Effect Of Gabapentin On Discharges Of Dorsal Root Ganglion Neurons And Its Mechanism

Neuropathic pain are a system of chronic pain sympotoms which result from peripheral nerve injury induced by trauma, ischemia, toxicosis or metabolic abnormality, including hyperalgesia, allodynia, spontaneous pain and paraesthesia. It is a clinical familiar sight that chronic compression of the dorsal root ganglion (DRG) or its near spinal root induced by herniated intervertebral disc, stenosis of intervertebral foramen or spur hyperplasia result in neuropathic pain. The therapy of neuropathic pain syndromes depends on two major classes of analgesics: nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids. NSAIDs induce various side-effects because its confined mechanisms, such as gastrointestinal reaction, gastric ulcer, gastric haemorrhage and allergy etc. The drug addiction of opiate analgesic limits its clinical application. On the other hand, chronic neuropathic pain after peripheral nerve injury represents low sensitivity to NSAIDs and opioids. So it become the important task to explore the pathogenesis of neuropathic pain and develop the new analgesic. With creating of animal experimental models and gradual understanding of pathophysiologic mechanism of neuropathic pain, researchers realized that neuropathic pain originated from both peripheral and central nerve system characterized the hyperexcitability of injured neurons. The characters and molecular changes of hyperexcitation neurons are similar to the molecular characters of "pacemaker" neurons in epilepsia. There are similarpathophysiologic and biochemical mechanism between epilepsia and neuropathic pain, which prompted people to treat neuropathic pain with anticonvulsants.Gabapentin (GBP), l-(aminomethyl) cyclohexane acetic acid, came into the market in England on May, 1993. Food and drugs administration (FDA) ratified GBP as the auxiliary medication of epilepsia treatment. GBP was known as a better anticonvulsant because it had not interaction with other drugs, did not metabolized in liver, did not induce or inhibit the microsome enzyme of liver and had low affinity for protein. Subsequent studies comfirmed that GBP was valid to chronic pain syndromes, especially to neuropathic pain, such as diabetic peripheral neuropathy, postherpetic neuralgia, neuropathic pain syndromes after cancer, trigeminal neuralgia. GBP has been shown to be more effective on neuropathic pain with lower side-effect than opiate, NSAIDs, antidepressants and other anticonvulsants. In addition, GBP had not drug-dependent. But, oral administration of GBP induced dizzy, ataxia, nausea and vomiting or even convulsion or neurotoxicity. In addition, the exact target and mechanism of GBP effect on neuropathic pain is largely unclear, which made GBP lack specific and pertinence in neuropathic pain treatment. Many neuropathic pain syndromes are induced by peripheral nerve injury, and abnormal excitability of primary sensory neurons become the originate signals of neuropathic pain. On the other hand, GBP attenuated pain syndromes only after a dose of 900-3,600 mg/day was reached in clinical trials, while the serum GBP concentrations reached 13.14 and 52.56 μM, respectively. It indicated that peripheral mechanisms might involve in the effect of GBP in systemic administration. So we attempt to observe whether GBP inhibit the generation of chronic pain signals after nerve injury. In addition, to explore the mechanism and target of GBP would help to enhance the pertinence of GBP in neuropathic pain treatment and provide the foundation for GBP acting as a peripheral analgetic.After never injury, abnormal spontaneous discharges generated in injured sensory nerve axons and their cell bodies in dorsal root ganglia (DRG). These spontaneous discharges enter the spinal cord and sensitize dorsal horn neurons and induce the neuropathic pain sympotom. Thus injured DRG neurons become the pacemaker of neuropathic pain. The animal experiments and clinical cases confirmed that spontaneous discharges of injured DRG neurons play a critical role for both initiation and maintenance of the neuropathic pain state. Although there are various mechanisms of neuropathic pain, to measure the properties of ectopic discharges can be regarded as one of valid means of exploring the mechanisms of pain and evaluating the effect of analgetic. The patterns and frenquency of ectopic discharges can be recorded, oberserved and analysed from injured afferent fibers or DRG neurons because its objectivity and quantification, which is help to judge kinds of types and degree of pain objectively.In the present study, we observed the effects of GBP on discharges and subthreshold membrane potential oscillations (SMPO) of DRG neurons by single fiber recording and intracellular recording in chronic compression of DRG (CCD) model made by our lab. In addition, we observed the effect of GBP on SMPO and persistent sodium current (I_NaP)of acute isolated DRG neurons by whole-cell patch clamp recording, in order to explore the ion channel mechanisms of GBP and look for a target of peripheral analgetic, and search for analgetic which inhibited pathological signals specifically without affect the physiological acute pain signals.Main Results:1. Main characters of ectopic spontaneous discharges in injured DRG neurons15.1 percent of units shew spontaneous discharges from the dorsal roots of the segments whose DRG had been compressed 2-8 days earlier. A total of 118 units showing spontaneous discharges were recorded from 42 CCDinjured DRG. The amplitude of action potential was 60³00 μV. The patterns of spontaneous discharges in these units were regular (8.93%); bursting (24.11%) or irregular (66.96%). spontaneous discharges of all tested units in this study persisted for 2-7hs (2.86±1.27h).2. GBP inhibited the ectopic spontaneous discharges of injured DRG neurons selectivelyThe results of present study indicated that 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. When the concentration was under 5 μM, only increase effect of GBP on spontaneous discharges was observed. When concentration was beyond 5 μM, GBP inhibited discharges of neurons in dose-dependent manner after a transient increase of discharges. The discharges suppressed fast and reversed slowly along with the concentration of GBP increased. When GBP (5, 10 and 20 μM) 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 2h washout. During the period of discharges suppression, it was still possible to evoke action potentials by a single electrical pulse delivered to the sciatic nerve. Conduction velocity was not noticeably affected before and after GBP administration.3. GBP inhibited various SMPO of DRG neuronsIn an attempt to further describe the membrane mechanism of the effectof GBP on spontaneous discharges, intracellular recording was performed on chronically compressed DRG neurons. A minority (8/180, 4.44%) displayed high-frequency, sinusoidal subthreshold membrane potential oscillation (SMPO) and spontaneous discharges. Some silent injured neurons exhibited discharges and SMPO during an intracellular depolarizing current injection. The results demonstrated that there was no significant change in the resting membrane potential after GBP administration. However, GBP (>5 μM) inhibited the spontaneous or evoked SMPO induced by intracellular stimulation.There were three kinds of discharges and SMPO of DRG neurons induced by ramp depolarization stimulates in whole-cell patch clamp recording. (1) Spindle SMPO and sporadic spike could be observed on the middle part of ramp under excitatory stimulation, (2) Under excitatory stimulation, repeated discharges exhibited with regular SMPO before discharges, (3) Under excitatory stimulation, bursting discharges exhibited with spindle SMPO on both ends of discharges. GBP inhibited all these three kinds of SMPO and discharges.4. GBP inhibited the sensitivity of norepinephrine in injured DRG neuronsExogenous norepinephrine (NE) affected the frequency of discharges of injured DRG neurons in the range of 1²00μ mol/L. The patterns of effect included excitation, excitation followed by suppression and alternation between excitation and suppression. During the period of discharges are eliminated after GBP application, NE could not exhibit its excitatory effect on discharges. The excitatory effect of NE reversed along with the discharges resumeing after GBP washout.5. GBP decreased the persistent sodium current of DRG neuronsUnder excitatory stimulation, a slow, wide duration inward current could...