tetrodotoxin and Peripheral-Nerve-Injuries

Topics: Animals; Botulinum Toxins; Bungarotoxins; Electromyography; Fatty Acids; Humans; Mice; Motor Neurons; Muscle Denervation; Muscles; Myasthenia Gravis; Nerve Degeneration; Nerve Regeneration; Neuromuscular Diseases; Neuromuscular Junction; Peripheral Nerve Injuries; Receptors, Cholinergic; Schwann Cells; Tetrodotoxin

The nine members of the voltage-gated sodium channel (Nav) family mediate inward sodium currents that depolarize neurons and lead to action potential firing. Increased Nav expression and function in sensory ganglia may drive ectopic action potentials and result in neuropathic pain. Using patch-clamp electrophysiology and molecular biology techniques, experiments were performed to elucidate the contribution of Nav channels to sodium currents in rat dorsal root ganglion (DRG) neurons following the L5/L6 spinal nerve ligation (SNL) model of neuropathic pain. The abundance of DRG neurons with fast, tetrodotoxin sensitive (TTX-S) currents was seven-fold higher whereas the abundance of DRG neurons with slow, tetrodotoxin resistant (TTX-R) currents was nearly thirty-fold lower when comparing ipsilateral (injured) to contralateral (uninjured) neurons. TTX-S currents were elevated in larger neurons while TTX-R currents were reduced in both small and large neurons. Among Nav transcripts encoding TTX-R channels, Scn10a (Nav1.8) and Scn11a (Nav1.9) expression was twenty- to thirty-fold lower, while among Nav transcripts encoding TTX-S channels, Scn3a (Nav1.3) expression was four-fold higher in injured compared to uninjured DRG by qRT-PCR analysis. In summary, the SNL model of neuropathic pain induced a phenotypic switch in Nav expression from TTX-R to TTX-S channels in injured DRG neurons. Transcriptional reprogramming of Nav genes may drive ectopic action potential firing and contribute to neuropathic pain.

Topics: Animals; Biophysical Phenomena; Electric Stimulation; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Peripheral Nerve Injuries; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Channel Blockers; Tetrodotoxin; Voltage-Gated Sodium Channels

Satellite glial cells in the dorsal root ganglion (DRG), like the better-studied glia cells in the spinal cord, react to peripheral nerve injury or inflammation by activation, proliferation, and release of messengers that contribute importantly to pathological pain. It is not known how information about nerve injury or peripheral inflammation is conveyed to the satellite glial cells. Abnormal spontaneous activity of sensory neurons, observed in the very early phase of many pain models, is one plausible mechanism by which injured sensory neurons could activate neighboring satellite glial cells. We tested effects of locally inhibiting sensory neuron activity with sodium channel blockers on satellite glial cell activation in a rat spinal nerve ligation (SNL) model. SNL caused extensive satellite glial cell activation (as defined by glial fibrillary acidic protein [GFAP] immunoreactivity) which peaked on day 1 and was still observed on day 10. Perfusion of the axotomized DRG with the Na channel blocker tetrodotoxin (TTX) significantly reduced this activation at all time points. Similar findings were made with a more distal injury (spared nerve injury model), using a different sodium channel blocker (bupivacaine depot) at the injury site. Local DRG perfusion with TTX also reduced levels of nerve growth factor (NGF) in the SNL model on day 3 (when activated glia are an important source of NGF), without affecting the initial drop of NGF on day 1 (which has been attributed to loss of transport from target tissues). Local perfusion in the SNL model also significantly reduced microglia activation (OX-42 immunoreactivity) on day 3 and astrocyte activation (GFAP immunoreactivity) on day 10 in the corresponding dorsal spinal cord. The results indicate that early spontaneous activity in injured sensory neurons may play important roles in glia activation and pathological pain.

Topics: Animals; Biomarkers; Bupivacaine; CD11b Antigen; Disease Models, Animal; Ganglia, Spinal; Glial Fibrillary Acidic Protein; Gliosis; Ligation; Male; Microglia; Nerve Growth Factor; Neuralgia; Peripheral Nerve Injuries; Peripheral Nerves; Peripheral Nervous System Diseases; Rats; Rats, Sprague-Dawley; Satellite Cells, Perineuronal; Sensory Receptor Cells; Sodium Channel Blockers; Tetrodotoxin; Time Factors

Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch-clamp recordings were obtained in vitro from L4 and/or L5 dissociated, small DRG neurons following in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na+ currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V1/2) and curve slope (k) of steady-state inactivation of Na+ currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX-S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD-induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na+ currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons.

Topics: Action Potentials; Animals; Electrophysiology; Ganglia, Spinal; Hyperalgesia; Neurons; Peripheral Nerve Injuries; Rats; Sodium; Sodium Channels; Spinal Cord Compression; Tetrodotoxin

Although anticonvulsant drugs that block voltage-dependent Na+ channels have been widely used for neuropathic pain, including peripheral nerve injury-induced pain, much less is known about the actions of these drugs on immature trigeminal ganglion (TG) neurons. Here we report the effects of carbamazepine (CBZ) and amitriptyline (ATL) on tetrodotoxin-resistant (TTX-R) Na' channels expressed on immature rat TG neurons. TTX-R Na+ currents (I(Na)) were recorded in the presence of 300 nM TTX by use of a conventional whole-cell patch clamp method. Both CBZ and ATL inhibited TTX-R I(Na) in a concentration-dependent manner, but ATL was more potent. While CBZ and ATL did not affect the overall voltage-activation relationship of TTX-R Na+ channels, both drugs shifted the voltage-activation relationship to the left, indicating that they inhibited TTX-R Na+ channels more efficiently at depolarized membrane potentials. ATL showed a profound use-dependent blockade of TTX-R I(Na), but CBZ had little effect. The present results suggest that both CBZ and ATL, common drugs used for treating neuropathic pain, efficiently inhibit TTX-R Na+ channels expressed on immature TG neurons, and that these drugs might be useful for the treatment of trigeminal nerve injury-induced neuropathic pain, as well as the inhibition of ongoing central sensitization, even during immature periods.

Topics: Amitriptyline; Animals; Anticonvulsants; Antidepressive Agents, Tricyclic; Carbamazepine; Drug Resistance; Electrophysiology; Neurons; Patch-Clamp Techniques; Peripheral Nerve Injuries; Rats; Rats, Sprague-Dawley; Sodium Channels; Tetrodotoxin; Trigeminal Ganglion

Some chronic pain conditions are maintained or enhanced by sympathetic activity. In animal models of pathological pain, abnormal sprouting of sympathetic fibers around large- and medium-sized sensory neurons is observed in dorsal root ganglia (DRGs). Large- and medium-sized cells are also more likely to be spontaneously active, suggesting that sprouting may be related to neuron activity. We previously showed that sprouting could be reduced by systemic or locally applied lidocaine. In the complete sciatic nerve transection model in rats, spontaneous activity initially originates in the injury site; later, the DRGs become the major source of spontaneous activity. In this study, spontaneous activity reaching the DRG soma was reduced by early nerve blockade (local perfusion of the transected nerve with TTX for the 1st 7 days after injury). This significantly reduced sympathetic sprouting. Conversely, increasing spontaneous activity by local nerve perfusion with K(+) channel blockers increased sprouting. The hyperexcitability and spontaneous activity of DRG neurons observed in this model were also significantly reduced by early nerve blockade. These effects of early nerve blockade on sprouting, excitability, and spontaneous activity were all observed 4-5 wk after the end of early nerve blockade, indicating that the early period of spontaneous activity in the injured nerve is critical for establishing the more long-lasting pathologies observed in the DRG. Individual spontaneously active neurons, labeled with fluorescent dye, were five to six times more likely than quiescent cells to be co-localized with sympathetic fibers, suggesting a highly localized correlation of activity and sprouting.

Topics: Action Potentials; Animals; Autonomic Nerve Block; Axotomy; Disease Models, Animal; Female; Fluorescent Dyes; Ganglia, Spinal; Growth Cones; Nerve Regeneration; Neural Conduction; Neuralgia; Neurons, Afferent; Organ Culture Techniques; Peripheral Nerve Injuries; Peripheral Nerves; Peripheral Nervous System Diseases; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Sodium Channel Blockers; Sympathetic Fibers, Postganglionic; Tetrodotoxin; Time Factors

It is generally believed that nerve injury results in neuronal hyperexcitability that reflects in part a change in Na+ currents. However, there are conflicting data on the nature of Na+ current changes and the association between alterations in Na+ currents and increases in excitability. One potential source of conflicting data is that injured and spared neurons may respond differently to nerve injury; these subpopulations of neurons have not been distinguished in previous studies with the axotomy model of nerve injury (complete transection of the sciatic nerve). The present study was performed to determine the relationship between changes in Na+ channels and changes in neuronal excitability in identified injured dorsal root ganglion neurons post-axotomy. Small (< 45 pF) neurons labeled with a DiI injection into the sciatic nerve were studied 10 days and 4 weeks post-axotomy. Ten days post-axotomy, tetrodotoxin-resistant (TTX-R) Na+ current (INa) was decreased and TTX-sensitive (TTX-S) INa was increased, however, excitability was unchanged. Four weeks post-axotomy, neurons had become hyperexcitable while TTX-R INa remained reduced and TTX-S INa had returned to control levels. Thus, axotomy-induced changes in Na+ currents were not correlated with an axotomy-induced change in excitability. Additional analysis of axotomized neurons suggested that concomitant changes in other ionic currents occurred. These results suggest that neuronal excitability following axotomy is dependent on the sum of changes in ionic currents, and the overall effect on excitability may not always correspond to that predicted by a change in a single class of voltage-gated ion channel.

Topics: Action Potentials; Animals; Axotomy; Carbocyanines; Cell Membrane; Cells, Cultured; Ganglia, Spinal; Male; Membrane Potentials; Neural Conduction; Neurons, Afferent; Patch-Clamp Techniques; Peripheral Nerve Injuries; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Topics: Animals; Chronic Disease; Drug Resistance; Ion Channel Gating; Ligation; Lumbosacral Region; NAV1.7 Voltage-Gated Sodium Channel; NAV1.8 Voltage-Gated Sodium Channel; Neurons, Afferent; Neuropeptides; Oligodeoxyribonucleotides, Antisense; Pain; Pain, Intractable; Peripheral Nerve Injuries; Peripheral Nerves; Rats; Sodium Channels; Spinal Nerves; Tetrodotoxin