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Systemic administration of lidocaine suppresses the excitability of rat cervical dorsal horn neurons and tooth-pulp-evoked jaw-opening reflex
European Journal of Pain 13 (2009) 929–934
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Systemic administration of lidocaine suppresses the excitability of rat cervical dorsal horn neurons and tooth-pulp-evoked jaw-opening reflex Mamoru Takeda a,*, Katsuo Oshima b, Masayuki Takahashi a, Shigeji Matsumoto a a b
Department of Physiology, School of Life Dentistry at Tokyo, Nippon Dental University, 1-9-20, Fujimi-cho, Chiyoda-ku, Tokyo 102-8159, Japan Division of General Dentistry, Nippon Dental University Hospital, 2-3-16, Fujimi-cho, Chiyoda-ku, Tokyo 102-8158, Japan
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Article history: Received 28 July 2008 Received in revised form 27 October 2008 Accepted 23 November 2008 Available online 31 December 2008 Keywords: Lidocaine Trigeminal system Upper cervical dorsal horn Wide dynamic rage neurons Referred pain Jaw-opening reflex
a b s t r a c t Although systemic lidocaine has been demonstrated to have analgesic actions in neuropathic pain conditions, the effect of intravenous lidocaine on trigeminal pain has not been elucidated. The aim of the present study is to investigate the effect of intravenous lidocaine administration on the excitability of the upper cervical dorsal horn (C1) neuron having convergent inputs from both tooth-pulp (TP) and facial skin as well as nociceptive jaw-opening reflex (JOR). After electrical stimulation of TP, extracellular single-unit recordings from 19 C1 neurons and the digastric muscle electromyogram (dEMG) were made in pentobarbital-anesthetized rats. These neurons also responded to non-noxious and noxious mechanical stimulation (touch and pinch) of facial skin, and every neuron was considered to be a wide dynamic range (WDR) neuron. The TP-evoked C1 neuronal and dEMG activities were dose-dependently inhibited by systematic administration of lidocaine (1–2 mg/kg, i.v.). After intravenous injection of lidocaine, the unit discharges induced by both touch and pinch stimuli were inhibited, and the size of the receptive field for pinch was also significantly decreased. The mean spontaneous discharge frequencies were significantly inhibited by the application of lidocaine. These changes were reversed within 20 min. These results suggest that in the absence of neuropathic pain intravenous lidocaine injection suppresses the trigeminal nociceptive reflex as well as the excitability of C1 neurons having convergent inputs from TP and somatic afferents. Systemic lidocaine administration, therefore, may contribute to the alleviation of trigeminalreferred pain associated with tooth pain. Ó 2008 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.
1. Introduction The voltage-gated sodium channel blocker, lidocaine, is the most representative and widely used form of local anesthetic agent, and systemic lidocaine has been very often used in the management of chronic/neuropathic pain (Kastrup et al., 1987; Bath et al., 1990; Rowbotham et al., 1991; Mao and Chen, 2000). In addition, there have been several observations that systemic lidocaine had little effect on the normal pain threshold, but had profound effects on acute pain conditions seen after tissue injury-induced hyperalgesia (Abram and Yaksh, 1994: Puig and Sorkin, 1996). On the other hand, systemic administrations of lidocaine suppress the nociceptive afferent fibre-evoked reflex in the spinal cord (Woolf and Wiesenfeld-Hallin, 1985). Ness (2000) reported that intravenous lidocaine inhibited wide dynamic range (WDR) neuronal activities and visceral nociceptive reflex. To date, it still remains to be determined whether intravenous administration of lidocaine acts on the acute pain states as well as on neuropathic * Corresponding author. Tel./fax: +81 3 3261 874. E-mail address: [email protected] (M. Takeda).
pain states (Hao et al., 1998; Sotgiu et al., 1991, 1992; Ness, 2000; Ness and Randich, 2006; Kawamata et al., 2006), including the trigeminal system. It should be noted that no studies to date have been conducted to address the trigeminal pain. Foreman (2000) reported that the upper cervical dorsal horn (C1) neurons play an important role in the integration of convergent inputs from somatic and visceral organs. We also reported that most C1 spinal WDR neurons responding to tooth-pulp (TP) received afferents inputs from the ipslilateral phrenic nerve, facial skin, Temporomandibular joint (TMJ) masseter muscle and superior sagittal sinus (SSS) (Matsumoto et al., 1999; Takeda et al., 1999, 2005, 2006; Tanimoto et al., 2002; Nishikawa et al., 2004; Fujimi et al., 2006). These results lead us to suggest that WDR neurons in the C1 region contribute to the mechanism of hyperalgesia and/or referred pain associated with dental pain. Previously we also reported that the iontophoretic application of either Nmethyl-D-aspartate (NMDA) or non-NMDA receptor blockers attenuates TP-/SSS-induced C1 neuronal excitation (Takeda et al., 1999; Fujimi et al., 2006). Interestingly, Biella and Sotgiu (1993) demonstrated that systemic injection of lidocaine inhibited the excitatory response to local iontophoretic application of glutamate
1090-3801/$34.00 Ó 2008 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpain.2008.11.017
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in the lumbar spinal cord, and this response was antagonized by co-application of strychnine. A more recent study shows that NMDA-induced inward currents were dose-dependently blocked by lidocaine application (Sugimoto et al., 2003). These findings lead us to suggest that intravenous injection of lidocaine contributes to modulate the trigeminal nociceptive response, and to innervate C1 neurons that have convergent inputs. However, no studies have been conducted to test this idea. Since the jaw-opening reflex (JOR) induced by electrical stimulation of the TP is a valid index for reflex response to noxious stimuli (trigeminal nociceptive reflex) (Mahan and Anderson, 1970; Mason et al., 1985; Takeda et al., 1998), the JOR threshold has been used as an indicator of the intensity of stimulus applied to the TP. The aim of the present study was, therefore, to investigate the effect of intravenous lidocaine on the excitability of the C1 WDR neurons having convergent inputs from both TP and facial skin and the TP-evoked nociceptive JOR. 2. Materials and methods The experiments were approved by the Animal Use and Care Committee of Nippon Dental University and were consistent with the ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983). Every effort was made to minimize the number of animals used and their suffering. 2.1. Animal preparation The experiments were performed on seven male Wistar rats (290–350 g, BW). They were initially anesthetized with pentobarbital (50 mg/Kg, i.v.), and the anesthesia was maintained with additional doses of 2–3 mg/Kg/h through a cannula in the jugular vein, as required. The trachea was cannulated, and the rectal temperature was maintained at 37 ± 0.5 °C with a radiant heater. Arterial blood pressure was measured by means of a pressure transducer through a cannula inserted into the femoral artery. Adequacy of the anesthesia was determined by the lack of response to pinching a paw. 2.2. TP stimulation Bipolar stimulating electrodes made from stainless steel wire (diameter 150 lm, enamel insulated except for the tip of 0.5– 1.0 mm) were inserted into the pulp of the upper incisors and were insulated from the surrounding tissue with dental cement to limit the current spread, as described in the previous studies (Takeda et al., 1998, 1999, 2005, 2006). TP electrical stimulation with constant current single pulses (0.1–3.8 mA, 0.1 ms, 1 Hz) was delivered by bipolar stimulating electrodes. 2.3. Receptive field Somatic receptive fields of C1 spinal neurons that responded to TP stimulation were examined by tactile stimulation with a small brush (<150 mN) and by pinching the skin with forceps. Noxious pinch stimulation was applied to the orofacial area with calibrated forceps at an intensity (4.0 N) that evoked painful sensation when applied to human subjects. The mechanical receptive field of neurons was mapped by probing the skin with von Frey filaments, and was then outlined on a life-sized drawing of rat on a tracing paper, as described in the previous studies (Takeda et al., 2000, 2005).
neuronal activity was recorded extracellulary from the C1 neurons by means of a glass micropipette (2–10 MX) filled with 2% pontamine sky blue and 0.5 M sodium acetate. The neuronal activity was amplified (WPI, DAM80), filtered (0.3–10 kHz) and monitored with an oscilloscope (Nihon Kohden, VC-10). By means of stainless steel electrodes (interpolar distance 2 mm, insulated except for the tip), the digastric electromyogram (dEMG) was recorded from the ipsilateral anterior belly of the digastric muscle to record the JOR. 2.5. Experimental protocols and data analysis Recordings of the C1 unit and the dEMG activity responding to TP stimulation and their data analysis were carried out in the following steps (Takeda et al., 1999, 2005, 2006). The threshold for JOR was determined from the dEMG, that is, pulse duration was 0.1 ms and pulse intensity at a stimulation frequency of 1 Hz was increased until three of five consecutive dEMG responses to TP stimulation were obtained. The peak-to-peak amplitudes in five stimuli trials were averaged. The poststimulus histogram of C1 unit activity induced by TP stimulation (the intensity of TP stimulation was set at 3.5 times dEMG threshold) was constructed (16–32 sweeps, bin width 1 ms). Following the identification of afferent TP inputs onto the C1 neurons, we tested whether the same C1 neurons received afferent input from the facial skin. Single-pulse electrical stimuli were applied to the center of receptive field to activate A-fiber afferent input (0.2 ms, <1 mA) and C-fiber afferent input (2 ms, <5 mA) (Chiang et al., 1998). The conduction velocity of afferent input was calculated by dividing the distance between the site of the facial skin/TP and the C1 region by the peak latency of PSTH between the stimulus artifact and evoked spike. Latency values were corrected for a 0.5-ms synaptic delay. Neuronal responses with estimated conduction velocity of less than 2 m/s and within 2–15 m/s were assumed to receive C-fiber and Ad-fiber afferent inputs, respectively (Bossut and Maixner, 1996; Takeda et al., 2005, 2006). If the changes from the control activity were more than 20%, the given drugs were considered effective. Concerning the effect of application of lidocaine on TP-evoked C1 spinal unit activity, the neuronal discharges were quantified by subtracting the background discharges from the evoked activities. The effect of the lidocaine was evaluated between 2 and 5 min after the end of the administration, because the peak effect of the anesthesia was thought to be observed in this period. The doses of lidocaine used in this study (1 and 2 mg/Kg) were determined by the existence of a significant inhibition on the WDR neuronal activities (Sotgiu et al., 1991, 1992; Ness 2000; Ness and Randich 2006). In this study, our attention was focused on the effect of lidocaine on the WDR neuronal activity, but we have not tested the nociceptive-specific (NS) neurons (Ness and Randich 2006). For this reason, we must state that (1) WDR neurons in the C1 region contribute to the mechanism of hyperalgesia and/or referred pain associated with dental pain (Matsumoto et al., 1999; Takeda et al., 1999, 2005, 2006; Tanimoto et al., 2002; Nishikawa et al., 2004; Fujimi et al., 2006) and (2) many reports suggest a preferential action of lidocaine on the WDR neurons (Sotgiu et al., 1991, 1992; Ness 2000). The number of spike discharge was counted by means of a spike counter (bin width 100 ms). The neuronal activity was recorded on a polygraph (NEC-Sanei 8 M 14) and was stored on a magnetic tape for off-line analysis. The statistical significance was analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s new multiple range test. A probability of less than 0.05 was considered statistically significant. Values were expressed as means ± SEM.
2.4. Recording of single-unit and dEMG activities 2.6. Identification of recording site The animals were then placed in a stereotaxic apparatus, and a laminectomy was performed to expose the C1 region of the spinal cord as described previously (Takeda et al., 2005, 2006). The single
At the end of the recording sessions, rats were deeply anesthetized, and cathodal DC currents (30 lA, 5 min) were passed
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through a recording micropipette. The animals were transcardially perfused with saline and 10% formalin. Frozen coronal sections were cut into 30 lm sections and stained with hematoxylin–eosin. Recording sites were identified from the blue spots, and construction of the electrode tracks was done by means of a combination with micromanipulator readings.
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3. Results
shown in Fig. 1D, recording sites were found in layers I–III (n = 12, 63%) and IV–V (n = 7, 37%)(depth: 190–790 lm) in the C1 dorsal horn (obex 2.1 to 3.2 mm). C1 neurons responding to TP stimulation exhibited a somatic receptive field in the orofacial area (see example, Fig. 2A). All these neurons also responded to mechanical stimulation of the receptive field innervated by ophthalmic and maxillary branches (touch and pinch). Every neuron that was recorded belonged to the category of WDR neurons.
3.1. General characteristics for the C1 neurons convergent inputs from TP and somatic afferents
3.2. Effect of intravenous application of lidocaine on tooth-pulpevoked C1 neuronal activity and amplitude of dEMG
TP electrical stimulation induced reflex responses of the digastric muscle at a latency of 5.9 ± 0.6 ms (n = 7), and the mean threshold intensity was 0.8 ± 0.3 mA (n = 7). Seven of 19 units (37%) showed spontaneous discharges (1.1–3.8 Hz). Typical examples of unit response to TP stimulation (3.5 times threshold for JOR) are shown in Fig. 1A. The mean latency of C1 neurons during TP stimulation was 8.6 ± 2.2 ms (n = 19). The average conduction velocity was 3.9 ± 2.1 m/s (Ad-range) (n = 19). The activity of 19 C1 neurons and the amplitude of 7 dEMG activities increased proportionally during an increase 1.0–3.5 times the JOR threshold, as described in the previous studies (Takeda et al., 1999, 2005). As
As shown in Fig. 1A, 2 min after the intravenous application of lidocaine (2 mg/kg), the TP-evoked C1 neuronal activity was inhibited and this inhibition returned to control level in approximately 20 min. After lidocaine application (2 mg/kg, i.v.), mean firing rates of TP-evoked C1 neuronal activity (3.5 T) significantly decreased, compared to control (control vs. lidocaine; 8.7 ± 0.8 spikes vs. 2.4 ± 0.4 spikes, p < 0.05). The suppression of TP-evoked C1 firings was dose-dependent (Fig. 1B). Systemic lidocaine administration also inhibited the JOR nociceptive reflex. The mean amplitude of dEMG activity (3.5 T) significantly decreased, compared to control (control vs. lidocaine; 1.9 ± 0.4 mV vs. 0.5 ± 0.1 mV, p < 0.05). The
Fig. 1. Effect of intravenous injection of lidocaine on the TP-evoked C1 neuronal activity. A: TP-evoked C1 response to poststimulus histogram (PSTH)(16 sweeps, 1 ms-bin width) corresponding to each sampling time after lidocaine (2 mg/kg, i.v.). Inset; C1 unit response and dEMG activity to TP stimulation (3.5 T). B: Summary of effects of intravenous lidocaine injection on TP-evoked C1 neuronal firing frequency. n = 5, *, p < 0.05. C: Summary of effects of intravenous lidocaine injection on TP-evoked dEMG amplitude. n = 5, *, p<0.05. D: Distribution of recorded neurons in the first cervical dorsal horn (n = 19).
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Fig. 2. Effect of intravenous application of lidocaine on mechanical receptive field properties of C1 neuron responding TP stimulation. A–C: Typical example of mechanoreceptive field properties of C1 neurons before (A) and after (B), and 20 min after (C) intravenous application of lidocaine (2 mg/kg, i.v.). Blackened area location and size of receptive fields responding to pinch stimulation. Hatched area location and size of receptive fields responding to touch stimulation. The poststimulus histogram shows the responses to touch and pinch stimuli applied to mechanoreceptive field. D–F: Summary of intravenous application of lidocaine effects on the firing frequency to mechanical stimulation (D), spontaneous discharges (E), and the size of receptive fields (F). *, p < 0.05.
amplitude of dEMG was dose-dependently significantly inhibited by intravenous application of lidocaine (1–2 mg/kg). The inhibitory effect returned to the control level within 20 min and the effect was dose-dependent (Fig. 1C). 3.3. Effect of intravenous application of lidocaine on the mechanical receptive field properties of C1 neurons A typical example of the effects of systemic administration of lidocaine on the response of C1 neuron to mechanical stimulation is shown in Fig. 2. Fig. 2A shows an example of C1 neuronal activity to mechanical stimulation with spontaneous activity. As shown in Fig. 2B, 2 min after lidocaine injection, decreases in the firing frequencies of C1 neurons to both pinch and touch stimuli and in size of the receptive field to pinch are observed. Furthermore, spontaneous discharges of C1 neurons decreased after lidocaine administration (Fig. 2B and E). The mean mechanical stimulation-evoked discharges were significantly inhibited by lidocaine administration, (pinch: 52.2 ± 3.8 vs. 11.2 ± 0.5 spikes, p < 0.05; touch; 13.4 ± 2.2 vs. 5.2 ± 0.7 spikes, p < 0.05), compared to control (Fig. 2D). On the other hand, the size of the mechanoreceptive field to pinch, but not to touch stimulation, significantly decreased 2 min after lidocaine injection (pinch: 0.55 ± 0.3 vs. 0.31 ±
0.2 cm2, p < 0.05: touch: 1.91 ± 0.5 vs. 1.55 ± 0.6 cm2, NS.) (Fig. 2C and F). These changes returned to the control level within approximately 20 min (Fig. 2C). 3.4. Effect of intravenous lidocaine injection on the arterial blood pressure and heart rate No significant effects were observed on the arterial blood pressure (before vs. after: 105 ± 12 vs. 98 ± 15 mmHg) and heart rate (before vs. after: 335 ± 22 vs. 341 ± 19 beat/min) following intravenous lidocaine injection (1–2 mg/kg). 4. Discussion 4.1. Systemic administration of lidocaine suppressed the C1 neuronal activities The aim of the present study was to investigate the effect of intravenous lidocaine administration on the trigeminal pain in the absence of neuropathic pain. Many reports have indeed shown that there was a selective inhibitory effect of systemic lidocaine on the activity evoked by noxious stimuli (Hao et al., 1998; Sotgiu
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et al., 1991; Ness, 2000; Ness and Randich, 2006) and on the hyperactivity following peripheral nerve injury in dorsal horn WDR neurons of rats (Sotgiu et al., 1992, 1994). In agreement, we found here that after intravenous injection of lidocaine, both electrical stimulation of TP (3.5 times the threshold for JOR: this threshold is close to the sensory threshold for humans) (Mason et al., 1985) and noxious pinch-evoked C1 WDR neuronal firings were suppressed. The effect of lidocaine was reversible and dose-dependent. However, there has been a report that systemic lidocaine injection did not show a significant inhibition on the spinal WDR neuronal activity evoked by both noxious and non-noxious stimuli in intact normal rats (Kawamata et al., 2006). Although the precise reason for the difference between our data and the report by Kawamata et al. (2006) is unclear, there are several possible explanations for the discrepancy. Our data were obtained from pentobarbital-anesthetized animals, whereas Kawamata et al. (2006) conducted the experiments under halothane anesthesia. This difference may be involved in the anesthetic conditions (e.g. inhaled anesthetics decrease the metabolic rate in the brain, whereas pentobarbital anesthesia potentiates the activation of GABAA receptor) (Trevor and Miller 1998; Akaike et al. 1990; Rho et al., 1996). On the other hand, Sotgiu et al. (1994) indicated that intravenous injection of lidocaine produced greater and more prolonged inhibition of spinal dorsal horn neurons than of simultaneously recorded primary afferents, suggesting that there is a central site for the action of lidocaine. In fact, Biella and Sotgiu (1993) demonstrated that central inhibitory effects of lidocaine could be mediated by spinal strychnine-sensitive glycine receptors, activated by lidocaine itself or, possibly, by its glycine residue-bearing metabolites. It is well known that glycine is a major inhibitory transmitter in the spinal cord and the trigeminal spinal nucleus (Todd and Sullivan 1990; Zarbin et al., 1981). Ressot et al. (2001) reported that intravenous administration of strychnine altered the response properties of trigeminal nociceptive neurons. It has also been generally accepted that the local glycinergic inhibitory interneurons modify the mechanical response and receptive field sizes of WDR neurons (Lin et al., 1994; Sorkin and Puig 1996; Biella and Sotgiu 1995). Microiontophoresis application of the glycine receptor antagonist strychnine to the spinal dorsal horn increased both spontaneous, and evoked (noxious and non-noxious) activities of dorsal horn neurons (Lin et al., 1994) which are accompanied by an increased size of the receptive field (Sorkin and Puig 1996). Interestingly enough, in the present study, after intravenous injection we noted the following: (1) the mean firing frequency of C1 WDR neurons to non-noxious stimulation (touch) was significantly inhibited; (2) the mean spontaneous discharges were also inhibited; and (3) the mean size of the mechanical receptive field was significantly inhibited. These changes were reversed within 20 min. We have previously reported that microiontophoretic application of a GABAA agonist, muscimol, inhibited the trigeminal spinal nucleus neuronal activities elicited by touch and pinch stimuli, spontaneous activity as well as the size of receptive field under urethane anesthesia (Takeda et al., 2000). Therefore, it can be speculated that a local mechanism acting via glycine receptors as well as via GABAA receptors normally exerts a tonic inhibition of mechanoreceptive transmission in the trigeminal spinal nucleus neurons and that such an inhibition may limit both the responsiveness and the size of receptive fields. Taken together, the results of the present study suggest that intravenous lidocaine may activate the strychnine-sensitive glycine receptors in the C1 WDR neurons, resulting in the inhibition of the conveying noxious and/or nonnoxious information from the area innervated by the trigeminal nerve at the level of C1 dorsal horn. Alternatively, recent studies indicate that non-specific cation channels of the transient receptor potential (TRP) family, such as transient receptor potential vanilloid 1 (TRPV1) and transient
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receptor potential ankyrin-1 (TRPA1), can modulate nociceptive transmission in the spinal cord (Ferrini et al., 2007; Kosugi et al., 2007; Horvath et al., 2008). There is also evidence that TRPV1 and TRPA1 are coexpressed in the small-diameter noxious sensory neurons (Ad-and C-neurons) (Kobayashi et al., 2005). Lefler et al. (2008) demonstrated that TRPV1 and TRPA1 channels expressed in the small-diameter sized dorsal root ganglion (DRG) neurons are activated and sensitized by lidocaine, suggesting that the activation of these receptors in the dorsal horn may contribute to the release of transmitters. It is generally accepted that there is a positive correlation between the neuronal size of isolated DRG neurons and the conduction velocity of these neurons (Harper and Lawson 1985). Since the conduction velocities of trigeminal primary neurons projected to the WDR neurons in the present study were in the Ad-range, it can be assumed that afferent fibers projecting to C1 neurons are of the small-sized type (Takeda et al., 2006). Ferrini et al. (2007) observed the following findings from mouse spinal dorsal horn slice preparation: (1) TRPV1 activation on primary afferent fibers releases substance P; and (2) substance P then excites inhibitory neurons in substantia gelatinosa neurons, leading to an increased release of GABA/glycine in lamina II via a parallel alternative pathway to glutamate. Given both conditions, it is possible to speculate from our findings that intravenous lidocaine may excite GABAergic/glycinergic inhibitory interneurons via TRPV1 receptor activation, and contributes to a suppressive effect on the C1 WDR neuronal activities. Further studies are required to fully elucidate the mechanism underlying lidocaine effects on the trigeminal pain. 4.2. Functional significance of inhibitory effect on the trigeminal nociception by systemic lidocaine Previously, we reported that most C1 spinal WDR neurons responding to TP received afferent input from the ipsilateral phrenic nerve, facial skin, TMJ, masseter muscle and superior sagittal sinus (Matsumoto et al., 1999; Takeda et al., 1999, 2006a, 2004 Tanimoto et al., 2002; Nishikawa et al., 2004; Fujimi et al., 2006), suggesting that WDR neurons in the C1 region contribute to the mechanism of hyperalgesia and/or referred pain associated with dental pain. It has been known that neurons in the upper cervical segments play an important role in the integration of convergent inputs from somatic and visceral organs (Foreman, 2000). Intravenous lidocaine was shown to be effective in reducing neuropathic pain, including trigeminal neuralgia (Boas et al., 1982). Since systemic lidocaine injection dose-dependently suppressed the C1 neuronal activities evoked by TP stimulation as well as noxious and non-noxious stimuli but had no significant effect on the cardiovascular responses, the results of this study suggest that lidocaine is one of the potential therapeutic agents for trigeminal referred pain. In the present study, we found that TP-evoked of lidocaine, which was consistent with the previous findings that systemic lidocaine inhibited the nociceptive reflex (Woolf and WiesenfeldHallin, 1985). The JOR evoked by electrical stimulation of the TP is a valid index for reflex response to noxious stimuli (Mahan and Anderson, 1970; Mason et al., 1985; Takeda et al., 1998). The majority of sensory neurons in the JOR arc are located in the trigeminal spinal nucleus oralis (Dubner et al., 1978); these neurons project to the trigeminal motor nucleus of the digastric muscle (Mizuno et al., 1975; Sugimoto and Takemura, 1993). The presence of a high density of glycine receptors in the trigeminal spinal nucleus oralis (Zarbin et al., 1981) and intravenous administration of strychnine altered response properties of trigeminal oralis neurons (Ressot et al., 2001), suggesting that activation of glycine receptors in the spinal nucleus oralis neurons by systemic lidocaine administration may contribute to the suppression of nocicep-
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tive JOR. Finally, this report is the first study to show that in normal rats, intravenous lidocaine injection can suppress the trigeminal nociceptive behavioral reflex. 4.3. Conclusion The present study herein provided evidence that in the absence of neuropathic pain, intravenous lidocaine suppressed both the trigeminal nociceptive reflex and the excitability of C1 neurons having convergent inputs from TP and somatic afferents. Therefore, systemic lidocaine treatment may contribute to the alleviation of trigeminal referred pain associated with tooth pain. References Abram SE, Yaksh TL. Systemic lidocaine blocks nerve injury-induced hyperalgesia and nociceptor driven spinal sensitization in the rat. Anesthesiology 1994;80:383–91. Akaike N, Tokutomi N, Ikemoto Y. Augmentation of GABA-induced currents in frog sensory neurons by pentobarbital. Am J Physiol 1990;258:C452–460. Bath FW, Jensen TS, Kastrup J, Stigsby B, Dejgard A. The effect of intravenous lidocaine on nociceptive processing in diabetic neuropathy. Pain 1990;40:29–34. Biella G, Sotgiu ML. Central effect of systemic lidocaine mediated by glycine spinal receptors: an iontophoretic study in the rat spinal cord. Brain Res 1993;603:201–6. Biella G, Sotgiu ML. Evidence that inhibitory mechanisms mask inappropriate somatotopic connections in the spinal cord of normal rat. J Neurophysiol 1995;74:495–505. Boas RA, Covino BG, Shahnarian A. Analgesic responses to I, V. Lidocaine. Br J Anexth 1982;54:501505. Bossut DF, Maixner W. Effects of cardiac vagal afferent electrostimulation on the responses of trigeminal and trigeminothalamic neurons to noxious orofacial stimulation. Pain 1996;65:101–9. Chiang CY, Park SJ, Kwan CL, Hu JW, Sessle BJ. NMDA receptor mechanisms contribute to neuoplasticity induced in caudaqlis nociceptive neurons by tooth pulp stimulation. Neurophysiol 1998;80:2621–31. Dubner R, Sessle BJ, Storey AT. Jaw and tongue reflex. In: Dubner R, Sessle BJ, Storey AT, editors. The neural basis of oral function. New York: Plenum; 1978. p. 246–310. Ferrini F, Salio C, Vergnano AM, Merighi A. Vanilloid receptor-1 (TRPV1)-dependent activation of inhibitory transmission in spinal substantia gelatinosa neurons of mouse. Pain 2007;129:195–209. Foreman RD. Integration of viscerosomatic sensory inputs at the spinal level. Prog Brain Res 2000;122:209–21. Fujimi Y, Takeda M, Tanimoto T, Matsumoto S. Effect of NMDA and non-NMDA receptor antagonists on the superior sagital sinus-evoked C1 spinal neurons responding to tooth-pulp stimulation in rats. Odontology 2006;94:22–8. Hao J-X, Kupers R, Wiesenfeld-Hallin Z. Systemic lidocaine induces expansion of the receptive field of spinal dorsal horn neurons in rats. Exp Brain Res 1998;118:431–4. Harper AA, Lawson SN. Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurons. J Physiol (Lond) 1985;359:31–46. Horvath G, Kekesi G, Nagy E, Benedek G. The role of TRPV1 receptors in the antinociceptive effect of anadamide at spinal level. Pain 2008;134:277–84. Kastrup J, Petersen P, Dejard A, Angelo HR, Hilsted J. Intravenous lidocaine infusion– a new treatment of chronic painful diabetic neuropathy. Pain 1987;28:69–75. Kawamata M, Sugino S, Narimatsu E, Yamauchi M, Kiya T, Furuse S, Namiki A. Effects of systemic administration of lidocaine and QX-314 on hyperexcitability of spinal dorsal horn neurons after incision in the rat. Pain 2006;122:68–80. Kosugi M, Nakatukasa T, Fujita T, Kuroda Y, Kumamoto E. Activation of TRPA1 channel faclilitates excitatory synaptic transmission in the substantia gelatinosa neurons of the adult spinal cord. J Neurosci 2007;27:4443–51. Kobayashi K, FukudaT ObataK, Yamanaka H, Dai Y, Tokunaga A, Noguchi K. Distinct expression of TRPM8, TRPA1 and TRPV1mRNAs in rat primary afferent neurons with Ad/C-fiber and colocalization with Trk receptors. J Comp Neurol 2005;493:596–606. Leffler A, Fischer MJ, Rehner D, Kienei S, Kistner K, Sauer S, et al. The vanilloid receptor TRPV1 is activated and sensitized by local anesthetics in rodent sensory neurons. J Clin Invest 2008;118:763–76. Lin Q, Peng Y, Willis WD. Glycine and GABAA antagonists reduce the inhibition of primate spinothalamic tract neurons produced by stimulation in periaqueductal gray. Brain Res 1994;654:286–302.
Mahan P, Anderson KV. Jaw depression elicited by tooth pulp stimulation. Exp Neurol 1970;29:439–48. Mao J, Chen LL. Systemic lidocaine for neuropathic pain relief. Pain 2000;87:7–17. Mason P, Strassman A, Maciewicz R. Is the jaw opening reflex a valid model of pain? Brain Res Rev 1985;10:137–46. Matsumoto S, Takeda M, Tanimoto T. Effects of electrical stimulation of the tooth pulp and phrenic nerve fibers on the C1 spinal neurons in the rats. Exp Brain Res 1999;126:351–8. Mizuno N, Konishi A, Sato M. Localization of masticatory motor neurons in the cat and rat by means of retrograde axonal transport of horse radish peroxydase. J Comp Neurol 1975;164:105–16. Ness TJ. Intravenous lidocaine inhibits visceral nociceptive reflex and spinal neurons in the rat. Anesthesiology 2000;92:1685–91. Ness TJ, Randich A. Which spinal cutaneous nociceptive neurons are inhibited by intravenous lidocaine in the rat ? Reg Anes Pain Med 2006;31:248–53. Nishikawa T, Takeda M, Tanimoto T, Matsumoto S. Convergence of nociceptive information from temporomandibular joint and tooth pulp afferents on C1 spinal neurons in the rats. Life Sci 2004;75:1465–78. Puig S, Sorkin LS. Formalin-evoked activity in identified primary afferent fibers: systemic lidocaine suppress phase-2 activity. Pain 1996;64:345–55. Ressot C, Collado V, Molat J-L, Dallel R. Strychnine alters responses properties of trigeminal nociceptive neurons in the rat. J Neurophysiol 2001;86:3069–72. Rho JM, Donevan SD, Rogawski MA. Direct activation of GABAA receptors by barbiturates in cultured rat hippocampal neurons. J Physiol 1996;497: 509–22. Rowbotham MC, Reisner-Keller LA, Fields HL. Both intravenous lidocaine and morphine reduce the pain of post therapeutic neuralgia. Neurology 1991;41:1024–8. Sorkin LS, Puig S. Neuonal model of tactile allodynia produced by spinal strychnine: effects of excitatory amino acid receptor antagonists and a l-opiate receptor agonist. Pain 1996;68:283–92. Sotgiu ML, Lacerenza M, Marchetti P. Selective inhibition by systemic lidocaine of noxious evoked activity in rat dorsal horn neurons. Neuroreport 1991;2:425–8. Sotgiu ML, Lacerenza M, Marchetti P. Effect of systemic lidocaine on dorsal horn neurons hyperactivity following chronic peripheral nerve injury in rats. Somatsenses Mot Res 1992;9:227–33. Sotgiu ML, Biella G, Castagna A, Lacerenza M, Marchetti P. Different time -courses of i.v. lidocaine effect on ganglionic and spinal units in neuropathc rats. Neuroreport 1994;5:873–6. Sugimoto T, Takemura M. Tooth pulp primary neurons: Cell size analysis, central connection and carbonic anhydrase activity. Brain Res Bull 1993;30:221–6. Sugimoto M, Uchida I, Mashimo T. Local anaesthetics have different mechanisms and sites of action at the recombinant N-methyl-D-aspartate (NMDA) receptors. Br J Pharmacol 2003;138:862–76. Takeda M, Tanimoto T, Matsumoto S. Supppressive effect of vagal afferents on the activity of trigeminal spinal neurons related to the jaw-opening reflex in rats: involvement of the endogenous opioid system. Brain Res Bull 1998;47:49–56. Takeda M, Tanimoto T, Matsumoto S. Effects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists on the tooth pulp-evoked C1 neurons in the rat. Exp Brain Res 1999;128:303–8. Takeda M, Tanimoto T, Matsumoto S. Change in mechanical receptive field properties induced by GABAA receptor activation in the trigeminal spinal nucleus caudalis neurons in rats. Exp Brain Res 2000;134:409–16. Takeda M, Tanimoto T, Ito M, Nasu M, Matsumoto S. Role of capsaicin sensitive primary afferent fiber input from masseter muscle into C1 spinal neuron responding to tooth-pulp stimulation in rats. Exp Brain Res 2005;160:107–17. Takeda M, Tanimoto T, Takahashi M, Kadoi J, Kadoi J, Matsumoto S. Activation of a2 adrenoreceptors suppresses the excitability of C1 spinal neurons having convergent inputs from tooth-pulp and superior sagital sinus in rats. Exp Brain Res 2006;74:210–20. Tanimoto T, Takeda M, Matsumoto S. Suppressive effect of vagal afferents on cervical dorsal horn neurons responding to tooth-pulp electrical stimulation in the rats. Exp Brain Res 2002;145:468–79. Todd AJ, Sullivan AC. Light microscope study of the co-existence of GABA-like immunoreactivity in the spinal cord of the rat. J Comp Neurol 1990;296:496–505. Trevor AJ, Miller RD. General anesthesthetics. Chapter 25. In: Katzung BG, editor. Basic and Clinical Pharmacology. 7th edition. Stamford: Appleton & Lange; 1998. Woolf CJ, Wiesenfeld-Hallin Z. The systemic administration of local anesthetics produces a selective depression of C-afferent fiber evoked activity in the spinal cord. Pain 1985;23:361–74. Zarbin MA, Wamsley JK, Kuhar MJ. Glycine receptor. Light microscopic autoradiographic localization with [3H] strychnine. J Neurosci 1981;1:532–47. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10.
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Systemic administration of lidocaine suppresses the excitability of rat cervical dorsal horn neurons and tooth-pulp-evoked jaw-opening reflex Mamoru Takeda a,*, Katsuo Oshima b, Masayuki Takahashi a, Shigeji Matsumoto a a b
Department of Physiology, School of Life Dentistry at Tokyo, Nippon Dental University, 1-9-20, Fujimi-cho, Chiyoda-ku, Tokyo 102-8159, Japan Division of General Dentistry, Nippon Dental University Hospital, 2-3-16, Fujimi-cho, Chiyoda-ku, Tokyo 102-8158, Japan
a r t i c l e
i n f o
Article history: Received 28 July 2008 Received in revised form 27 October 2008 Accepted 23 November 2008 Available online 31 December 2008 Keywords: Lidocaine Trigeminal system Upper cervical dorsal horn Wide dynamic rage neurons Referred pain Jaw-opening reflex
a b s t r a c t Although systemic lidocaine has been demonstrated to have analgesic actions in neuropathic pain conditions, the effect of intravenous lidocaine on trigeminal pain has not been elucidated. The aim of the present study is to investigate the effect of intravenous lidocaine administration on the excitability of the upper cervical dorsal horn (C1) neuron having convergent inputs from both tooth-pulp (TP) and facial skin as well as nociceptive jaw-opening reflex (JOR). After electrical stimulation of TP, extracellular single-unit recordings from 19 C1 neurons and the digastric muscle electromyogram (dEMG) were made in pentobarbital-anesthetized rats. These neurons also responded to non-noxious and noxious mechanical stimulation (touch and pinch) of facial skin, and every neuron was considered to be a wide dynamic range (WDR) neuron. The TP-evoked C1 neuronal and dEMG activities were dose-dependently inhibited by systematic administration of lidocaine (1–2 mg/kg, i.v.). After intravenous injection of lidocaine, the unit discharges induced by both touch and pinch stimuli were inhibited, and the size of the receptive field for pinch was also significantly decreased. The mean spontaneous discharge frequencies were significantly inhibited by the application of lidocaine. These changes were reversed within 20 min. These results suggest that in the absence of neuropathic pain intravenous lidocaine injection suppresses the trigeminal nociceptive reflex as well as the excitability of C1 neurons having convergent inputs from TP and somatic afferents. Systemic lidocaine administration, therefore, may contribute to the alleviation of trigeminalreferred pain associated with tooth pain. Ó 2008 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.
1. Introduction The voltage-gated sodium channel blocker, lidocaine, is the most representative and widely used form of local anesthetic agent, and systemic lidocaine has been very often used in the management of chronic/neuropathic pain (Kastrup et al., 1987; Bath et al., 1990; Rowbotham et al., 1991; Mao and Chen, 2000). In addition, there have been several observations that systemic lidocaine had little effect on the normal pain threshold, but had profound effects on acute pain conditions seen after tissue injury-induced hyperalgesia (Abram and Yaksh, 1994: Puig and Sorkin, 1996). On the other hand, systemic administrations of lidocaine suppress the nociceptive afferent fibre-evoked reflex in the spinal cord (Woolf and Wiesenfeld-Hallin, 1985). Ness (2000) reported that intravenous lidocaine inhibited wide dynamic range (WDR) neuronal activities and visceral nociceptive reflex. To date, it still remains to be determined whether intravenous administration of lidocaine acts on the acute pain states as well as on neuropathic * Corresponding author. Tel./fax: +81 3 3261 874. E-mail address: [email protected] (M. Takeda).
pain states (Hao et al., 1998; Sotgiu et al., 1991, 1992; Ness, 2000; Ness and Randich, 2006; Kawamata et al., 2006), including the trigeminal system. It should be noted that no studies to date have been conducted to address the trigeminal pain. Foreman (2000) reported that the upper cervical dorsal horn (C1) neurons play an important role in the integration of convergent inputs from somatic and visceral organs. We also reported that most C1 spinal WDR neurons responding to tooth-pulp (TP) received afferents inputs from the ipslilateral phrenic nerve, facial skin, Temporomandibular joint (TMJ) masseter muscle and superior sagittal sinus (SSS) (Matsumoto et al., 1999; Takeda et al., 1999, 2005, 2006; Tanimoto et al., 2002; Nishikawa et al., 2004; Fujimi et al., 2006). These results lead us to suggest that WDR neurons in the C1 region contribute to the mechanism of hyperalgesia and/or referred pain associated with dental pain. Previously we also reported that the iontophoretic application of either Nmethyl-D-aspartate (NMDA) or non-NMDA receptor blockers attenuates TP-/SSS-induced C1 neuronal excitation (Takeda et al., 1999; Fujimi et al., 2006). Interestingly, Biella and Sotgiu (1993) demonstrated that systemic injection of lidocaine inhibited the excitatory response to local iontophoretic application of glutamate
1090-3801/$34.00 Ó 2008 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpain.2008.11.017
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in the lumbar spinal cord, and this response was antagonized by co-application of strychnine. A more recent study shows that NMDA-induced inward currents were dose-dependently blocked by lidocaine application (Sugimoto et al., 2003). These findings lead us to suggest that intravenous injection of lidocaine contributes to modulate the trigeminal nociceptive response, and to innervate C1 neurons that have convergent inputs. However, no studies have been conducted to test this idea. Since the jaw-opening reflex (JOR) induced by electrical stimulation of the TP is a valid index for reflex response to noxious stimuli (trigeminal nociceptive reflex) (Mahan and Anderson, 1970; Mason et al., 1985; Takeda et al., 1998), the JOR threshold has been used as an indicator of the intensity of stimulus applied to the TP. The aim of the present study was, therefore, to investigate the effect of intravenous lidocaine on the excitability of the C1 WDR neurons having convergent inputs from both TP and facial skin and the TP-evoked nociceptive JOR. 2. Materials and methods The experiments were approved by the Animal Use and Care Committee of Nippon Dental University and were consistent with the ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983). Every effort was made to minimize the number of animals used and their suffering. 2.1. Animal preparation The experiments were performed on seven male Wistar rats (290–350 g, BW). They were initially anesthetized with pentobarbital (50 mg/Kg, i.v.), and the anesthesia was maintained with additional doses of 2–3 mg/Kg/h through a cannula in the jugular vein, as required. The trachea was cannulated, and the rectal temperature was maintained at 37 ± 0.5 °C with a radiant heater. Arterial blood pressure was measured by means of a pressure transducer through a cannula inserted into the femoral artery. Adequacy of the anesthesia was determined by the lack of response to pinching a paw. 2.2. TP stimulation Bipolar stimulating electrodes made from stainless steel wire (diameter 150 lm, enamel insulated except for the tip of 0.5– 1.0 mm) were inserted into the pulp of the upper incisors and were insulated from the surrounding tissue with dental cement to limit the current spread, as described in the previous studies (Takeda et al., 1998, 1999, 2005, 2006). TP electrical stimulation with constant current single pulses (0.1–3.8 mA, 0.1 ms, 1 Hz) was delivered by bipolar stimulating electrodes. 2.3. Receptive field Somatic receptive fields of C1 spinal neurons that responded to TP stimulation were examined by tactile stimulation with a small brush (<150 mN) and by pinching the skin with forceps. Noxious pinch stimulation was applied to the orofacial area with calibrated forceps at an intensity (4.0 N) that evoked painful sensation when applied to human subjects. The mechanical receptive field of neurons was mapped by probing the skin with von Frey filaments, and was then outlined on a life-sized drawing of rat on a tracing paper, as described in the previous studies (Takeda et al., 2000, 2005).
neuronal activity was recorded extracellulary from the C1 neurons by means of a glass micropipette (2–10 MX) filled with 2% pontamine sky blue and 0.5 M sodium acetate. The neuronal activity was amplified (WPI, DAM80), filtered (0.3–10 kHz) and monitored with an oscilloscope (Nihon Kohden, VC-10). By means of stainless steel electrodes (interpolar distance 2 mm, insulated except for the tip), the digastric electromyogram (dEMG) was recorded from the ipsilateral anterior belly of the digastric muscle to record the JOR. 2.5. Experimental protocols and data analysis Recordings of the C1 unit and the dEMG activity responding to TP stimulation and their data analysis were carried out in the following steps (Takeda et al., 1999, 2005, 2006). The threshold for JOR was determined from the dEMG, that is, pulse duration was 0.1 ms and pulse intensity at a stimulation frequency of 1 Hz was increased until three of five consecutive dEMG responses to TP stimulation were obtained. The peak-to-peak amplitudes in five stimuli trials were averaged. The poststimulus histogram of C1 unit activity induced by TP stimulation (the intensity of TP stimulation was set at 3.5 times dEMG threshold) was constructed (16–32 sweeps, bin width 1 ms). Following the identification of afferent TP inputs onto the C1 neurons, we tested whether the same C1 neurons received afferent input from the facial skin. Single-pulse electrical stimuli were applied to the center of receptive field to activate A-fiber afferent input (0.2 ms, <1 mA) and C-fiber afferent input (2 ms, <5 mA) (Chiang et al., 1998). The conduction velocity of afferent input was calculated by dividing the distance between the site of the facial skin/TP and the C1 region by the peak latency of PSTH between the stimulus artifact and evoked spike. Latency values were corrected for a 0.5-ms synaptic delay. Neuronal responses with estimated conduction velocity of less than 2 m/s and within 2–15 m/s were assumed to receive C-fiber and Ad-fiber afferent inputs, respectively (Bossut and Maixner, 1996; Takeda et al., 2005, 2006). If the changes from the control activity were more than 20%, the given drugs were considered effective. Concerning the effect of application of lidocaine on TP-evoked C1 spinal unit activity, the neuronal discharges were quantified by subtracting the background discharges from the evoked activities. The effect of the lidocaine was evaluated between 2 and 5 min after the end of the administration, because the peak effect of the anesthesia was thought to be observed in this period. The doses of lidocaine used in this study (1 and 2 mg/Kg) were determined by the existence of a significant inhibition on the WDR neuronal activities (Sotgiu et al., 1991, 1992; Ness 2000; Ness and Randich 2006). In this study, our attention was focused on the effect of lidocaine on the WDR neuronal activity, but we have not tested the nociceptive-specific (NS) neurons (Ness and Randich 2006). For this reason, we must state that (1) WDR neurons in the C1 region contribute to the mechanism of hyperalgesia and/or referred pain associated with dental pain (Matsumoto et al., 1999; Takeda et al., 1999, 2005, 2006; Tanimoto et al., 2002; Nishikawa et al., 2004; Fujimi et al., 2006) and (2) many reports suggest a preferential action of lidocaine on the WDR neurons (Sotgiu et al., 1991, 1992; Ness 2000). The number of spike discharge was counted by means of a spike counter (bin width 100 ms). The neuronal activity was recorded on a polygraph (NEC-Sanei 8 M 14) and was stored on a magnetic tape for off-line analysis. The statistical significance was analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s new multiple range test. A probability of less than 0.05 was considered statistically significant. Values were expressed as means ± SEM.
2.4. Recording of single-unit and dEMG activities 2.6. Identification of recording site The animals were then placed in a stereotaxic apparatus, and a laminectomy was performed to expose the C1 region of the spinal cord as described previously (Takeda et al., 2005, 2006). The single
At the end of the recording sessions, rats were deeply anesthetized, and cathodal DC currents (30 lA, 5 min) were passed
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through a recording micropipette. The animals were transcardially perfused with saline and 10% formalin. Frozen coronal sections were cut into 30 lm sections and stained with hematoxylin–eosin. Recording sites were identified from the blue spots, and construction of the electrode tracks was done by means of a combination with micromanipulator readings.
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shown in Fig. 1D, recording sites were found in layers I–III (n = 12, 63%) and IV–V (n = 7, 37%)(depth: 190–790 lm) in the C1 dorsal horn (obex 2.1 to 3.2 mm). C1 neurons responding to TP stimulation exhibited a somatic receptive field in the orofacial area (see example, Fig. 2A). All these neurons also responded to mechanical stimulation of the receptive field innervated by ophthalmic and maxillary branches (touch and pinch). Every neuron that was recorded belonged to the category of WDR neurons.
3.1. General characteristics for the C1 neurons convergent inputs from TP and somatic afferents
3.2. Effect of intravenous application of lidocaine on tooth-pulpevoked C1 neuronal activity and amplitude of dEMG
TP electrical stimulation induced reflex responses of the digastric muscle at a latency of 5.9 ± 0.6 ms (n = 7), and the mean threshold intensity was 0.8 ± 0.3 mA (n = 7). Seven of 19 units (37%) showed spontaneous discharges (1.1–3.8 Hz). Typical examples of unit response to TP stimulation (3.5 times threshold for JOR) are shown in Fig. 1A. The mean latency of C1 neurons during TP stimulation was 8.6 ± 2.2 ms (n = 19). The average conduction velocity was 3.9 ± 2.1 m/s (Ad-range) (n = 19). The activity of 19 C1 neurons and the amplitude of 7 dEMG activities increased proportionally during an increase 1.0–3.5 times the JOR threshold, as described in the previous studies (Takeda et al., 1999, 2005). As
As shown in Fig. 1A, 2 min after the intravenous application of lidocaine (2 mg/kg), the TP-evoked C1 neuronal activity was inhibited and this inhibition returned to control level in approximately 20 min. After lidocaine application (2 mg/kg, i.v.), mean firing rates of TP-evoked C1 neuronal activity (3.5 T) significantly decreased, compared to control (control vs. lidocaine; 8.7 ± 0.8 spikes vs. 2.4 ± 0.4 spikes, p < 0.05). The suppression of TP-evoked C1 firings was dose-dependent (Fig. 1B). Systemic lidocaine administration also inhibited the JOR nociceptive reflex. The mean amplitude of dEMG activity (3.5 T) significantly decreased, compared to control (control vs. lidocaine; 1.9 ± 0.4 mV vs. 0.5 ± 0.1 mV, p < 0.05). The
Fig. 1. Effect of intravenous injection of lidocaine on the TP-evoked C1 neuronal activity. A: TP-evoked C1 response to poststimulus histogram (PSTH)(16 sweeps, 1 ms-bin width) corresponding to each sampling time after lidocaine (2 mg/kg, i.v.). Inset; C1 unit response and dEMG activity to TP stimulation (3.5 T). B: Summary of effects of intravenous lidocaine injection on TP-evoked C1 neuronal firing frequency. n = 5, *, p < 0.05. C: Summary of effects of intravenous lidocaine injection on TP-evoked dEMG amplitude. n = 5, *, p<0.05. D: Distribution of recorded neurons in the first cervical dorsal horn (n = 19).
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Fig. 2. Effect of intravenous application of lidocaine on mechanical receptive field properties of C1 neuron responding TP stimulation. A–C: Typical example of mechanoreceptive field properties of C1 neurons before (A) and after (B), and 20 min after (C) intravenous application of lidocaine (2 mg/kg, i.v.). Blackened area location and size of receptive fields responding to pinch stimulation. Hatched area location and size of receptive fields responding to touch stimulation. The poststimulus histogram shows the responses to touch and pinch stimuli applied to mechanoreceptive field. D–F: Summary of intravenous application of lidocaine effects on the firing frequency to mechanical stimulation (D), spontaneous discharges (E), and the size of receptive fields (F). *, p < 0.05.
amplitude of dEMG was dose-dependently significantly inhibited by intravenous application of lidocaine (1–2 mg/kg). The inhibitory effect returned to the control level within 20 min and the effect was dose-dependent (Fig. 1C). 3.3. Effect of intravenous application of lidocaine on the mechanical receptive field properties of C1 neurons A typical example of the effects of systemic administration of lidocaine on the response of C1 neuron to mechanical stimulation is shown in Fig. 2. Fig. 2A shows an example of C1 neuronal activity to mechanical stimulation with spontaneous activity. As shown in Fig. 2B, 2 min after lidocaine injection, decreases in the firing frequencies of C1 neurons to both pinch and touch stimuli and in size of the receptive field to pinch are observed. Furthermore, spontaneous discharges of C1 neurons decreased after lidocaine administration (Fig. 2B and E). The mean mechanical stimulation-evoked discharges were significantly inhibited by lidocaine administration, (pinch: 52.2 ± 3.8 vs. 11.2 ± 0.5 spikes, p < 0.05; touch; 13.4 ± 2.2 vs. 5.2 ± 0.7 spikes, p < 0.05), compared to control (Fig. 2D). On the other hand, the size of the mechanoreceptive field to pinch, but not to touch stimulation, significantly decreased 2 min after lidocaine injection (pinch: 0.55 ± 0.3 vs. 0.31 ±
0.2 cm2, p < 0.05: touch: 1.91 ± 0.5 vs. 1.55 ± 0.6 cm2, NS.) (Fig. 2C and F). These changes returned to the control level within approximately 20 min (Fig. 2C). 3.4. Effect of intravenous lidocaine injection on the arterial blood pressure and heart rate No significant effects were observed on the arterial blood pressure (before vs. after: 105 ± 12 vs. 98 ± 15 mmHg) and heart rate (before vs. after: 335 ± 22 vs. 341 ± 19 beat/min) following intravenous lidocaine injection (1–2 mg/kg). 4. Discussion 4.1. Systemic administration of lidocaine suppressed the C1 neuronal activities The aim of the present study was to investigate the effect of intravenous lidocaine administration on the trigeminal pain in the absence of neuropathic pain. Many reports have indeed shown that there was a selective inhibitory effect of systemic lidocaine on the activity evoked by noxious stimuli (Hao et al., 1998; Sotgiu
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et al., 1991; Ness, 2000; Ness and Randich, 2006) and on the hyperactivity following peripheral nerve injury in dorsal horn WDR neurons of rats (Sotgiu et al., 1992, 1994). In agreement, we found here that after intravenous injection of lidocaine, both electrical stimulation of TP (3.5 times the threshold for JOR: this threshold is close to the sensory threshold for humans) (Mason et al., 1985) and noxious pinch-evoked C1 WDR neuronal firings were suppressed. The effect of lidocaine was reversible and dose-dependent. However, there has been a report that systemic lidocaine injection did not show a significant inhibition on the spinal WDR neuronal activity evoked by both noxious and non-noxious stimuli in intact normal rats (Kawamata et al., 2006). Although the precise reason for the difference between our data and the report by Kawamata et al. (2006) is unclear, there are several possible explanations for the discrepancy. Our data were obtained from pentobarbital-anesthetized animals, whereas Kawamata et al. (2006) conducted the experiments under halothane anesthesia. This difference may be involved in the anesthetic conditions (e.g. inhaled anesthetics decrease the metabolic rate in the brain, whereas pentobarbital anesthesia potentiates the activation of GABAA receptor) (Trevor and Miller 1998; Akaike et al. 1990; Rho et al., 1996). On the other hand, Sotgiu et al. (1994) indicated that intravenous injection of lidocaine produced greater and more prolonged inhibition of spinal dorsal horn neurons than of simultaneously recorded primary afferents, suggesting that there is a central site for the action of lidocaine. In fact, Biella and Sotgiu (1993) demonstrated that central inhibitory effects of lidocaine could be mediated by spinal strychnine-sensitive glycine receptors, activated by lidocaine itself or, possibly, by its glycine residue-bearing metabolites. It is well known that glycine is a major inhibitory transmitter in the spinal cord and the trigeminal spinal nucleus (Todd and Sullivan 1990; Zarbin et al., 1981). Ressot et al. (2001) reported that intravenous administration of strychnine altered the response properties of trigeminal nociceptive neurons. It has also been generally accepted that the local glycinergic inhibitory interneurons modify the mechanical response and receptive field sizes of WDR neurons (Lin et al., 1994; Sorkin and Puig 1996; Biella and Sotgiu 1995). Microiontophoresis application of the glycine receptor antagonist strychnine to the spinal dorsal horn increased both spontaneous, and evoked (noxious and non-noxious) activities of dorsal horn neurons (Lin et al., 1994) which are accompanied by an increased size of the receptive field (Sorkin and Puig 1996). Interestingly enough, in the present study, after intravenous injection we noted the following: (1) the mean firing frequency of C1 WDR neurons to non-noxious stimulation (touch) was significantly inhibited; (2) the mean spontaneous discharges were also inhibited; and (3) the mean size of the mechanical receptive field was significantly inhibited. These changes were reversed within 20 min. We have previously reported that microiontophoretic application of a GABAA agonist, muscimol, inhibited the trigeminal spinal nucleus neuronal activities elicited by touch and pinch stimuli, spontaneous activity as well as the size of receptive field under urethane anesthesia (Takeda et al., 2000). Therefore, it can be speculated that a local mechanism acting via glycine receptors as well as via GABAA receptors normally exerts a tonic inhibition of mechanoreceptive transmission in the trigeminal spinal nucleus neurons and that such an inhibition may limit both the responsiveness and the size of receptive fields. Taken together, the results of the present study suggest that intravenous lidocaine may activate the strychnine-sensitive glycine receptors in the C1 WDR neurons, resulting in the inhibition of the conveying noxious and/or nonnoxious information from the area innervated by the trigeminal nerve at the level of C1 dorsal horn. Alternatively, recent studies indicate that non-specific cation channels of the transient receptor potential (TRP) family, such as transient receptor potential vanilloid 1 (TRPV1) and transient
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receptor potential ankyrin-1 (TRPA1), can modulate nociceptive transmission in the spinal cord (Ferrini et al., 2007; Kosugi et al., 2007; Horvath et al., 2008). There is also evidence that TRPV1 and TRPA1 are coexpressed in the small-diameter noxious sensory neurons (Ad-and C-neurons) (Kobayashi et al., 2005). Lefler et al. (2008) demonstrated that TRPV1 and TRPA1 channels expressed in the small-diameter sized dorsal root ganglion (DRG) neurons are activated and sensitized by lidocaine, suggesting that the activation of these receptors in the dorsal horn may contribute to the release of transmitters. It is generally accepted that there is a positive correlation between the neuronal size of isolated DRG neurons and the conduction velocity of these neurons (Harper and Lawson 1985). Since the conduction velocities of trigeminal primary neurons projected to the WDR neurons in the present study were in the Ad-range, it can be assumed that afferent fibers projecting to C1 neurons are of the small-sized type (Takeda et al., 2006). Ferrini et al. (2007) observed the following findings from mouse spinal dorsal horn slice preparation: (1) TRPV1 activation on primary afferent fibers releases substance P; and (2) substance P then excites inhibitory neurons in substantia gelatinosa neurons, leading to an increased release of GABA/glycine in lamina II via a parallel alternative pathway to glutamate. Given both conditions, it is possible to speculate from our findings that intravenous lidocaine may excite GABAergic/glycinergic inhibitory interneurons via TRPV1 receptor activation, and contributes to a suppressive effect on the C1 WDR neuronal activities. Further studies are required to fully elucidate the mechanism underlying lidocaine effects on the trigeminal pain. 4.2. Functional significance of inhibitory effect on the trigeminal nociception by systemic lidocaine Previously, we reported that most C1 spinal WDR neurons responding to TP received afferent input from the ipsilateral phrenic nerve, facial skin, TMJ, masseter muscle and superior sagittal sinus (Matsumoto et al., 1999; Takeda et al., 1999, 2006a, 2004 Tanimoto et al., 2002; Nishikawa et al., 2004; Fujimi et al., 2006), suggesting that WDR neurons in the C1 region contribute to the mechanism of hyperalgesia and/or referred pain associated with dental pain. It has been known that neurons in the upper cervical segments play an important role in the integration of convergent inputs from somatic and visceral organs (Foreman, 2000). Intravenous lidocaine was shown to be effective in reducing neuropathic pain, including trigeminal neuralgia (Boas et al., 1982). Since systemic lidocaine injection dose-dependently suppressed the C1 neuronal activities evoked by TP stimulation as well as noxious and non-noxious stimuli but had no significant effect on the cardiovascular responses, the results of this study suggest that lidocaine is one of the potential therapeutic agents for trigeminal referred pain. In the present study, we found that TP-evoked of lidocaine, which was consistent with the previous findings that systemic lidocaine inhibited the nociceptive reflex (Woolf and WiesenfeldHallin, 1985). The JOR evoked by electrical stimulation of the TP is a valid index for reflex response to noxious stimuli (Mahan and Anderson, 1970; Mason et al., 1985; Takeda et al., 1998). The majority of sensory neurons in the JOR arc are located in the trigeminal spinal nucleus oralis (Dubner et al., 1978); these neurons project to the trigeminal motor nucleus of the digastric muscle (Mizuno et al., 1975; Sugimoto and Takemura, 1993). The presence of a high density of glycine receptors in the trigeminal spinal nucleus oralis (Zarbin et al., 1981) and intravenous administration of strychnine altered response properties of trigeminal oralis neurons (Ressot et al., 2001), suggesting that activation of glycine receptors in the spinal nucleus oralis neurons by systemic lidocaine administration may contribute to the suppression of nocicep-
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