Classification Of Excitability In Drg Neurons And The Inhibition Of Riluzole On I_NaP

Neuropathic pain indicates neurological damage by surgical trauma, ischemia, intoxication as well as abnomal-metabolism, which has symptoms as hyperalgesia, allodynia, paraesthesia and spontaneous pain. The dorsal root ganglia (DRG) is the cell aggregation of the afferent sensory fibers which has a faithful role of transduction by passing the afferent signal to spinal cord in normal condition. Under pathological conditions, such as direct or indirect injury on DRG, the status of soma would be in a condition of hyperexcitability.and resulting in many types of firing pattern which have been proved to be the chronic pain signal.Excitability is the ability that excitable cells are able to produce the action potential (AP) when stimulated. Traditionally, we estimated it just through the intensity of stimulus threshold and the number of AP. The change of excitability after injury has the components of threshold and the number of AP, while based on the theory of Hodgkin-Huxley, there would be the change of excitability classification. It is Hodgkin (1948) who first classified the excitability into three classes according to the relationship of firing frequency and applied current intensity, in a study of crustacean axon. The class 1 is sensitive to the injected current, class 2 respond with a consistent frequency during the increase of injected current intensity, while class 3 hardly firing with the high intensity of injected current. Our previous work on Mesencephalic V neurons proved this kind of classification, also an additional standard for class 2 has been given. For DRG neuron, the periodic and bursting firing has been found with the activity of subthreshold membrane oscillation (SMPO) and the incidence increase after the injury which suggest a possible exist of above mentioned neuronal excitability classification.Rinzel's work show that the production of different type of excitability is due to different bifurcation from resting to firing status. The ascertain of classicifiication help us to understand how the cell response to outside stimulation, namely, how the cell ecode the outside information. On the other side, it was believed that the change of the parameter of the ion current could lead to the change of the excitability classification which mean the ratio of different ion concentration might result in the change of excitability cloassification. It was shown that in CCD model, a kind of sodium current- INap has close relationship with the hyperexcitability. So, there might be a corresponding change of the ratio for excitability classification, which need further investigation.Main results:Part1:Classification of Excitability in DRG neurons and its ion channel mechanism in CCD model The present study is going to detect the neuron's excitability to see whether the classification exist. The next, the ratio change of the classification would be checked to permit the further investigation of the chronic pain signal from this new angle. In the end, the ion current mechanism were carried on.Chronic compressed of dorsal root ganglion model was created , prepared to integrated DRG specimen in vitro, and be classified excitability by characteristics of nonlinear dynamics with whole-cell patch clamp.1. Injection of depolarizing current steps or ramps through the recording electrodes produced spike discharges in most of the 179 DRG neurons examined.,these neurons could be classified into three classes. Class 1 neurons (n=23, 13%) showed a low threshold in response to depolarizing current steps and fring frequencies that varied almost linearly with the intensity of injected current. A similar dependency of spike frequency on current intensity was also observed in this class of cells when current steps were replaced by current ramps. Class 2 neurons (n=48, 27%) showed a higher spiking threshold and a constant fring frequency that was independent of the intensity of injected current. The intensity of current steps required to evoke action potentials in this group is clearly higher than that for class 1. Further increases in current intensity evoked more action potentials, but the frequency of the spikes remained constant .Current ramps caused this class of cells to fire spikes with long delays, again at a constant frequency. It is noted that all these cells exhibited subthreshold membrane oscillations when ramp depolarizing currents were injected. Class 3 neurons (n=108, 60%) showed the highest spiking threshold. We never observed any spontaneous firing, and the current intensities required to evoke action potentials were much higher than those sufficient for cells of the other two classes. 2. We measure INaP in DRG neurons and found that there is different distribution in 3 excitability classes neurons.3. Compare the ratio change after CCD model and possible reason. We find that disposition of 3 excitability classes neurons has changed significantly. Class 1 rise up and class 3 decrease. It suggestion that class 3 transit to class 2 or class 1, and class 2 transit to class1.4. Compare the INaP of normal and CCD neurons, find that INaP of class 1 and class 2 increases after Neuropathology injure.Main conclusions:5. There exist three excitability classes in DRG neuron. The distributed of INaP in three classification is obviously different.6. In the CCD group classification of neurons in the proportion of significant changes have taken place; which 1 and type 2 neurons INaP increased in varying degrees.7. DRG neuron injury classification changes and the ratio of the increase in tips INaP neuropathic injury induced electrophysiological changes may eventually change with INaP closely related, and this results prompt us to adjust INaP changes may be affected after the call of neuropathic injury important means of change.Part2:Anti-allodynic effects and mechanisms of riluzole in a rat model with chronically compressed dorsal root ganglion neuronsRiluzole (2-amino-6-trifluoromethoxybenzothiazole) has been used clinically in the treatment of several neurological disorders, including amyotrophic lateral sclerosis , It has also been used to treat spinal and brain injury as a neuroprotective drug and were widely used for relieving the neuropathic pain.However, the mechanisms underlying these clinical applications are far from clear. Recently, a number of reports have focused on the effects of riluzole on INaP in central neurons, and have proposed riluzole as a relatively specific persistent sodium channel blocker.. However, clinical research has shown that oral administration of riluzole does not effect thermal and mechanical hyperalgesia in patients with inflammatory pain or alleviate allodynia and mechanical hyperalgesia in neuropathic pain patients. These controversial effects of riluzole in clinic application and animal research call for simplified model systems to explore its basic mechanisms at the cellular and molecular levels. To avoid the complexity of riluzole's effects on the CNS, we have investigated its cellular effects on dorsal root ganglion (DRG) neurons. In our animal model, a chronic and steady compression is ipsilaterally applied to the DRG in rats (CCD model), causing the animals to show typical mechanical allodynia behaviors ipisilaterally. TWe focused on the effect of riluzole on the chronic pain signal to provide the theoretical support for its primary analgesia.Main results:1. Riluzole can inhibit spontaneous discharge after CCD by inhibit SMPO. To further evaluate the effects of riluzole on SMPOs of injured DRG neurons, recordings using whole-cell patch-clamp methods were performed in 28 large- and medium-sized neurons (≥35μm in diameter) in in vitro DRG preparations of CCD animals. During the injection of 800 ms depolarizing current pulses, about a third of the recorded DRG cells (10/28, 35.7%) displayed high frequency sinusoidal SMPOs and repetitive discharges. The effects of riluzole were examined in five of these neurons by bath application of the drug (10μM). It was found that the SMPOs were eliminated and that the repetitive firing was slowed down and eventually stopped 3 min after the drug application. It is interesting to note that although riluzole largely abolished the evoked SMPOs and the repetitive firing, these DRG neurons still responded to the same depolarizing current pulses with a single action potential at the initial phase of the current pulse, indicating that these cells are still capable of generating action potentials under the presence of riluzole.2. Riluzole is large inhibition for persistent sodium current (INaP) on a low dosage. Under a holding potential of -60 mV, INaP was recorded in normal DRG neurons by applying a 3 s depolarization ramp current from -80 to 0 mV. The inward sodium current was induced at potentials of -60 to -50 mV, reached a peak at -35 mV, and was sensitive to a low dose of TTX. The enhanced INaP in injured Peripheral analgesia of riluzole A-type DRG neurons was significantly inhibited by riluzole in a dose-dependent manner.3. Riluzole is low effect for transient sodium current (INaT) on a high dosage. We examined the effects of the drug on the TTX-sensitive transient sodium current (INaT) in a similar preparation of injured A-type DRG neurons. We found that INaT was significantly altered only in the presence of high dosage (500μM) riluzole。Main conclusions:Riluzole may inhibit the injured dorsal root ganglion neurons INaP, thereby inhibiting the generation of ectopic spontaneous discharges, to achieve peripheral analgesic effect.