Suppression of the excitability of rat nociceptive secondary sensory neurons following local administration of the Phytochemical, (-)-Epigallocatechin-3-gallate.

The phytochemical, polyphenolic compound, (-)-epigallocatechin-3-gallate (EGCG), is the main catechin found in green tea. Although a modulatory effect of EGCG on voltage-gated sodium and potassium channels has been reported in excitable tissues, the in vivo effect of EGCG on the excitability of nociceptive sensory neurons remains to be determined. Our aim was to investigate whether local administration of EGCG to rats attenuates the excitability of nociceptive spinal trigeminal nucleus caudalis (SpVc) neurons in response to mechanical stimulation in vivo. Extracellular single unit recordings were made from SpVc neurons in response to orofacial mechanical stimulation of anesthetized rats. The mean firing frequency of SpVc wide-dynamic range neurons following both non-noxious and noxious mechanical stimuli was significantly inhibited by EGCG in a dose-dependent and reversible manner. The mean magnitude of inhibition by EGCG on SpVc neuronal discharge frequency was similar to that of the local anesthetic, 1% lidocaine. Local injection of half-dose of lidocaine replaced the half-dose of EGCG. These results suggest that local injection of EGCG suppresses the excitability of nociceptive SpVc neurons, possibly via the inhibition of voltage-gated sodium channels and opening of voltage-gated potassium channels in the trigeminal ganglion. Therefore, administration of EGCG as a local anesthetic may provide relief from trigeminal nociceptive pain without side effects.

Modulatory mechanism underlying how dietary constituents attenuate orofacial pain.

Physiological pain protects the body and its systems from damage, but pathological pain has no obvious biological role. Complementary alternative medicine (CAM) agents are being increasingly studied in the treatment of clinical pain, and some dietary constituents (polyphenol, carotenoids, and fatty acids) and supplements may modify pain pathways. Because these substances modulate neuronal excitability-including the trigeminal pain pathway via various voltage-gated ionic channels and transient receptor potential and ligand-gated channels, dietary constituents could contribute to CAM as therapeutic agents for attenuating orofacial noxious sensory information. This review summarizes the current understanding of the mechanisms by which dietary constituents might attenuate excitability of trigeminal nociceptive neurons implicated in blocking pain, particularly in relation to the authors' recent experimental data, and discusses the development of functional foods and the contribution of dietary constituents in the relief of clinical dental pain without the side effects of nonsteroidal anti-inflammatory drugs.

Effect of lutein on the acute inflammation-induced c-Fos expression of rat trigeminal spinal nucleus caudalis and C1 dorsal horn neurons.

Although lutein is known to inhibit chronic inflammation, its effect on acute inflammation-induced nociceptive processing in the trigeminal system remains to be determined. The aim of the present study was to investigate whether pretreatment with lutein attenuates acute inflammation-induced sensitization of nociceptive processing in rat spinal trigeminal nucleus caudalis (SpVc) and upper cervical (C1) dorsal horn neurons, via c-Fos immunoreactivity. Mustard oil, a transient receptor potential ankyrin-1 channel agonist, was injected into the whisker pads to induce inflammation. Pretreatment of rats with lutein resulted in significant decreases in the inflammation-induced mean times of face grooming and the thickness of inflammation-induced edema in whisker pads relative to those features in inflamed rats (i.e., rats with no lutein pretreatment). In both the ipsilateral superficial and deep laminae of the SpVc and C1 dorsal horn, there were significantly larger numbers of c-Fos-positive neurons in inflamed rats than in naïve rats, and lutein pretreatment significantly decreased that number relative to inflamed rats. These results suggest that systemic administration of lutein attenuates acute inflammation-induced nocifensive behavior and augmented nociceptive processing of SpVc and C1 neurons that send stimulus localization and intensity information to higher pain centers. These findings support lutein as a potential therapeutic agent for use as an alternative, complementary medicine to attenuate, or even prevent, acute inflammatory pain.

Effect of resveratrol on c-fos expression of rat trigeminal spinal nucleus caudalis and C1 dorsal horn neurons following mustard oil-induced acute inflammation.

The dietary constituent, resveratrol, was recently identified as a transient receptor potential ankyrin 1 (TRPA1) antagonist, voltage-dependent sodium ion (Na+ ) channel, and cyclooxygenase-2 (COX-2) inhibitor. The aim of the present study was to investigate whether pretreatment with resveratrol attenuates acute inflammation-induced sensitization of nociceptive processing in rat spinal trigeminal nucleus caudalis (SpVc) and upper cervical (C1) dorsal horn neurons, via c-fos immunoreactivity. Mustard oil (MO), a TRPA1 channel agonist, was injected into the whisker pads of rats to induce inflammation. Pretreatment with resveratrol significantly decreased the mean thickness of inflammation-induced edema in whisker pads compared with those of untreated, inflamed rats. Ipsilateral of both the superficial and deep laminae of SpVc and C1 dorsal horn, there were significantly more c-fos-immunoreactive SpVc/C1 neurons in inflamed rats compared with naïve rats, and resveratrol pretreatment significantly decreased that number relative to untreated, inflamed rats. These results suggest that systemic administration of resveratrol attenuates acute inflammation-induced augmented nociceptive processing of trigeminal SpVc and C1 neurons. These findings support resveratrol as a potential therapeutic agent for use in alternative, complementary medicine to attenuate, or even prevent, acute trigeminal inflammatory pain.

Modulatory Mechanism of Nociceptive Neuronal Activity by Dietary Constituent Resveratrol.

Changes to somatic sensory pathways caused by peripheral tissue, inflammation or injury can result in behavioral hypersensitivity and pathological pain, such as hyperalgesia. Resveratrol, a plant polyphenol found in red wine and various food products, is known to have several beneficial biological actions. Recent reports indicate that resveratrol can modulate neuronal excitability, including nociceptive sensory transmission. As such, it is possible that this dietary constituent could be a complementary alternative medicine (CAM) candidate, specifically a therapeutic agent. The focus of this review is on the mechanisms underlying the modulatory effects of resveratrol on nociceptive neuronal activity associated with pain relief. In addition, we discuss the contribution of resveratrol to the relief of nociceptive and/or pathological pain and its potential role as a functional food and a CAM.

Systemic administration of resveratrol suppress the nociceptive neuronal activity of spinal trigeminal nucleus caudalis in rats.

Although a modulatory role has been reported for the red wine polyphenol resveratrol on several types of ion channels and excitatory synaptic transmission in the nervous system, the acute effects of resveratrol in vivo, particularly on nociceptive transmission of the trigeminal system, remain to be determined. The aim of the present study was to investigate whether acute intravenous resveratrol administration to rats attenuates the excitability of wide dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo. Extracellular single unit recordings were made from 18 SpVc neurons in response to orofacial mechanical stimulation of pentobarbital-anesthetized rats. Responses to both non-noxious and noxious mechanical stimuli were analyzed in the present study. The mean firing frequency of SpVc WDR neurons in response to both non-noxious and noxious mechanical stimuli was inhibited by resveratrol (0.5-2 mg/kg, i.v.) and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 10 min. These inhibitory effects were reversed after approximately 20 min. The relative magnitude of inhibition by resveratrol of SpVc WDR neuronal discharge frequency was significantly greater for noxious than non-noxious stimulation. These results suggest that, in the absence of inflammatory or neuropathic pain, acute intravenous resveratrol administration suppresses trigeminal sensory transmission, including nociception, and so resveratrol may be used as a complementary and alternative medicine therapeutic agent for the treatment of trigeminal nociceptive pain, including hyperalgesia.

Suppression of ATP-induced excitability in rat small-diameter trigeminal ganglion neurons by activation of GABAB receptor.

The aim of the present study was to investigate whether a GABAB receptor agonist could modulate ATP-activated neuronal excitability of nociceptive TRG neurons using perforated whole-cell patch-clamp and immunohistochemical techniques. Immunohistochemical analysis revealed that 86% of P2X3 receptor-immunoreactive, small-diameter TRG neurons co-expressed GABAB receptor. Under voltage-clamp conditions (Vh=-60mV), application of ATP activated the inward current in acutely isolated rat TRG neurons in a dose-dependent manner (10-50 µM) and this current could be blocked by pyridoxal-phosphate-6-azophenyl-27,47-disulfonic acid (PPADS) (10 µM), a selective P2 purinoreceptor antagonist. The peak amplitude of ATP-activated currents was significantly inhibited after application of GABAB receptor agonist, baclofen (10-50 µM), in a concentration-dependent and reversible manner. The baclofen-induced inhibition of ATP-activated current was abolished by co-application of 3-amino-2 (4-chlorophenyl)-2hydroxypropysufonic acid) saclofen, a GABAB receptor antagonist (50 µM). Under current-clamp conditions, application of 20 µM ATP significantly depolarized the membrane potential resulting in increased mean action potential frequencies, and these ATP-induced effects were significantly inhibited by baclofen and these effects were antagonized by co-application of saclofen. Together, the results suggested that GABAB receptor activation could inhibit the ATP-induced excitability of small-diameter TRG neurons activated through the P2X3 receptor. Thus, the interaction between P2X3 and GABAB receptors of small-diameter TRG neuronal cell bodies is a potential therapeutic target for the treatment of trigeminal nociception.

Direct inhibition of the transient voltage-gated K(+) currents mediates the excitability of tetrodotoxin-resistant neonatal rat nodose ganglion neurons after ouabain application.

The purpose of the present study was to determine the relationship between the responses of transient and sustained K(+) currents, and action potentials to ouabain, and to compare the immunoreactive expression of alpha Na(+)-K(+)-ATPase isoforms (α(1), α(2) and α(3)) in neonatal rat small-diameter nodose ganglion neurons. We used perforated patch-clamp techniques. We first confirmed that the neurons (n=20) were insensitive to 0.5 µM tetrodotoxin (TTX). Application of 1 µM ouabain 1) decreased the transient K(+) currents in 60% of neurons and the sustained K(+) currents in 20%, 2) increased voltage-gated transient and sustained K(+) currents in 20% of neurons, and 3) had no effect on transient K(+) currents in 20% of neurons and on sustained K(+) currents in 60%. Thirteen of the neurons were of a rapidly adapting type, and the remaining 7 were of a slowly adapting type. In 6 rapidly adapting type neurons (46%), their activity was not significantly altered by ouabain application, but in 4 rapidly adapting type neurons, the activity increased. In the remaining 3 rapidly adapting type neurons, ouabain application hyperpolarized the resting membrane potential. The slowly adapting type 7 neurons each showed increased activity after 1 µM ouabain application. The α(1) isoform of Na(+)-K(+)-ATPase was identified as the predominant immunoreactive isoforms in small-diameter nodose ganglion neurons. These results suggest that the increased activity of small-diameter nodose ganglion neurons seen after application of 1 µM ouabain is mediated by direct inhibition of the transient K(+) current.

Alteration of primary afferent activity following inferior alveolar nerve transection in rats.

BACKGROUND: In order to evaluate the neural mechanisms underlying the abnormal facial pain that may develop following regeneration of the injured inferior alveolar nerve (IAN), the properties of the IAN innervated in the mental region were analyzed. RESULTS: Fluorogold (FG) injection into the mental region 14 days after IAN transection showed massive labeling of trigeminal ganglion (TG). The escape threshold to mechanical stimulation of the mental skin was significantly lower (i.e. mechanical allodynia) at 11-14 days after IAN transection than before surgery. The background activity, mechanically evoked responses and afterdischarges of IAN Adelta-fibers were significantly higher in IAN-transected rats than naive. The small/medium diameter TG neurons showed an increase in both tetrodotoxin (TTX)-resistant (TTX-R) and -sensitive (TTX-S) sodium currents (INa) and decrease in total potassium current, transient current (IA) and sustained current (IK) in IAN-transected rats. The amplitude, overshoot amplitude and number of action potentials evoked by the depolarizing pulses after 1 muM TTX administration in TG neurons were significantly higher, whereas the threshold current to elicit spikes was smaller in IAN-transected rats than naive. Resting membrane potential was significantly smaller in IAN-transected rats than that of naive. CONCLUSIONS: These data suggest that the increase in both TTX-S INa and TTX-R INa and the decrease in IA and Ik in small/medium TG neurons in IAN-transected rats are involved in the activation of spike generation, resulting in hyperexcitability of Adelta-IAN fibers innervating the mental region after IAN transection.

Effect of 8-bromo-cAMP on the tetrodotoxin-resistant sodium (Nav 1.8) current in small-diameter nodose ganglion neurons.

We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current in neonatal rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0mM, 8-Br-cAMP at 0.1mM showed a maximal increase in the peak TTX-R Na(+) (Nav1.8) current and produced a hyperpolarizing shift in the conductance-voltage (G-V) curve. The PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5microM) decreased the peak Nav 1.8 current. The Ro-31-8425-induced modulation of the G(V)(1/2) baseline (a percent change in G at baseline V1/2) was not affected by additional 8-Br-cAMP application (0.1mM). The maximal increase in Nav 1.8 currents was seen at 0.1microM after the application of a PKC activator, phorbol 12-myristate 13-acetate (PMA) and forskolin. The PMA-induced increase in Nav 1.8 currents was not significantly affected by additional 0.1mM 8-Br-cAMP application. Intracellular application of a PKA inhibitor, protein kinase inhibitor (PKI, 0.01mM), inhibited the baseline Nav 1.8 current, significantly attenuated the 8-Br-cAMP-and PMA-induced increase in the peak Nav 1.8 current, and caused a significant increase in the slope factor of the inactivation curve. The PKI application at a higher concentration (0.5mM) greatly inhibited the PMA (0.1microM)-induced increase in the peak Nav 1.8 current amplitude and further enhanced the Ro-31-8425-induced decrease in the current. These results suggest that the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated by activation of both PKA and PKC.

Effect of ouabain on the afterhyperpolarization of slowly adapting pulmonary stretch receptors in the rat lung.

In anesthetized, artificially ventilated rats with one vagus nerve section, the purposes of the present study were to investigate whether release from phasic consecutive hyperinflations (inflation volume=3 tidal volumes) results in the afterhyperpolarization (AHP) of the slowly adapting pulmonary stretch receptor (SAR) activity and whether the effect of ouabain, a Na+-K+ ATPase inhibitor, alters AHP of the SAR activity seen after release from maintained inflations. Release from 10 consecutive phasic hyperinflations did not cause any significant inhibition of SAR activity. Release from maintained inflations (for approximately 10 and 15 cmH2O) for 5 s produced the induction of disappearance of SAR activity, corresponding with the AHP. Intravenous administration of ouabain (20 and 40 microg/kg) had no significant effects on the responses of SAR activity and SAR adaptation index (AI) to maintained inflations, but ouabain treatment with at 40 microg/kg resulted in a significant increase in the SAR activity after stopping the respirator and significantly attenuated the AHP of the SAR activity. In the immunohistochemical study, we found Na+-K+ ATPase alpha3-subunit-isoforms-like immunoreactivity in SAR terminals, forming leaflike extensions in the intrapulmonary bronchioles at different diameters, and those terminals were buried in the smooth muscle. In the same sections, the alpha1 subunit immunoreactivity of SAR terminals was not found. These results suggest that the mechanism of generating the AHP of SARs is mainly mediated by the activation of Na+-K+ ATPase alpha3 subunit isoform.

Prostaglandin E2-induced modification of tetrodotoxin-resistant Na+ currents involves activation of both EP2 and EP4 receptors in neonatal rat nodose ganglion neurones.

1 The aim of the present study was to investigate which EP receptor subtypes (EP1-EP4) act predominantly on the modification of the tetrodotoxin-resistant Na+ current (I(NaR)) in acutely isolated neonatal rat nodose ganglion (NG) neurones. 2 Of the four EP receptor agonists ranging from 0.01 to 10 muM, the EP2 receptor agonist (ONO-AE1-259, 0.1-10 microM) and the EP4 receptor agonist (ONO-AE1-329, 1 microM) significantly increased peak I(NaR). The responses were associated with a hyperpolarizing shift in the activation curve. 3 Neither the EP1 receptor agonist ONO-DI-004 nor the EP3 receptor agonist ONO-AE-248 significantly modified the properties of I(NaR). 4 In PGE2 applications ranging from 0.01 to 10 microM, 1 microM PGE2 produced a maximal increase in the peak I(NaR) amplitude. The PGE2 (1 microM)-induced increase in the GV(1/2) baseline (% change in G at baseline V(1/2)) was significantly attenuated by either intracellular application of the PKA inhibitor PKI or extracellular application of the protein kinase C inhibitor staurosporine (1 microM). However, the slope factor k was not significantly altered by PGE2 applications at 0.01-10 microM. In addition, the hyperpolarizing shift of V(1/2) by PGE2 was not significantly altered by either PKI or staurosporine. 5 In other series of experiments, reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from nodose ganglia indicated that all four EP receptors were present. 6 The NG contained many neuronal cell bodies (diameter <30 microm) with intense or moderate EP2, EP3, and EP4 receptor-immunoreactivities. 7 These results suggest that the PGE2-induced modification of I(NaR) is mainly mediated by activation of both EP2 and EP4 receptors.