRM-induced descending modulation, but further reveal that responses to deep A-and C-fiber inputs are also subject to descending modulation from PBA and NRM.
It has been shown that glial cell line-derived neurotrophic factor (GDNF) exhibits analgesic effe... more It has been shown that glial cell line-derived neurotrophic factor (GDNF) exhibits analgesic effects on the neuropathic pain, although the site of action of GDNF is largely unknown. We, therefore, examined the effect of site-directed overexpression of GDNF on neuropathic pain by means of a lentiviral vector system expressing GDNF. Mice were treated with spinal nerve ligation (SNL) at the fifth lumbar (L5) spinal nerve and the lentiviral vector was locally injected on the same day. In SNL mice, the GDNF-expressing virus injection to the dorsal spinal cord or uninjured L4 dorsal root ganglion (DRG) significantly reduced mechanical allodynia, whereas the injection to the injured L5 DRG or plantar skin showed no analgesic or hyperalgesic effects. These results suggest that GDNF exerts the analgesic effect on neuropathic pain by acting on the uninjured DRG neurons and/or their innervated spinal cells.
In the present study, the hypothesis that sex-related differences in glutamate-evoked rat massete... more In the present study, the hypothesis that sex-related differences in glutamate-evoked rat masseter muscle afferent discharge may result from estrogen-related modulation of peripheral NMDA receptor activity and/or expression was tested by examining afferent fiber discharge in response to masseter injection of NMDA and the expression of NR2A/B subunits by masseter ganglion neurons in male and female rats. The results showed that injection of NMDA into the masseter muscle evoked discharges in putative mechanonociceptive afferent fibers and increased blood pressure that was concentration-dependent, however, a systemic action of NMDA appeared responsible for increased blood pressure. NMDA-evoked afferent discharge was significantly greater in female than in male rats, was positively correlated with plasma estrogen levels in females and was significantly greater in ovariectomized female rats treated with a high dose (5 μg/day) compared to a low dose (0.5 μg/ day) of estrogen. Pre-treatment of high dose estrogen-treated-ovariectomized female rats with the Src tyrosine kinase inhibitor PP2 did not affect NMDA-evoked afferent discharge. NMDA-evoked afferent discharge was attenuated by the antagonists ketamine and ifenprodil, which is selective for NR2B containing NMDA receptors. Fewer masseter ganglion neurons expressed the NR2A (16%) subunit as compared with the NR2B subunit (38%), which was expressed at higher frequencies in intact female (46%) and high dose estrogen-treated ovariectomized female (60%) rats than in male (31%) rats. Taken together, these results suggest that sex-related differences in NMDA-evoked masseter afferent discharge are due, at least in part, to an estrogen-mediated increase in expression of peripheral NMDA receptors by masseter ganglion neurons in female rats.
Central sensitization is a crucial process underlying the increased neuronal excitability of noci... more Central sensitization is a crucial process underlying the increased neuronal excitability of nociceptive pathways following peripheral tissue injury and inflammation. Our previous findings have suggested that extracellular ATP molecules acting at purinergic receptors located on presynaptic terminals (e.g., P2X2/3, P2X3 subunits) and glial cells are involved in the glutamatergic-dependent central sensitization induced in medullary dorsal horn (MDH) nociceptive neurons by application to the tooth pulp of the inflammatory irritant mustard oil (MO). Since growing evidence indicates that activation of P2X7 receptors located on glia is involved in chronic inflammatory and neuropathic pain, the aim of the present study was to test in vivo for P2X7 receptor involvement in this acute inflammatory pain model. Experiments were carried out in anesthetized Sprague-Dawley male rats. Single unit recordings were made in MDH functionally identified nociceptive neurons for which mechanoreceptive field, mechanical activation threshold and responses to noxious stimuli were tested. We found that continuous intrathecal (i.t.) superfusion over MDH of the potent P2X7 receptor antagonists brilliant blue G and periodated oxidized ATP could each significantly attenuate the MO-induced MDH central sensitization. MDH central sensitization could also be produced by i.t. superfusion of ATP and even more effectively by the P2X7 receptor agonist benzoylbenzoyl ATP. Superfusion of the microglial blocker minocycline abolished the MO-induced MDH central sensitization, consistent with reports that dorsal horn P2X7 receptors are mostly expressed on microglia. In control experiments, superfusion over MDH of vehicle did not produce any significant changes. These novel findings suggest that activation of P2X7 receptors in vivo may be involved in the development of central sensitization in an acute inflammatory pain model.
Mustard oil application to tooth pulp produces central sensitization in rat medullary dorsal horn... more Mustard oil application to tooth pulp produces central sensitization in rat medullary dorsal horn (MDH) nociceptive neurons, which has been implicated in persistent pain mechanisms. We found that superfusion onto MDH of methylaminoisobutyric acid, a competitive inhibitor of the neuronal system A transporter for presynaptic uptake of glutamine (a glutamate precursor released from astroglia), significantly depressed development of mustard oil-induced central sensitization in rat MDH nociceptive neurons. This finding indicates that the system A transporter is required for the expression of central sensitization and confirms the important roles of astroglia, glutamine and presynaptic modulation of glutamate release in the development of central sensitization.
Background: To evaluate whether P2X receptors are involved in responses to noxious pulp stimulati... more Background: To evaluate whether P2X receptors are involved in responses to noxious pulp stimulation, the P2X3 and P2X2/3 receptor agonist α,β-methyleneATP (α,β-meATP) was applied to the molar tooth pulp and nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation in trigeminal spinal subnucleus caudalis (Vc), trigeminal spinal subnucleus interpolaris (Vi), upper cervical spinal cord (C1/C2) and paratrigeminal nucleus (Pa5) neurons were analyzed in rats. Results: Genioglossus (GG) muscle activity was evoked by pulpal application of 100 mM α,β-meATP and was significantly larger than GG activity following vehicle (phosphate-buffered saline PBS) application (p < 0.01). The enhanced GG muscle activity following 100 mM α,β-meATP was significantly reduced (p < 0.05) by co-application of 1 mM TNP-ATP (P2X1, P2X3 and, P2X2/3 antagonist). A large number of pERK-LI cells were expressed in the Vc, Vi/Vc, C1/C2 and Pa5 at 5 min following pulpal application of 10...
An electromyographic (EMG) study was carried out in 51 anesthetized rats to assess if neurokinin,... more An electromyographic (EMG) study was carried out in 51 anesthetized rats to assess if neurokinin, NK-1 and NK-2, receptor mechanisms and tachykinins were involved in the increased jaw muscle activity which can be reflexly evoked by injection of the small-fiber excitant and inflammatory irritant mustard oil (MO) into the temporomandibular joint (TMJ) region. A baseline level of EMG activity was recorded bilaterally for 20 min from digastric (DIG) and masseter (MASS) muscles and then each animal was treated with NK-1 or NK-2 antagonist or vehicle. In one series of experiments either the NK-1 antagonist CP-99,994 (20 microg approximately 54 nmol), the NK-2 antagonist MEN-10,376 (10 microg approximately 9 nmol or 20 microg approximately 18 nmol) or vehicle (control) was administrated into the lateral ventricle (i.c.v.); in another series the NK-1 antagonist (4 mg/kg approximately 3-4 micromol/rat) or vehicle (control) was given intravenously (i.v.). After 10 min, MO (20 microl, 20%) was...
Currently available methods for studying the morphology of physiologically characterized primary ... more Currently available methods for studying the morphology of physiologically characterized primary afferents are limited by difficulties inherent in impaling thin fibers and by the limited distances over which conventional tracers move during the course of a recording session. We have encountered an alternative method that overcomes these limitations. Neurobiotin (NB; Vector) injections into rat trigeminal (V) primary afferents in the brain stem or V ganglion provided rapid, long-range staining with recording and electrophoretic parameters that are commonly used to eject horseradish peroxidase (HRP) or Phaseolus vulgaris leucoagglutinin (PHA-L). When NB was injected into brain stem fibers responsive to vibrissal deflection with A-beta conduction velocities, collaterals were darkly stained in each of the 4 V subnuclei, as well as the cervical dorsal horn. Labeled fibers were also seen in the V root and peripherally in the infra-orbital nerve for a distance up to 15 mm from the injectio...
The effect of food hardness during mastication on nociceptive transmission in the spinal cord was... more The effect of food hardness during mastication on nociceptive transmission in the spinal cord was studied by analyzing complete Freund's adjuvant (CFA) induced nocifensive behavior and Fos expression. The behavioral study showed that the shortening of the withdrawal latency following CFA injection into the hind paw was depressed after a change in the given food hardness from soft to hard. The depression of nocifensive behavior in the rats with hard food was reversed after i.v. injection of naloxone. Fos protein-like immunoreactive cells (Fos protein-LI cells) were expressed in the superficial and deep laminae of the L4 -6 spinal dorsal horn after s.c. injection of CFA into the hind paw during soft food mastication. The number of Fos protein-LI cells was decreased in the rats with hard food mastication followed by soft food. This reduction of Fos protein-LI cells following change in food hardness was reversed after i.v. application of naloxone. Furthermore, the depression of Fos protein-LI cells following hard food intake was significantly inhibited after bilateral inferior alveolar nerve transection or bilateral ablation of the somatosensory cortex. These findings suggest that the change in food hardness during mastication might drive an opioid descending system through the trigeminal sensory pathway and somatosensory cortex resulting in an antinociceptive effect on chronic pain. However, IAN transection and cortical ablation did not induce 100% reversal of Fos expression, suggesting other than trigeminal sensory system may be involved in this phenomena, such as the pathway through the brainstem reticular formation.
We have previously shown that injection of the excitatory amino glutamate into the rat temporoman... more We have previously shown that injection of the excitatory amino glutamate into the rat temporomandibular joint (TMJ) evokes reflex activity in both anterior digastric (DIG) and masseter (MASS) muscles that can be attenuated by prior TMJ injection of a N-methyl-Daspartate (NMDA) receptor antagonist. The aim of the present study was to test if jaw muscle activity could also be evoked by P2X receptor agonist injection into the rat TMJ region and if the reflex activity could be modulated by TMJ injection of P2X receptor antagonist or NMDA receptor antagonist. The selective P2X subtype agonist α,β-methylene adenosine 5′-triphosphate (α,β-me ATP) and vehicle (phosphate-buffered saline) or the selective P2X antagonist, 2′-(or-3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) or selective NMDA antagonist (±)-D-2-amino-5-phosphonovalerate(APV) were injected into the rat TMJ region. Electromyographic (EMG) reflex activity was recorded in both DIG and MASS muscles. Compared with the baseline EMG activity, α,β-me-ATP injection into the TMJ (but not its systemic administration) following pre-injection of the vehicle significantly increased the magnitude and the duration of ipsilateral DIG and MASS EMG activity in a dose-dependent manner. The α,β-me-ATP-evoked responses could be antagonized by pre-injection of TNP-ATP into the same TMJ site but contralateral TMJ injection of TNP-ATP proved ineffective. Furthermore, the α,β-me-ATP-evoked responses could also be antagonized by APV injected into the same TMJ site but not by its systemic injection. These results indicate the interaction of peripheral purinergic as well as glutamatergic receptor mechanisms in the processing of TMJ nociceptive afferent inputs that evoke reflex activity in jaw muscles.
We have examined the effect of the peripheral application of glutamate and capsaicin to deep cran... more We have examined the effect of the peripheral application of glutamate and capsaicin to deep craniofacial tissues in influencing the activation and peripheral sensitization of deep craniofacial nociceptive afferents. The activity of single trigeminal nociceptive afferents with receptive fields in deep craniofacial tissues were recorded extracellularly in 55 halothane-anesthetized rats. The mechanical activation threshold (MAT) of each afferent was assessed before and after injection of 0.5M glutamate (or vehicle) and 1% capsaicin (or vehicle) into the receptive field. A total of 68 afferents that could be activated by blunt noxious mechanical stimulation of the deep craniofacial tissues (23 masseter, 5 temporalis, 40 temporomandibular joint) were studied. When injected alone, glutamate and capsaicin activated and induced peripheral sensitization reflected as MAT reduction in many afferents. Following glutamate injection, capsaicin-evoked activity was greater than that evoked by capsaicin alone, whereas following capsaicin injection, glutamate-evoked responses were similar to glutamate alone. These findings indicate that peripheral application of glutamate or capsaicin may activate or induce peripheral sensitization in a subpopulation of trigeminal nociceptive afferents innervating deep craniofacial tissues, as reflected in changes in MAT and other afferent response properties. The data further suggest that peripheral glutamate and capsaicin receptor mechanisms may interact to modulate the activation and peripheral sensitization in some deep craniofacial nociceptive afferents.
receptors have been suggested to be expressed on the central terminals of A␦-afferent fibers inne... more receptors have been suggested to be expressed on the central terminals of A␦-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X 2ϩ3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X 1 , P2X 3 homomers, and P2X 2ϩ3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined ␣,-methylene-ATP (␣meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. ␣meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 M TNP-ATP (n ϭ 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 M TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 M ␣meATP could be largely inhibited by 10 M iso-pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A␦-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.
Growing evidence suggests that astroglia are involved in pain states, but no studies have tested ... more Growing evidence suggests that astroglia are involved in pain states, but no studies have tested their possible involvement in modulating the activity of nociceptive neurons per se. This study has demonstrated that the central sensitization induced in functionally identified nociceptive neurons in trigeminal subnucleus caudalis (the medullary dorsal horn) by application of an inflammatory irritant to the rat's tooth pulp can be significantly attenuated by continuous intrathecal superfusion of methionine sulfoximine (MSO; 0.1 mm), an inhibitor of the astroglial enzyme glutamine synthetase that is involved in the glutamate–glutamine shuttle. Simultaneous superfusion of MSO and glutamine (0.25 mm) restored the irritant-induced central sensitization. In control experiments, superfusion of either MSO or glutamine alone, or vehicle, did not produce any significant changes in neuronal properties. These findings suggest that the astroglial glutamate–glutamine shuttle is essential for th...
The aim of this study was to determine if lingual nerve trauma affects the features of face prima... more The aim of this study was to determine if lingual nerve trauma affects the features of face primary motor cortex (MI) defined by intracortical microstimulation (ICMS). The left lingual nerve was transected in adult male rats by an oral surgical procedure; sham rats (oral surgery but no nerve transection) as well as naive intact rats served as control groups. ICMS was applied at post-operative days 0, 7, 14, 21, and 28 to map the jaw and tongue motor representations in face MI by analyzing ICMS-evoked movements and electromyographic activity recorded in the genioglossus (GG) and anterior digastric (AD) muscles. There were no statistically significant effects of acute (day 0) nerve transection or sham procedure (p40.05). The surgery in the sham animals was associated with limited post-operative change; this was reflected in a significant (p50.05) increase in the number of GG sites in left MI at post-operative day 14 compared to day 0. However, nerve transection was associated with significant increases in the total number of AD and GG sites in left or right MI or specifically the number of GG sites in rats at post-operative days 21 or 28 compared to earlier time periods. There were also significant differences between nerve-transected and sham groups at post-operative days 7, 14, or 21. These findings suggest that lingual nerve transection is associated with significant time-dependent neuroplastic changes in the tongue motor representations in face MI.
The aim of this study was to examine the possible role of N-methyl-D-aspartate (NMDA)receptor mec... more The aim of this study was to examine the possible role of N-methyl-D-aspartate (NMDA)receptor mechanismsin responsesinducedby the small-fibreexcitantand inflammatoryirritantmustardoil injectedinto the temporomandibular joint (TMJ)region of rats. The effectsof the non-competitiveNMDAantagonistMK-801weretested on the mustardoil-evoked increases in electromyog- rapbic (EMG) activity of the masseter and digastric muscles and Evans Blue plasma extravasation. Five minutes before the mustard oil injection, MK-801 or its vehicle was administered systemically (iv.), into the third ventricle (i.c.v.), or locally into the TMJ region. Compared with control animals receiving vehicle, the rats receiving MK-801 at an iv. dose of 0.5 mgikg (n= 5) showed a significant reduction in the incidence and magnitude of EMG responsesas wellas in the plasmaextravasationevoked by mustard oil; MK-801 at an iv. dose of 0.1 mg/kg (n= 5) had no significant effect on plasma extravasation or on the incidence and magnitude of EMG responsesbut did significantly increase the latency of EMG responses. An i.c.v. dose of 0.1 mglkg (n = 5) or 0.01 mgllcg(n = 5) had no significant effect on plasma extravasation or incidence of EMG responses but did significantly reduce the magnitudes of the masseter EMG response; the 0.01 mgkg dose also significantly increased the latency of the digastric EMG response. The magnitudes of both the masseter and digastric EMG responses were also significantly reduced by MK-801 administered into the TMJ region at a dose of 0.1 mgkg (n= 5) but not by 0.01 mg/kg (n= 5); neither dose significantly affected the incidence of EMG responses or the plasma extravasation. These data suggest that both central and peripheral NMDA receptor mechanisms may play an important role in EMG responses evoked by the small-fibre excitant and inflammatory irritant mustard oil, but that different neurochemical mechanisms may be involved in the plasma extravasation induced by mustard oil.
The aim of this study was to determine if the application of mustard oil (MO), a small-fiber exci... more The aim of this study was to determine if the application of mustard oil (MO), a small-fiber excitant and inflammatory irritant, or other algesic chemicals (capsaicin, CAP, and bradykinin, BK) to the rat maxillary molar tooth pulp induces electromyographic (EMG) activity of the masseter and digastric muscles, and also to determine if endogenous opioid mechanisms may be involved in any documented EMG changes. Application of MO to the tooth pulp induced a significant increase in EMG activity of the ipsilateral masseter up to 30 min. The application of mineral oil to the pulp or MO application to the pulp-extirpated tooth did not induce any significant EMG increases. The application of CAP or BK to the pulp in contrast had much weaker effects on EMG activity of the jaw muscles. CAP produced a small but prolonged increase in masseter EMG activity, and BK induced a short-lasting increase in digastric EMG activity. The systemic administration of the opiate antagonist naloxone significantly reactivated (i.e. rekindled) the EMG response evoked by MO application to the pulp. Naloxone did not produce any such significant rekindling effect on EMG activity following CAP, BK or mineral oil application to the pulp or following MO application to the pulp-extirpated tooth. The MO, BK and especially CAP groups showed histological evidence of vasodilatation and polymorphonuclear leukocyte infiltration in the pulp tissue and a significant increase in plasma extravasation of Evans Blue dye, whereas mineral oil did not induce these changes. These findings suggest that pulp afferent inputs to the central nervous system evoked by BK, CAP and especially MO may induce enhanced jaw muscle activity. In addition, the naloxone data suggest that an opioid suppressive mechanism may be induced by the pulpal afferent inputs evoked by MO, and may serve to limit the jaw muscle activity elicited by these inputs.
Modulatory effects of the new antimigraine drug naratriptan, a 5-HT 1 -receptor-agonist, on neuro... more Modulatory effects of the new antimigraine drug naratriptan, a 5-HT 1 -receptor-agonist, on neurons of the nucleus raphe  magnus were examined in rat by extracellular recordings. In the nucleus raphe  magnus neuronal activity decreased in on-cells and increased in off-cells after intravenous administration of naratriptan. The modulatory effects of naratriptan were similar to the well-known effects of morphine on neurons in the nucleus raphe  magnus. The results of this study suggest central actions of naratriptan and may point to an involvement of the endogenous pain control system in the antinociceptive effects of the 5-HT 1 -receptor-agonist.
The aim of this study was to determine whether trigeminal subnucleus caudalis (Vc) neurons show d... more The aim of this study was to determine whether trigeminal subnucleus caudalis (Vc) neurons show differential inhibitory effects produced by parabrachial area (PBA) or nucleus raphe magnus (NRM) conditioning stimulation (CS) on their A-fiber versus C-fiber inputs electrically evoked from cutaneous and deep tissues. A total of 55 Vc neurons in urethane/chloralose-anesthetized rats were functionally classified as low-threshold mechanoreceptive (LTM; n = 12), nociceptive (wide-dynamic-range, WDR, and nociceptive-specific, NS; n = 111, deep (D; n = 6), or nociceptive convergent neurons receiving skin and deep inputs (S + D; n = 26). Most neuronal responses were tested for both PBA and NRM CS (20-40 PA) delivered 50 msec prior to test stimulation (1.1-1.5 x threshold) of their mechanoreceptive field (RF) or the hypoglossal nerve (X11). Inhibition of responses to both cutaneous and deep A-fiber and C-fiber inputs occurred in over two-thirds of the Vc neurons tested in each neuronal class, and facilitation of responses was only occasionally found. In general, both PBA (ipsi-and contralateral) and NRM CS produced profound inhibition of electrically evoked responses in al1 neuron types, e.g., responses reduced to mean values of 50-53% of control in LTM neurons, 35-46% in WDR, NS, and S + D neurons, and 61-63% in D neurons. NO significant differente in the magnitude of their inhibitory effects was found between PBA and NRM, and between Vc neuronal responses to A-and C-fiber inputs irrespective of their origin from skin or deep tissues. However, in a group of 6 S + D Vc neurons, responses elicited by deep inputs were more powerfully inhibited by PBA stimulation than those elicited by cutaneous inputs, although the magnitude of NRM-induced inhibition of the responses to deep and cutaneous inputs were comparable. These data confirm earlier findings that Vc neuronal responses to cutaneous A-and C-fiber inputs are subject to PBA-and NRM-induced descending modulation, but further reveal that responses to deep A-and C-fiber inputs are also subject to descending modulation from PBA and NRM.
It has been shown that glial cell line-derived neurotrophic factor (GDNF) exhibits analgesic effe... more It has been shown that glial cell line-derived neurotrophic factor (GDNF) exhibits analgesic effects on the neuropathic pain, although the site of action of GDNF is largely unknown. We, therefore, examined the effect of site-directed overexpression of GDNF on neuropathic pain by means of a lentiviral vector system expressing GDNF. Mice were treated with spinal nerve ligation (SNL) at the fifth lumbar (L5) spinal nerve and the lentiviral vector was locally injected on the same day. In SNL mice, the GDNF-expressing virus injection to the dorsal spinal cord or uninjured L4 dorsal root ganglion (DRG) significantly reduced mechanical allodynia, whereas the injection to the injured L5 DRG or plantar skin showed no analgesic or hyperalgesic effects. These results suggest that GDNF exerts the analgesic effect on neuropathic pain by acting on the uninjured DRG neurons and/or their innervated spinal cells.
In the present study, the hypothesis that sex-related differences in glutamate-evoked rat massete... more In the present study, the hypothesis that sex-related differences in glutamate-evoked rat masseter muscle afferent discharge may result from estrogen-related modulation of peripheral NMDA receptor activity and/or expression was tested by examining afferent fiber discharge in response to masseter injection of NMDA and the expression of NR2A/B subunits by masseter ganglion neurons in male and female rats. The results showed that injection of NMDA into the masseter muscle evoked discharges in putative mechanonociceptive afferent fibers and increased blood pressure that was concentration-dependent, however, a systemic action of NMDA appeared responsible for increased blood pressure. NMDA-evoked afferent discharge was significantly greater in female than in male rats, was positively correlated with plasma estrogen levels in females and was significantly greater in ovariectomized female rats treated with a high dose (5 μg/day) compared to a low dose (0.5 μg/ day) of estrogen. Pre-treatment of high dose estrogen-treated-ovariectomized female rats with the Src tyrosine kinase inhibitor PP2 did not affect NMDA-evoked afferent discharge. NMDA-evoked afferent discharge was attenuated by the antagonists ketamine and ifenprodil, which is selective for NR2B containing NMDA receptors. Fewer masseter ganglion neurons expressed the NR2A (16%) subunit as compared with the NR2B subunit (38%), which was expressed at higher frequencies in intact female (46%) and high dose estrogen-treated ovariectomized female (60%) rats than in male (31%) rats. Taken together, these results suggest that sex-related differences in NMDA-evoked masseter afferent discharge are due, at least in part, to an estrogen-mediated increase in expression of peripheral NMDA receptors by masseter ganglion neurons in female rats.
Central sensitization is a crucial process underlying the increased neuronal excitability of noci... more Central sensitization is a crucial process underlying the increased neuronal excitability of nociceptive pathways following peripheral tissue injury and inflammation. Our previous findings have suggested that extracellular ATP molecules acting at purinergic receptors located on presynaptic terminals (e.g., P2X2/3, P2X3 subunits) and glial cells are involved in the glutamatergic-dependent central sensitization induced in medullary dorsal horn (MDH) nociceptive neurons by application to the tooth pulp of the inflammatory irritant mustard oil (MO). Since growing evidence indicates that activation of P2X7 receptors located on glia is involved in chronic inflammatory and neuropathic pain, the aim of the present study was to test in vivo for P2X7 receptor involvement in this acute inflammatory pain model. Experiments were carried out in anesthetized Sprague-Dawley male rats. Single unit recordings were made in MDH functionally identified nociceptive neurons for which mechanoreceptive field, mechanical activation threshold and responses to noxious stimuli were tested. We found that continuous intrathecal (i.t.) superfusion over MDH of the potent P2X7 receptor antagonists brilliant blue G and periodated oxidized ATP could each significantly attenuate the MO-induced MDH central sensitization. MDH central sensitization could also be produced by i.t. superfusion of ATP and even more effectively by the P2X7 receptor agonist benzoylbenzoyl ATP. Superfusion of the microglial blocker minocycline abolished the MO-induced MDH central sensitization, consistent with reports that dorsal horn P2X7 receptors are mostly expressed on microglia. In control experiments, superfusion over MDH of vehicle did not produce any significant changes. These novel findings suggest that activation of P2X7 receptors in vivo may be involved in the development of central sensitization in an acute inflammatory pain model.
Mustard oil application to tooth pulp produces central sensitization in rat medullary dorsal horn... more Mustard oil application to tooth pulp produces central sensitization in rat medullary dorsal horn (MDH) nociceptive neurons, which has been implicated in persistent pain mechanisms. We found that superfusion onto MDH of methylaminoisobutyric acid, a competitive inhibitor of the neuronal system A transporter for presynaptic uptake of glutamine (a glutamate precursor released from astroglia), significantly depressed development of mustard oil-induced central sensitization in rat MDH nociceptive neurons. This finding indicates that the system A transporter is required for the expression of central sensitization and confirms the important roles of astroglia, glutamine and presynaptic modulation of glutamate release in the development of central sensitization.
Background: To evaluate whether P2X receptors are involved in responses to noxious pulp stimulati... more Background: To evaluate whether P2X receptors are involved in responses to noxious pulp stimulation, the P2X3 and P2X2/3 receptor agonist α,β-methyleneATP (α,β-meATP) was applied to the molar tooth pulp and nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation in trigeminal spinal subnucleus caudalis (Vc), trigeminal spinal subnucleus interpolaris (Vi), upper cervical spinal cord (C1/C2) and paratrigeminal nucleus (Pa5) neurons were analyzed in rats. Results: Genioglossus (GG) muscle activity was evoked by pulpal application of 100 mM α,β-meATP and was significantly larger than GG activity following vehicle (phosphate-buffered saline PBS) application (p < 0.01). The enhanced GG muscle activity following 100 mM α,β-meATP was significantly reduced (p < 0.05) by co-application of 1 mM TNP-ATP (P2X1, P2X3 and, P2X2/3 antagonist). A large number of pERK-LI cells were expressed in the Vc, Vi/Vc, C1/C2 and Pa5 at 5 min following pulpal application of 10...
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Papers by James Wei Hu