tetrodotoxin and Sciatica

Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN channels) have large influences upon neuronal excitability. However, the participation of spinal HCN channels in chronic pain states, where pathological conditions are related to altered neuronal excitability, has not been clarified. Intraperitoneally (i.p.) or intrathecally (i.t.) administered ZD7288, a selective blocker of Ih channels, reduced thermal and mechanical hypersensitivity in mice under neuropathic conditions induced by the partial ligation of the sciatic nerve, while no analgesic effect was observed in naïve animals. Moreover, in the mouse formalin test, ZD7288 (i.p. and i.t.) reduced the licking/biting behavior observed during the second phase without affecting the first phase. To further explore the pain-modulatory action of spinal HCN channels, whole-cell patch clamp recordings were made from the visually identified substantia gelatinosa neurons in adult mouse spinal cord slices with an attached dorsal root, and A-fiber- and/or C-fiber-mediated monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the L4 or L5 dorsal root using a suction electrode. Bath-applied ZD7288 reduced A-fiber- and C-fiber-mediated monosynaptic EPSCs more preferentially in slices prepared from mice after peripheral nerve injury. In addition, ZD7288 reduced the frequency of miniature EPSCs without affecting their amplitude in cells receiving monosynaptic afferent inputs, indicating that it inhibits EPSCs via presynaptic mechanisms. The present behavioral and electrophysiological data suggest that spinal HCN channels, most likely at the primary afferent terminals, contribute to the maintenance of chronic pain.

Topics: Animals; Cardiotonic Agents; Chronic Disease; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; Hyperalgesia; In Vitro Techniques; Male; Membrane Potentials; Mice; Mice, Neurologic Mutants; Nerve Fibers; Pain Measurement; Patch-Clamp Techniques; Presynaptic Terminals; Pyrimidines; Sciatica; Sodium Channel Blockers; Spinal Cord; Tetrodotoxin

A large body of evidence has demonstrated that the ectopic discharges of action potentials in primary afferents, resulted from the abnormal expression of voltage gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons following peripheral nerve injury are important for the development of neuropathic pain. However, how nerve injury affects the expression of VGSCs is largely unknown. Here, we reported that selective injury of motor fibers by L5 ventral root transection (L5-VRT) up-regulated Nav1.3 and Nav1.8 at both mRNA and protein level and increased current densities of TTX-S and TTX-R channels in DRG neurons, suggesting that nerve injury may up-regulate functional VGSCs in sensory neurons indirectly. As the up-regulated Nav1.3 and Nav1.8 were highly co-localized with TNF-α, we tested the hypothesis that the increased TNF-α may lead to over-expression of the sodium channels. Indeed, we found that peri-sciatic administration of recombinant rat TNF-α (rrTNF) without any nerve injury, which produced lasting mechanical allodynia, also up-regulated Nav1.3 and Nav1.8 in DRG neurons in vivo and that rrTNF enhanced the expression of Nav1.3 and Nav1.8 in cultured adult rat DRG neurons in a dose-dependent manner. Furthermore, inhibition of TNF-α synthesis, which prevented neuropathic pain, strongly inhibited the up-regulation of Nav1.3 and Nav1.8. The up-regulation of the both channels following L5-VRT was significantly lower in TNF receptor 1 knockout mice than that in wild type mice. These data suggest that increased TNF-α may be responsible for up-regulation of Nav1.3 and Nav1.8 in uninjured DRG neurons following nerve injury.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Electric Stimulation; Functional Laterality; Ganglia, Spinal; Immunosuppressive Agents; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Neurons; NAV1.3 Voltage-Gated Sodium Channel; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor, Type I; RNA, Messenger; Sciatica; Sensory Receptor Cells; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Thalidomide; Tumor Necrosis Factor-alpha; Up-Regulation

The biophysical properties of a tetrodotoxin resistant (TTXr) sodium channel, Na(V)1.8, and its restricted expression to the peripheral sensory neurons suggest that blocking this channel might have therapeutic potential in various pain states and may offer improved tolerability compared with existing sodium channel blockers. However, the role of Na(V)1.8 in nociception cannot be tested using a traditional pharmacological approach with small molecules because currently available sodium channel blockers do not distinguish between sodium channel subtypes. We sought to determine whether small interfering RNAs (siRNAs) might be capable of achieving the desired selectivity. Using Northern blot analysis and membrane potential measurement, several siRNAs were identified that were capable of a highly-selective attenuation of Na(V)1.8 message as well as functional expression in clonal ND7/23 cells which were stably transfected with the rat Na(V)1.8 gene. Functional knockdown of the channel was confirmed using whole-cell voltage-clamp electrophysiology. One of the siRNA probes showing a robust knockdown of Na(V)1.8 current was evaluated for in vivo efficacy in reversing an established tactile allodynia in the rat chronic constriction nerve-injury (CCI) model. The siRNA, which was delivered to lumbar dorsal root ganglia (DRG) via an indwelling epidural cannula, caused a significant reduction of Na(V)1.8 mRNA expression in lumbar 4 and 5 (L4-L5) DRG neurons and consequently reversed mechanical allodynia in CCI rats. We conclude that silencing of Na(V)1.8 channel using a siRNA approach is capable of producing pain relief in the CCI model and further support a role for Na(V)1.8 in pathological sensory dysfunction.

Topics: Anesthetics, Local; Animals; Blotting, Northern; Cell Line, Tumor; Disease Models, Animal; Drug Interactions; Electric Stimulation; Hyperalgesia; Male; Membrane Potentials; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neuroblastoma; Patch-Clamp Techniques; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sciatica; Sodium Channels; Tetrodotoxin; Time Factors; Transfection

Abnormal impulses caused by very slowly inactivating Na channels of peripheral nerve have been proposed to play a critical role in neuropathic pain. Low concentrations of local anesthetics, often effective in treating experimental and clinical neuropathic pain, are also known to potently suppress the long after-depolarizations induced by these persistently open Na channels. However, these drug actions on impulses that have propagated away from such sites are undetermined. In the present study, the focal application of anemone toxin II (ATX, 300 nM), which slows Na-channel inactivation, produced prolonged depolarizing after-potentials and, coincidentally, induced spontaneous bursting impulse activity that propagated away from the site of ATX application in the frog sciatic nerve in vitro. The application of low concentrations of lidocaine (1-10 microM), both at the site of ATX exposure and at a distant site, selectively and reversibly inhibited the spontaneous bursting, while having no effect on the electrically stimulated initial spike of the compound action potential. Inhibition occurred as a shortening of burst episodes rather than a reduction in frequency of impulses within a burst or a reduction of intraburst impulse amplitude. Tetrodotoxin also inhibited the induced spontaneous activity, but only at concentrations that also depressed the compound action potential spike. These findings show that low concentrations of lidocaine can restore normal firing patterns in nerve where hyperexcitability has been caused by delayed Na-channel inactivation, without acting directly at the site where ectopic impulses are generated. Thus, it appears that the pattern of abnormal activity rather than an abnormally gating Na channel per se can be a target for lidocaine's therapeutic action.

Topics: 4-Aminopyridine; Action Potentials; Anesthetics, Local; Animals; Cnidarian Venoms; Electric Stimulation; Lidocaine; Potassium Channel Blockers; Rana catesbeiana; Rana pipiens; Sciatica; Tetrodotoxin