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Effects of gabapentin on spontaneous discharges and subthreshold membrane potential oscillation of type A neurons in injured DRG
Pain 116 (2005) 187–193 www.elsevier.com/locate/pain
Effects of gabapentin on spontaneous discharges and subthreshold membrane potential oscillation of type A neurons in injured DRG Rui-Hua Yang, Jun-Ling Xing, Jian-Hong Duan, San-Jue Hu* Institute of Neuroscience, The Fourth Military Medical University, Chang-le-xi Road, Xi’an, Shaanxi 710032, People’s Republic of China Received 8 July 2004; received in revised form 30 March 2005; accepted 4 April 2005
Abstract Ectopic spontaneous discharges play a critical role for both initiation and maintenance of the neuropathic pain state. Gabapentin (GBP) has been shown to be effective in animal models of neuropathic pain as well as in chronic pain patients. To investigate the peripheral mechanisms of GBP, the effects of GBP on spontaneous discharges and subthreshold membrane potential oscillation (SMPO) of chronically compressed dorsal root ganglion (DRG) were examined electrophysiolocally in vitro. The rate of spontaneous discharges was transitorily enhanced when GBP was applied to the DRG. When the concentration was under 5 mM, only enhanced effect was observed, while spontaneous discharges were completely suppressed when the concentration of GBP was beyond 5 mM. The similar doses of GBP blocking the spontaneous discharges failed to block the propagation of impulses by electrical nerve stimulation. Furthermore, we found that the SMPO of injured DRG cells can be selectively abolished by GBP without interrupting spike propagation. The results suggest that the inhibitory effect of GBP on SMPO might be one of the membrane mechanisms of action of GBP. This may partially explain the antinociceptive action of GBP by directly suppression nociceptive afferent input to the spinal cord. q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Gabapentin; Spontaneous discharge; Subthreshold membrane potential oscillation; Dorsal root ganglion; Neuropathic pain
1. Introduction Classical anticonvulsant drugs have a long history of use in treatment of chronic pain, particularly in neuropathic pain (Jensen, 2002). Gabapentin (GBP, 1-(aminomethyl)-cyclohexaneacetic acid) is an anticonvulsant with a much lower incidence of side effects compared with other drugs used for neuropathic pain (Rosenberg et al., 1997; Rosner et al., 1996). A number of animal studies have shown a dosedependent reversal of tactile allodynia (Fox et al., 2003; Gillin and Sorkin, 1998; Gustafsson et al., 2003; Wallin et al., 2002), mechanical and thermal hyperalgesia (Cho et al., 2002; Hunter et al., 1997), and formalin-induced nociception (Carlton and Zhou, 1998; Kaneko et al., 2000; Shimoyama et al., 1997) after systemic and/or intrathecal
* Corresponding author. Tel.: C86 29 83374590; fax: C86 29 83246270. E-mail address: [email protected] (S.-J. Hu).
administration of GBP. Analgesic effects of GBP have also been presented in case reports as well as in controlled clinical trials of painful diabetic neuropathy or postherpetic neuropathy (Hemstreet and Lupointe, 2001; Singh et al., 2003). However, the mechanisms and site(s) of its antinociceptive action are largely unclear. Abnormal spontaneous discharges are known to be generated in injured sensory nerve axons and their cell bodies in dorsal root ganglia (DRG) (Devor and Seltzer, 1999; Hu and Xing, 1998; Obata et al., 2003; Sheen et al., 1993; Sukhotinsky et al., 2004). These spontaneous discharges enter the spinal cord and sensitize dorsal horn neurons. The amount of spontaneous discharge is generally well correlated with the degree of pain behavior in neuropathic rats (Han et al., 2000). In fact, blocking spontaneous discharges attenuates pain behaviors both in neuropathic animals and clinical cases (Devor et al., 1992; Gracely et al., 1992; Sheen et al., 1993; Yoon et al., 1996). Therefore, spontaneous discharges play a critical role for
0304-3959/$20.00 q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2005.04.001
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both initiation and maintenance of the neuropathic pain state. In addition, subthreshold membrane potential oscillations (SMPO) were observed recently in DRG neurons, and proved to be a fundamental factor in the generation of abnormal spontaneous discharge (Amir et al., 1999; Hu et al., 1997; Liu et al., 2000; Xing et al., 2001). SMPO gives rise to action potentials when the amplitude of oscillation reaches threshold. The DRG neurons without SMPO are incapable of spontaneously generating repetitive discharges even with greater depolarization (Amir et al., 1999; Xing et al., 2003). Therefore, the spontaneous discharges and SMPO may be an objective and quantitative index of searching for alternative treatment of neuropathic pain and studying mechanisms of antinociceptive action of drugs. Using the chronic compression of DRG model of neuropathic pain, the present study was conducted to examine the effect of GBP on spontaneous discharge as well as SMPO of injured DRG neurons.
2. Method 2.1. Surgery Experiments were conducted on adult Sprague–Dawley rats (150–180 g) of both sexes under an institutionally approved protocol. A chronically compressed-ganglion model was prepared according to the method described previously (Hu and Xing, 1998). Briefly, after induction of anesthesia with pentobarbital sodium (40 mg/kg, i.p.), and the L4 or L5 intervertebral foramen on one side was clearly exposed and a fine, L-shaped stainless steel rod (about 4 mm in length and 0.5–0.8 mm in diameter) was inserted into the foramen and left there to produce a steady compression of the DRG. In some rats the rod was inserted into each foramen so that both ganglia were compressed. 2.2. In vitro tissue preparation The 2–8 days after stainless rod insertion, rats were anesthetized again with pentobarbital sodium. The laminectomy was performed at L1–L6 level, then the L5 DRG were sufficiently exposed, and the rod was carefully taken out to prevent the reinjury to DRG. The L4 or L5 DRG with dorsal root and the sciatic nerve were carefully and quickly removed and kept in oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF content in mM: NaCl 130, KCl 3.5, NaH2PO4 1.25, NaHCO3 24, glucose 10, MgCl2 1.2, CaCl2 1.2; pH 7.2–7.4, Sigma, USA) at room temperature for 30 min. The removed tissue was mounted on an in vitro recording chamber, which had three compartments (the sciatic nerve, the DRG and the dorsal root) separated by Vaseline barriers. The compartment containing DRG was continuously perfused with oxygenated ACSF at a flow rate of 2 ml/min. The ACSF temperature was kept at 35G1 8C by running the perfusion tube through a temperature-controlled water circulator. In other two compartments, the dorsal root and sciatic nerve were covered with
warm paraffin oil. Then, the capsule was carefully removed from the surface of DRG under a dissecting microscope. 2.3. Electrophysiological recording 2.3.1. Single-fiber recording Under a microscope, a microfilament, about 30–50 mm in diameter, was teased away from the dorsal root of DRG by a pair of sharpened forceps. Each fascicle was in turn placed on a fine platinum hook recording electrode (30 mm in diameter) until a spontaneously active single unit could be found. Discharges were assigned to be a single unit if they were of the same shape and amplitude (Hu and Zhu, 1989). The discharges were amplified, displayed on a memory oscilloscope (VC-11, Nihon Kohden Corporation, Japan) and recorded by a computer. Spontaneous discharges were recorded for at least 5 min and then GBP was applied to the DRG compartment to examine its effect on spontaneous discharges. Electrical stimulation was applied to sciatic nerve through an isolator to measure conduction velocity and to examine the nerve propagation after GBP administration. Fibers were classified as A or C fibers when conduction velocities were in the ranges of O2 m/s or !2 m/s, respectively (Xie et al., 1995). Only A fibers were studied in the present experiment. 2.3.2. Intracellular recording In order to observe the effect of GBP on SMPO, intracellular recording was performed on chronically compressed DRG neurons. Intracellular recordings from DRG neurons were obtained using sharp microelectrodes made from filament-containing borosilicate glass tubing. The microelectrodes were pulled with a glass microelectrode puller (model PN-3, Narishige Scientific instrument, Japan) and filled with 3 M potassium acetate, and had a final resistance of 40–60 MU. Neurons were impaled by advancing the microelectrode in 4- or 8-mm steps and applying a small capacitance buzz. The conduction velocity was measured via electrical stimulation of the sciatic nerve through an isolator. DRG neurons were categorized by their conduction velocities (Xie et al., 1995). Only A-type neurons with conduction velocities of 4.0–35.1 m/s were studied in the present experiment. Cells that had stable membrane potentials more negative than K40 mV for more than 3 min after initial penetration were further investigated. GBP (Sigma, USA) is dissolved in artificial cerebrospinal fluid. Different doses of GBP (1, 2, 5, 10 and 20 mM) were diluted from stock solutions before application. After GBP washout and the spontaneous discharges returned to its initial basal rate, the second application was made or another fiber was tested from the same DRG. 2.4. Data analysis Data are presented as meanGSEM, unless otherwise specified. The mean control discharge rate was computed as the mean number of impulses/s (GSEM) for 3 min before GBP application. For each fiber, the effect of GBP on the discharges was defined as the mean discharge rate per minute occurring during GBP application to the DRG. The drug response was compared by using a Student’s t-test, P!0.05 was considered to be statistically significant.
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3. Results 3.1. Effects of GBP on spontaneous discharges A total of 112 units showing spontaneous discharges were recorded from the dorsal roots of the segments whose DRG had been compressed 2–8 days earlier. The conduction velocities of 48 units tested were within the range of 4.1– 37.5 m/s, indicating that all the recorded spontaneous active units were A fibers. The patterns of spontaneous discharges in these units were regular (8.9%); bursting (24.1%) or irregular (67.0%). spontaneous discharges of all tested units in this study persisted for 2–7 h (2.86G1.27 h). Different concentrations of GBP were applied to the DRG from which spontaneous discharges originated. When the concentration was under 5 mM, only increase effect of GBP on spontaneous discharges was observed. Among the 38 units investigated, 33 (86.8%) units displayed this excitatory effect with the other 5 units not changed. For some units, we were able to apply four graded doses of GBP (1, 2, 5 and 10 mM).
Fig. 2. Time course of the excitation and subsequent dose-dependent inhibition of spontaneous discharges by GBP. Each data point is the mean discharge rate per minute occurring during GBP application to the DRG and after GBP washout. The spontaneous discharges were suppressed in response to all doses of GBP except the lowest. The horizontal line above the graph, and the numbers just beneath, indicate the duration of delivery and the concentration of GBP, respectively. The number of units in each groups are 11, 8, 6 and 6, respectively.
As shown in Fig. 1, the rate of discharges increased significantly in earlier period of GBP application, and the mean firing rate showed a higher degree as dose of GBP increased. The most intriguing result of GBP was the inhibitory effect on spontaneous discharges when concentration was beyond 5 mM. There was a significant effect of GBP on the frequency of the spontaneous discharges (Fig. 2). When GBP (5, 10 and 20 mM) was applied, the spontaneous discharge was completely suppressed after 12, 11 and 7 min followed a transient excitatory effect, and the depression was reversed at 7, 19 and 21 min after GBP washout, respectively. Of all 74 inhibited units, the inhibitory effects persisted for 3–40 min with different concentrations and application durations of GBP. The activity of other 12 units did not resume within 40 min washout. 3.2. Persistent impulse propagation after GBP administration
Fig. 1. Transient excitatory effects of gabapentin on ectopic discharges. (A) Shows the effects of gabapentin on a spontaneous active unit. GBP was applied to the DRG at specified doses for the duration indicated by horizontal bars. (B) Shows dose–response relationship for excitatory effect of GBP. The mean firing rates showed a higher degree as doses of GBP increased. Blank column means firing rates in vehicle ACSF. Bias column means firing rates at the maximum GBP effect. Data are presented as meanGSEM (nZ6), * means t-test, P!0.05
Within 5–15 min of applying GBP (O5 mM.), the discharges of 74 fibers tested was completely suppressed as shown for a typical fiber in Fig. 3A. During the period of suppression, it was still possible to evoke action potentials by a single electrical pulse delivered to the sciatic nerve (Fig. 3B and C). Conduction velocity was not noticeably affected. That is, under conditions used in present study, GBP blocked the generation of spontaneous discharge but not the conduction of action potentials.
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Fig. 4. One example of inhibitory effect of GBP on spontaneous discharges and SMPO in injured DRG neuron. The second line represent the increase of discharges after GBP administration, follow three lines represent the first 800 ms of successive 2 min intervals. The lowest line represents the recovery of SMPO and discharges. The arrows mark the onset of 10 mM GBP application to the DRG and washout, respectively. Inset shows the action potentials evoked by intracellular stimulation after the discharges and SMPO abolished. Fig. 3. The excitatory and subsequent inhibitory effect of gabapentin on the ectopic discharges. (A) Shows the course of the excitation and subsequent complete suppression of 10 mM GBP on spontaneous discharge followed by a gradual return to the initial basal rate after washout. GBP was applied to the DRG for the duration indicated by horizontal bars. (B) Left trace is oscilloscope trace of an initial spontaneous discharge rate of about 8 Hz prior to application of GBP. An action potential evoked in the same fiber by a single electrical pulse delivered to the sciatic nerve. The conduction velocity was 25.0 m/s. The action potential after evoked-spike is spontaneous activity. (C) A period of complete suppression of spontaneous discharge. During the period of discharge suppression, an action potential could still be evoked by nerve stimulation (right trace).
3.4. Inhibitory effect of GBP on evoked SMPO The effect of GBP on the SMPO of silent injured neurons was examined during an intracellular depolarizing current injection for 300 ms. In eight neurons, after bath application of GBP (10 mM) for 10 min, the SMPO was eliminated and the number of spikes decreased. In all of neurons tested,
3.3. Inhibitory effect of GBP on spontaneous SMPO In an attempt to further describe the membrane mechanism of the effect of GBP on spontaneous discharges, intracellular recording was performed on chronically compressed DRG neurons. When penetrated, most DRG neurons had a relatively stable membrane potential. However, a minority (8/180, 4.4%) displayed highfrequency, sinusoidal SMPO and spontaneous discharges. Among eight neurons which had SMPO and fired repetitively at resting membrane potential, six neurons maintained stably for more than 5 min and could be used to examine the effect of GBP. There was no significant change in the resting membrane potential after GBP administration. However, GBP (O5 mM) gradually decreased the frequency of discharges. GBP abolished spontaneous SMPO after discharges completely suppressed. During the period of discharges and SMPO are eliminated, the action potentials could still be evoked by intracellular stimulation. The depression was reversed about 24 min after GBP washout (Fig. 4).
Fig. 5. Effect of GBP on evoked SMPO and discharges induced by injecting depolarizing currents (300 ms) in silent injured DRG neurons. (A) In control ACSF, oscillations are triggered and 18 spikes are induced. (B) After bath application of 10 mM of GBP for 10 min, the oscillations are eliminated and the induced spikes decreased to 1. (C) 20 min after washout of the GBP, the SMPO was restored and the number of spikes recovered to 10. Spike height is truncated.
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the SMPO and firing recovered after GBP washout (Fig. 5). In one of eight neurons, the rheobase was slightly elevated after GBP administration.
4. Discussion Neuropathic pain syndromes after peripheral nerve injuries are often poorly relieved by two major classes of analgesics: nonsteroidal anti-inflammatory drugs and opioids (MacFarlane et al., 1997). In the search for alternative treatment, anticonvulsants have become the more commonly used interventions (MacFarlane et al., 1997; McQuay et al., 1995). Among these agents, GBP has been shown to be effective in animal models of neuropathic pain as well as in chronic pain patients (Backonja, 2000; Field et al., 1997; Hunter et al., 1997; Kaneko et al., 2000; Rosenberg et al., 1997; Rosner et al., 1996). Chapman et al. (1998) reported that GBP inhibits the spontaneous discharges of spinal dorsal horn neurons in rats with L5/L6 spinal nerve ligation, suggesting a spinal site of action of this agent. Pan et al. (1999) also indicated that systemic administration of GBP inhibits peripheral ectopic afferent discharges from injured nerve sites, which mediates the antiallodynic effect of GBP. However, previous studies have not clarified the site(s), time course and mechanisms of antinociceptive action of GBP. In the present study, GBP was directly applied to the chronic compressed DRG neurons, which was considered as a critical source of triggering hyperalgesia and spontaneous pain (Xing et al., 2001; Yoon et al., 1996). The result indicated that high concentration of GBP (O5 mM) suppressed the spontaneous discharges in dose-dependent manner. The discharges suppressed fast and reversed slowly along with the concentration of GBP increased. It suggested that DRG neurons might be the peripheral action site of GBP. In clinical trials, GBP attenuated pain syndromes only after a dose of 900–3600 mg/day was reached (Backonja, 2000; Jensen, 2002). As mentioned above, the spontaneous discharges originated from injured DRG neurons were suppressed by GBP only when the concentration was beyond 5 mM, which may explain why a relatively high dose of GBP can relieve the pain syndrome while a lower one cannot. In practice, serum GBP concentrations reached 17.5 and 52.6 mM in patients after 1 h of ingestion with 1200 and 3600 mg, respectively (Berry et al., 2003). It indicated that peripheral mechanisms might involve in the effect of GBP in systemic administration. In addition, GBP (O5 mM) selectively silenced the spontaneous discharges of injured DRG neurons but failed to block the propagation of impulses by electrical nerve stimulation in the present study. This selective sensitivity of spontaneous discharges may account for the effectiveness of GBP in the management of neuropathic pain. We also observed the transient increase in discharge rate in earlier period of GBP application. Previous study had
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demonstrated that the expression of a2/d subunit of voltagegated Ca channel is up-regulated significantly in DRG of rat neuropathic pain models, and the binding of GBP to a2/d subunit occurs at a relatively high affinity (KDZ38 nM) (Gee et al., 1996; Luo et al., 2001; Newton et al., 2001; Suman-Chauhan et al., 1993). GBP significantly decreased Ca2C influx currents in injured DRG cells through the a2/d subunit (Alden and Garcia, 2001). In addition, our previous study confirmed that decrease of intracellular Ca2C facilitated the spontaneous discharges of injured DRG neurons (Xing et al, 1999). Therefore, The mechanisms underlying the initial overshoot may be related to the reduction of intracellular Ca2C ions. Although a transient increase in discharge rate was observed in early period after GBP application in the present study, an early increase in pain level has not been reported either in patient treatment or in animal experiments. As mentioned above, although many studies have attempted to establish the cellular and molecular targets of the actions of GBP, a clear consensus still has not been obtained. It is quite likely that several different cellular actions account for various aspects of GBP pharmacology. On the other hand, C fibers may play a more important role in neuropathic pain. Excitatory impulses in primary afferent C fibers, e.g. during peripheral trauma, may induce long-term changes in the spinal dorsal horn, which is believed to contribute to some forms of hyperalgesia (Liu et al., 1997). In addition, more attention should be paid to pain during the course of GBP treatment as a minor early increase in pain level might be detected. Further studies are needed to explore the mechanism and functional implication of this excitatory effect. In order to explore the membrane mechanisms of action of GBP, the effect of GBP on SMPO was observed by intracellular recording. The result indicated that GBP suppressed the discharges and SMPO of injured DRG neurons. During the period of suppression, it was still possible to evoke action potentials by an intracellular depolarized stimulation. After GBP washout, the discharges reversed following SMPO reappeared. The oscillatory behaviors of primary sensory neurons have been proved to be a fundamental factor in the generation of abnormal spontaneous activity (Amir et al., 1999; Liu et al., 2000; Xing et al., 2001). Ca channel blocker was not effective at blocking the SMPO. The oscillation sinusoids of DRG neurons are due to an interaction between voltagedependent, tetrodotoxin-sensitive NaC conductance and passive, voltage-independent KC leak. (Amir et al., 1999, 2002). It was confirmed that the voltage-gated persistent sodium channel activity causes pronounced inward rectification in the depolarizing direction and mediates the generation of subthreshold oscillatory activity (Agrawal et al., 2001). These data have led us to hypothesize that GBP may suppresses the SMPO through the direct or indirect action on the sodium channels. In summary, GBP administration induced a transient excitation and subsequent dose-dependent inhibitory effect
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on spontaneous discharges of injured DRG neurons in vitro. High, but clinically relevant concentrations of GBP inhibited the spontaneous discharges of injured DRG neurons without interrupting spike propagation. By intracellular recording, we found that GBP abolished the SMPO at the same dose of inhibited spontaneous discharges. It suggested that the inhibitory effect of GBP on SMPO might be one of the membrane mechanisms of action of GBP. These results may partially explain the antinociceptive action of GBP by directly eliminating nociceptive afferent input to the spinal cord.
Acknowledgements This work was supported by the NSFC (30030040, 30200084) grants of China.
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Effects of gabapentin on spontaneous discharges and subthreshold membrane potential oscillation of type A neurons in injured DRG Rui-Hua Yang, Jun-Ling Xing, Jian-Hong Duan, San-Jue Hu* Institute of Neuroscience, The Fourth Military Medical University, Chang-le-xi Road, Xi’an, Shaanxi 710032, People’s Republic of China Received 8 July 2004; received in revised form 30 March 2005; accepted 4 April 2005
Abstract Ectopic spontaneous discharges play a critical role for both initiation and maintenance of the neuropathic pain state. Gabapentin (GBP) has been shown to be effective in animal models of neuropathic pain as well as in chronic pain patients. To investigate the peripheral mechanisms of GBP, the effects of GBP on spontaneous discharges and subthreshold membrane potential oscillation (SMPO) of chronically compressed dorsal root ganglion (DRG) were examined electrophysiolocally in vitro. The rate of spontaneous discharges was transitorily enhanced when GBP was applied to the DRG. When the concentration was under 5 mM, only enhanced effect was observed, while spontaneous discharges were completely suppressed when the concentration of GBP was beyond 5 mM. The similar doses of GBP blocking the spontaneous discharges failed to block the propagation of impulses by electrical nerve stimulation. Furthermore, we found that the SMPO of injured DRG cells can be selectively abolished by GBP without interrupting spike propagation. The results suggest that the inhibitory effect of GBP on SMPO might be one of the membrane mechanisms of action of GBP. This may partially explain the antinociceptive action of GBP by directly suppression nociceptive afferent input to the spinal cord. q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Gabapentin; Spontaneous discharge; Subthreshold membrane potential oscillation; Dorsal root ganglion; Neuropathic pain
1. Introduction Classical anticonvulsant drugs have a long history of use in treatment of chronic pain, particularly in neuropathic pain (Jensen, 2002). Gabapentin (GBP, 1-(aminomethyl)-cyclohexaneacetic acid) is an anticonvulsant with a much lower incidence of side effects compared with other drugs used for neuropathic pain (Rosenberg et al., 1997; Rosner et al., 1996). A number of animal studies have shown a dosedependent reversal of tactile allodynia (Fox et al., 2003; Gillin and Sorkin, 1998; Gustafsson et al., 2003; Wallin et al., 2002), mechanical and thermal hyperalgesia (Cho et al., 2002; Hunter et al., 1997), and formalin-induced nociception (Carlton and Zhou, 1998; Kaneko et al., 2000; Shimoyama et al., 1997) after systemic and/or intrathecal
* Corresponding author. Tel.: C86 29 83374590; fax: C86 29 83246270. E-mail address: [email protected] (S.-J. Hu).
administration of GBP. Analgesic effects of GBP have also been presented in case reports as well as in controlled clinical trials of painful diabetic neuropathy or postherpetic neuropathy (Hemstreet and Lupointe, 2001; Singh et al., 2003). However, the mechanisms and site(s) of its antinociceptive action are largely unclear. Abnormal spontaneous discharges are known to be generated in injured sensory nerve axons and their cell bodies in dorsal root ganglia (DRG) (Devor and Seltzer, 1999; Hu and Xing, 1998; Obata et al., 2003; Sheen et al., 1993; Sukhotinsky et al., 2004). These spontaneous discharges enter the spinal cord and sensitize dorsal horn neurons. The amount of spontaneous discharge is generally well correlated with the degree of pain behavior in neuropathic rats (Han et al., 2000). In fact, blocking spontaneous discharges attenuates pain behaviors both in neuropathic animals and clinical cases (Devor et al., 1992; Gracely et al., 1992; Sheen et al., 1993; Yoon et al., 1996). Therefore, spontaneous discharges play a critical role for
0304-3959/$20.00 q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2005.04.001
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both initiation and maintenance of the neuropathic pain state. In addition, subthreshold membrane potential oscillations (SMPO) were observed recently in DRG neurons, and proved to be a fundamental factor in the generation of abnormal spontaneous discharge (Amir et al., 1999; Hu et al., 1997; Liu et al., 2000; Xing et al., 2001). SMPO gives rise to action potentials when the amplitude of oscillation reaches threshold. The DRG neurons without SMPO are incapable of spontaneously generating repetitive discharges even with greater depolarization (Amir et al., 1999; Xing et al., 2003). Therefore, the spontaneous discharges and SMPO may be an objective and quantitative index of searching for alternative treatment of neuropathic pain and studying mechanisms of antinociceptive action of drugs. Using the chronic compression of DRG model of neuropathic pain, the present study was conducted to examine the effect of GBP on spontaneous discharge as well as SMPO of injured DRG neurons.
2. Method 2.1. Surgery Experiments were conducted on adult Sprague–Dawley rats (150–180 g) of both sexes under an institutionally approved protocol. A chronically compressed-ganglion model was prepared according to the method described previously (Hu and Xing, 1998). Briefly, after induction of anesthesia with pentobarbital sodium (40 mg/kg, i.p.), and the L4 or L5 intervertebral foramen on one side was clearly exposed and a fine, L-shaped stainless steel rod (about 4 mm in length and 0.5–0.8 mm in diameter) was inserted into the foramen and left there to produce a steady compression of the DRG. In some rats the rod was inserted into each foramen so that both ganglia were compressed. 2.2. In vitro tissue preparation The 2–8 days after stainless rod insertion, rats were anesthetized again with pentobarbital sodium. The laminectomy was performed at L1–L6 level, then the L5 DRG were sufficiently exposed, and the rod was carefully taken out to prevent the reinjury to DRG. The L4 or L5 DRG with dorsal root and the sciatic nerve were carefully and quickly removed and kept in oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF content in mM: NaCl 130, KCl 3.5, NaH2PO4 1.25, NaHCO3 24, glucose 10, MgCl2 1.2, CaCl2 1.2; pH 7.2–7.4, Sigma, USA) at room temperature for 30 min. The removed tissue was mounted on an in vitro recording chamber, which had three compartments (the sciatic nerve, the DRG and the dorsal root) separated by Vaseline barriers. The compartment containing DRG was continuously perfused with oxygenated ACSF at a flow rate of 2 ml/min. The ACSF temperature was kept at 35G1 8C by running the perfusion tube through a temperature-controlled water circulator. In other two compartments, the dorsal root and sciatic nerve were covered with
warm paraffin oil. Then, the capsule was carefully removed from the surface of DRG under a dissecting microscope. 2.3. Electrophysiological recording 2.3.1. Single-fiber recording Under a microscope, a microfilament, about 30–50 mm in diameter, was teased away from the dorsal root of DRG by a pair of sharpened forceps. Each fascicle was in turn placed on a fine platinum hook recording electrode (30 mm in diameter) until a spontaneously active single unit could be found. Discharges were assigned to be a single unit if they were of the same shape and amplitude (Hu and Zhu, 1989). The discharges were amplified, displayed on a memory oscilloscope (VC-11, Nihon Kohden Corporation, Japan) and recorded by a computer. Spontaneous discharges were recorded for at least 5 min and then GBP was applied to the DRG compartment to examine its effect on spontaneous discharges. Electrical stimulation was applied to sciatic nerve through an isolator to measure conduction velocity and to examine the nerve propagation after GBP administration. Fibers were classified as A or C fibers when conduction velocities were in the ranges of O2 m/s or !2 m/s, respectively (Xie et al., 1995). Only A fibers were studied in the present experiment. 2.3.2. Intracellular recording In order to observe the effect of GBP on SMPO, intracellular recording was performed on chronically compressed DRG neurons. Intracellular recordings from DRG neurons were obtained using sharp microelectrodes made from filament-containing borosilicate glass tubing. The microelectrodes were pulled with a glass microelectrode puller (model PN-3, Narishige Scientific instrument, Japan) and filled with 3 M potassium acetate, and had a final resistance of 40–60 MU. Neurons were impaled by advancing the microelectrode in 4- or 8-mm steps and applying a small capacitance buzz. The conduction velocity was measured via electrical stimulation of the sciatic nerve through an isolator. DRG neurons were categorized by their conduction velocities (Xie et al., 1995). Only A-type neurons with conduction velocities of 4.0–35.1 m/s were studied in the present experiment. Cells that had stable membrane potentials more negative than K40 mV for more than 3 min after initial penetration were further investigated. GBP (Sigma, USA) is dissolved in artificial cerebrospinal fluid. Different doses of GBP (1, 2, 5, 10 and 20 mM) were diluted from stock solutions before application. After GBP washout and the spontaneous discharges returned to its initial basal rate, the second application was made or another fiber was tested from the same DRG. 2.4. Data analysis Data are presented as meanGSEM, unless otherwise specified. The mean control discharge rate was computed as the mean number of impulses/s (GSEM) for 3 min before GBP application. For each fiber, the effect of GBP on the discharges was defined as the mean discharge rate per minute occurring during GBP application to the DRG. The drug response was compared by using a Student’s t-test, P!0.05 was considered to be statistically significant.
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3. Results 3.1. Effects of GBP on spontaneous discharges A total of 112 units showing spontaneous discharges were recorded from the dorsal roots of the segments whose DRG had been compressed 2–8 days earlier. The conduction velocities of 48 units tested were within the range of 4.1– 37.5 m/s, indicating that all the recorded spontaneous active units were A fibers. The patterns of spontaneous discharges in these units were regular (8.9%); bursting (24.1%) or irregular (67.0%). spontaneous discharges of all tested units in this study persisted for 2–7 h (2.86G1.27 h). Different concentrations of GBP were applied to the DRG from which spontaneous discharges originated. When the concentration was under 5 mM, only increase effect of GBP on spontaneous discharges was observed. Among the 38 units investigated, 33 (86.8%) units displayed this excitatory effect with the other 5 units not changed. For some units, we were able to apply four graded doses of GBP (1, 2, 5 and 10 mM).
Fig. 2. Time course of the excitation and subsequent dose-dependent inhibition of spontaneous discharges by GBP. Each data point is the mean discharge rate per minute occurring during GBP application to the DRG and after GBP washout. The spontaneous discharges were suppressed in response to all doses of GBP except the lowest. The horizontal line above the graph, and the numbers just beneath, indicate the duration of delivery and the concentration of GBP, respectively. The number of units in each groups are 11, 8, 6 and 6, respectively.
As shown in Fig. 1, the rate of discharges increased significantly in earlier period of GBP application, and the mean firing rate showed a higher degree as dose of GBP increased. The most intriguing result of GBP was the inhibitory effect on spontaneous discharges when concentration was beyond 5 mM. There was a significant effect of GBP on the frequency of the spontaneous discharges (Fig. 2). When GBP (5, 10 and 20 mM) was applied, the spontaneous discharge was completely suppressed after 12, 11 and 7 min followed a transient excitatory effect, and the depression was reversed at 7, 19 and 21 min after GBP washout, respectively. Of all 74 inhibited units, the inhibitory effects persisted for 3–40 min with different concentrations and application durations of GBP. The activity of other 12 units did not resume within 40 min washout. 3.2. Persistent impulse propagation after GBP administration
Fig. 1. Transient excitatory effects of gabapentin on ectopic discharges. (A) Shows the effects of gabapentin on a spontaneous active unit. GBP was applied to the DRG at specified doses for the duration indicated by horizontal bars. (B) Shows dose–response relationship for excitatory effect of GBP. The mean firing rates showed a higher degree as doses of GBP increased. Blank column means firing rates in vehicle ACSF. Bias column means firing rates at the maximum GBP effect. Data are presented as meanGSEM (nZ6), * means t-test, P!0.05
Within 5–15 min of applying GBP (O5 mM.), the discharges of 74 fibers tested was completely suppressed as shown for a typical fiber in Fig. 3A. During the period of suppression, it was still possible to evoke action potentials by a single electrical pulse delivered to the sciatic nerve (Fig. 3B and C). Conduction velocity was not noticeably affected. That is, under conditions used in present study, GBP blocked the generation of spontaneous discharge but not the conduction of action potentials.
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Fig. 4. One example of inhibitory effect of GBP on spontaneous discharges and SMPO in injured DRG neuron. The second line represent the increase of discharges after GBP administration, follow three lines represent the first 800 ms of successive 2 min intervals. The lowest line represents the recovery of SMPO and discharges. The arrows mark the onset of 10 mM GBP application to the DRG and washout, respectively. Inset shows the action potentials evoked by intracellular stimulation after the discharges and SMPO abolished. Fig. 3. The excitatory and subsequent inhibitory effect of gabapentin on the ectopic discharges. (A) Shows the course of the excitation and subsequent complete suppression of 10 mM GBP on spontaneous discharge followed by a gradual return to the initial basal rate after washout. GBP was applied to the DRG for the duration indicated by horizontal bars. (B) Left trace is oscilloscope trace of an initial spontaneous discharge rate of about 8 Hz prior to application of GBP. An action potential evoked in the same fiber by a single electrical pulse delivered to the sciatic nerve. The conduction velocity was 25.0 m/s. The action potential after evoked-spike is spontaneous activity. (C) A period of complete suppression of spontaneous discharge. During the period of discharge suppression, an action potential could still be evoked by nerve stimulation (right trace).
3.4. Inhibitory effect of GBP on evoked SMPO The effect of GBP on the SMPO of silent injured neurons was examined during an intracellular depolarizing current injection for 300 ms. In eight neurons, after bath application of GBP (10 mM) for 10 min, the SMPO was eliminated and the number of spikes decreased. In all of neurons tested,
3.3. Inhibitory effect of GBP on spontaneous SMPO In an attempt to further describe the membrane mechanism of the effect of GBP on spontaneous discharges, intracellular recording was performed on chronically compressed DRG neurons. When penetrated, most DRG neurons had a relatively stable membrane potential. However, a minority (8/180, 4.4%) displayed highfrequency, sinusoidal SMPO and spontaneous discharges. Among eight neurons which had SMPO and fired repetitively at resting membrane potential, six neurons maintained stably for more than 5 min and could be used to examine the effect of GBP. There was no significant change in the resting membrane potential after GBP administration. However, GBP (O5 mM) gradually decreased the frequency of discharges. GBP abolished spontaneous SMPO after discharges completely suppressed. During the period of discharges and SMPO are eliminated, the action potentials could still be evoked by intracellular stimulation. The depression was reversed about 24 min after GBP washout (Fig. 4).
Fig. 5. Effect of GBP on evoked SMPO and discharges induced by injecting depolarizing currents (300 ms) in silent injured DRG neurons. (A) In control ACSF, oscillations are triggered and 18 spikes are induced. (B) After bath application of 10 mM of GBP for 10 min, the oscillations are eliminated and the induced spikes decreased to 1. (C) 20 min after washout of the GBP, the SMPO was restored and the number of spikes recovered to 10. Spike height is truncated.
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the SMPO and firing recovered after GBP washout (Fig. 5). In one of eight neurons, the rheobase was slightly elevated after GBP administration.
4. Discussion Neuropathic pain syndromes after peripheral nerve injuries are often poorly relieved by two major classes of analgesics: nonsteroidal anti-inflammatory drugs and opioids (MacFarlane et al., 1997). In the search for alternative treatment, anticonvulsants have become the more commonly used interventions (MacFarlane et al., 1997; McQuay et al., 1995). Among these agents, GBP has been shown to be effective in animal models of neuropathic pain as well as in chronic pain patients (Backonja, 2000; Field et al., 1997; Hunter et al., 1997; Kaneko et al., 2000; Rosenberg et al., 1997; Rosner et al., 1996). Chapman et al. (1998) reported that GBP inhibits the spontaneous discharges of spinal dorsal horn neurons in rats with L5/L6 spinal nerve ligation, suggesting a spinal site of action of this agent. Pan et al. (1999) also indicated that systemic administration of GBP inhibits peripheral ectopic afferent discharges from injured nerve sites, which mediates the antiallodynic effect of GBP. However, previous studies have not clarified the site(s), time course and mechanisms of antinociceptive action of GBP. In the present study, GBP was directly applied to the chronic compressed DRG neurons, which was considered as a critical source of triggering hyperalgesia and spontaneous pain (Xing et al., 2001; Yoon et al., 1996). The result indicated that high concentration of GBP (O5 mM) suppressed the spontaneous discharges in dose-dependent manner. The discharges suppressed fast and reversed slowly along with the concentration of GBP increased. It suggested that DRG neurons might be the peripheral action site of GBP. In clinical trials, GBP attenuated pain syndromes only after a dose of 900–3600 mg/day was reached (Backonja, 2000; Jensen, 2002). As mentioned above, the spontaneous discharges originated from injured DRG neurons were suppressed by GBP only when the concentration was beyond 5 mM, which may explain why a relatively high dose of GBP can relieve the pain syndrome while a lower one cannot. In practice, serum GBP concentrations reached 17.5 and 52.6 mM in patients after 1 h of ingestion with 1200 and 3600 mg, respectively (Berry et al., 2003). It indicated that peripheral mechanisms might involve in the effect of GBP in systemic administration. In addition, GBP (O5 mM) selectively silenced the spontaneous discharges of injured DRG neurons but failed to block the propagation of impulses by electrical nerve stimulation in the present study. This selective sensitivity of spontaneous discharges may account for the effectiveness of GBP in the management of neuropathic pain. We also observed the transient increase in discharge rate in earlier period of GBP application. Previous study had
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demonstrated that the expression of a2/d subunit of voltagegated Ca channel is up-regulated significantly in DRG of rat neuropathic pain models, and the binding of GBP to a2/d subunit occurs at a relatively high affinity (KDZ38 nM) (Gee et al., 1996; Luo et al., 2001; Newton et al., 2001; Suman-Chauhan et al., 1993). GBP significantly decreased Ca2C influx currents in injured DRG cells through the a2/d subunit (Alden and Garcia, 2001). In addition, our previous study confirmed that decrease of intracellular Ca2C facilitated the spontaneous discharges of injured DRG neurons (Xing et al, 1999). Therefore, The mechanisms underlying the initial overshoot may be related to the reduction of intracellular Ca2C ions. Although a transient increase in discharge rate was observed in early period after GBP application in the present study, an early increase in pain level has not been reported either in patient treatment or in animal experiments. As mentioned above, although many studies have attempted to establish the cellular and molecular targets of the actions of GBP, a clear consensus still has not been obtained. It is quite likely that several different cellular actions account for various aspects of GBP pharmacology. On the other hand, C fibers may play a more important role in neuropathic pain. Excitatory impulses in primary afferent C fibers, e.g. during peripheral trauma, may induce long-term changes in the spinal dorsal horn, which is believed to contribute to some forms of hyperalgesia (Liu et al., 1997). In addition, more attention should be paid to pain during the course of GBP treatment as a minor early increase in pain level might be detected. Further studies are needed to explore the mechanism and functional implication of this excitatory effect. In order to explore the membrane mechanisms of action of GBP, the effect of GBP on SMPO was observed by intracellular recording. The result indicated that GBP suppressed the discharges and SMPO of injured DRG neurons. During the period of suppression, it was still possible to evoke action potentials by an intracellular depolarized stimulation. After GBP washout, the discharges reversed following SMPO reappeared. The oscillatory behaviors of primary sensory neurons have been proved to be a fundamental factor in the generation of abnormal spontaneous activity (Amir et al., 1999; Liu et al., 2000; Xing et al., 2001). Ca channel blocker was not effective at blocking the SMPO. The oscillation sinusoids of DRG neurons are due to an interaction between voltagedependent, tetrodotoxin-sensitive NaC conductance and passive, voltage-independent KC leak. (Amir et al., 1999, 2002). It was confirmed that the voltage-gated persistent sodium channel activity causes pronounced inward rectification in the depolarizing direction and mediates the generation of subthreshold oscillatory activity (Agrawal et al., 2001). These data have led us to hypothesize that GBP may suppresses the SMPO through the direct or indirect action on the sodium channels. In summary, GBP administration induced a transient excitation and subsequent dose-dependent inhibitory effect
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on spontaneous discharges of injured DRG neurons in vitro. High, but clinically relevant concentrations of GBP inhibited the spontaneous discharges of injured DRG neurons without interrupting spike propagation. By intracellular recording, we found that GBP abolished the SMPO at the same dose of inhibited spontaneous discharges. It suggested that the inhibitory effect of GBP on SMPO might be one of the membrane mechanisms of action of GBP. These results may partially explain the antinociceptive action of GBP by directly eliminating nociceptive afferent input to the spinal cord.
Acknowledgements This work was supported by the NSFC (30030040, 30200084) grants of China.
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